Novel anti-NaPi2b antibodies and antibody-drug conjugates based thereon, therapeutic methods, and their use

Abstract

This invention relates to a novel anti-NaPi2b antibody, an antibody-drug conjugate (ADC) based thereon, and therapeutic methods and their use, particularly in relation to cancer treatment.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to European Patent Application No. 22202150, filed with the European Patent Office on 18 October 2022, which for all purposes is incorporated herein by reference in its entirety.

[0002] Sequence List This application includes a sequence listing in a computer-readable form, which is incorporated herein by reference.

[0003] Technical field of inventions This invention relates to a novel anti-NaPi2b antibody, an antibody-drug conjugate (ADC) based thereon, and therapeutic methods and their use, particularly in relation to cancer treatment. [Background technology]

[0004] Background of the Invention NaPi2b, encoded by the SLC34A2 gene, is a multi-pass transmembrane sodium-dependent phosphate transporter expressed in human lung, ovarian, and thyroid cancers, as well as in the normal tissues from which these tumors originate. As a member of the SLC34 solute transporter protein family, it is responsible for transcellular inorganic phosphate uptake and maintenance of phosphate homeostasis, and is associated with cell differentiation and tumorigenesis. Napi2b mRNA / protein expression has been found in non-squamous non-small cell lung cancer, non-mucinous ovarian cancer, and papillary thyroid carcinoma; expression in normal tissues has been reported in the lungs, bronchi, and kidneys (Lin et al., 2015). In normal lung tissue, NaPi2b is involved in phosphate transport, and mutations in SLC34A2 have been associated with pulmonary and testicular microlithiasis (Corut et al., 2006). Napi2b is a promising target for ADC therapy due to its differential expression in tumors compared to most normal tissues, its prominent localization to the cell surface, and its high endocytosis rate. Although several first-generation antibody-drug conjugates have been developed in the past, these either do not provide long-term survival benefits or are associated with dose-limiting toxicity.

[0005] Therefore, there is a need for novel antibodies with improved characteristics, and ADCs based on them that have improved toxicity profiles and higher functionality. Accordingly, the technical objective of the present invention is to satisfy this need.

[0006] The present invention satisfies this need by providing, in particular, a novel anti-NaPi2b antibody that reduces the tendency for antibody modification by post-translational or post-expression and post-purification modifications (e.g., deamide), improves on-target binding, improves target-dependent internal transit rates, limits the tendency for aggregation and HMWS formation, and exhibits cross-reactivity with rat NaPi2b and cynomolgus monkey NaPi2b having similar binding ability to human NaPi2b (thus enabling better toxicity analysis and intercomparison); a novel NaPi2b-targeted antibody-drug conjugate (ADC) based thereon, produced by applying P5 conjugation technology (e.g., WO2018 / 041985) (based on modification of interchain cysteine ​​residues with unsaturated phosphoamidate reagents); and therapeutic methods and their use, particularly relating to cancer treatment. [Overview of the project]

[0007] This technical challenge is resolved by the provisions defined in the claims.

[0008] Therefore, the present invention relates to an anti-NaPi2b antibody (for example, an antibody against NPT2B_human sodium-dependent phosphate transporter protein 2B (e.g., having UniProt accession number: O95436 or SEQ ID NO: 1) and / or an antibody against NPT2B_rat sodium-dependent phosphate transporter protein 2B (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO: 2)), wherein (a) the NaPi2b antibody is capable of binding to human Napi2b (e.g., SEQ ID NO: 1) and / or rat Napi2b (e.g., SEQ ID NO: 2), and preferably, the binding to human Napi2b and rat Napi2b is substantially the same K D The antibody is selected from the group consisting of AV-25 antibody, AV-15 antibody, AV-18 antibody, AV-21 antibody, and AV-29 antibody, and most preferably the same K D(b) The NaPi2b antibody has a difference of up to 50% (e.g., a difference of up to 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 7%, 5%, 4%, 3%, 2%, or 1%); (c) The NaPi2b antibody is cross-reactive with rat Napi2b (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO: 2); (d) The NaPi2b antibody is cross-reactive with cynomolgus monkey (e.g., Macaca fascicularis) Napi2b (e.g., UniProtKB accession number: A0A2K5UHY1 or SEQ ID NO: (d) The NaPi2b antibody is capable of cross-reactivity with (3) the antigen-mediated internal transfer of the antibody; (e) The NaPi2b antibody optionally has a heavy chain variable region (V H It is possible for the CDR2 of ) to not have a dipeptide deamide moiety, and preferably, the absent dipeptide deamide moiety is V H This is an NG (Asn-Gly) within CDR2.

[0009] The present invention further relates to the monoclonal human or humanized IgG1 anti-Napi2b antibody of the present invention, preferably comprising a kappa (κ) light chain.

[0010] The present invention also relates to a hybridoma that produces the antibody of the present invention.

[0011] The present invention also relates to nucleic acids encoding the antibodies of the present invention.

[0012] The present invention also relates to an expression vector comprising at least one of the nucleic acid molecules of the present invention.

[0013] The present invention also relates to isolated host cells (e.g., isolated recombinant host cells) containing the vector and / or nucleic acids of the present invention.

[0014] The present invention also relates to an antibody-drug conjugate (ADC) comprising the anti-Napi2b antibody of the present invention.

[0015] The present invention further relates to an antibody-drug conjugate (ADC) of the present invention, comprising an anti-NaPi2b antibody of the present invention (e.g., a humanized monoclonal NaPi2b-specific IgG1 antibody) conjugated to a cytotoxic payload / drug, wherein (a) the cytotoxic payload is selected from the group consisting of camptothecin, mytansinoids, calicheamycin, duochamycin, tubulisin, amatoxin, drastatin, and auristatin, e.g., monomethyl auristatin E (MMAE), pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogs or prodrugs thereof; and / or (b) the cytotoxic payload is exatecan, DXD, SN38, camptothecin, topotecan, irinotecan, berotecan, (c) The cytotoxic payload is a camptothecin moiety C selected from the group consisting of lulutotecan, rubitecan, silatecan, cositecan, and gimatecan; and / or (c) the cytotoxic payload is conjugated via a cleavable linker (L), preferably the linker L is cleavable by a protease, glucuronidase, sulfatase, phosphatase, esterase, or disulfide reduction, and more preferably the linker is cleavable by a protease, preferably a cathepsin such as cathepsin B; and / or (d) the linker (L) comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; and / or (e) the cytotoxic payload is an exatecan conjugated via a chemical valine-citrulline-PAB releasing unit or a valine-alanine-PAB releasing unit, the releasing unit being cleavable by a protease.Preferably, the drug-to-antibody ratio (DAR) is in the range of 0 to 20, more preferably in the range of 4 to 8, and most preferably 4 or 8.

[0016] The present invention also relates to compositions or kits comprising the anti-NaPi2b, antibody-drug conjugate (ADC), hybridoma, nucleic acid, expression vector, and / or host cell of the present invention.

[0017] The present invention also relates to a method for synthesizing antibody-drug conjugates (ADCs) of the present invention.

[0018] The present invention further relates to methods of treatment, as well as to the use of the antibodies, antibody-drug conjugates (ADCs), nucleic acids, expression vectors, host cells, compositions, and / or kits of the present invention.

[0019] Sequence List Overview As described herein, unless otherwise specified, UniProtKB accession number ( https: / / www.uniprot.org / release-notes / 2022-08-03-release For example, see the UniProt release 2022_03, which was released on August 3, 2022.

[0020] This application includes a sequence listing in a computer-readable form, which is incorporated herein by reference. [Brief explanation of the drawing]

[0021] The present invention will be better understood by referring to the detailed description and considering in conjunction with the non-limiting embodiments and accompanying drawings, respectively. The drawings are shown below: [Figure 1-1] The following is an example sequence of the anti-NaPi2b antibody of the present invention. [Figure 1-2] This is a continuation of Figure 1-1. [Figure 1-3] This is a continuation of Figure 1-2. [Figure 2] Charge variant analysis of an exemplary antibody of the present invention is shown. [Figure 3] The graph shows the binding to human and rat NaPi2B, as evaluated by flow cytometry. The graph shows the mean (n=2) ± SEM. [Figure 4] The graph shows the binding to immobilized human NaPi2B as evaluated by ELISA. The graph shows the mean (n=2) ± SEM. [Figure 5] This shows internal migration evaluated by flow cytometry. [Figure 6] The melting curve evaluated by NanoDSF is shown. [Figure 7] This graph shows in vitro cytotoxicity in NaPi2B-positive cells as evaluated by the resazurin assay. The graph shows the mean (n=2) ± SEM. [Figure 8] This demonstrates the bystander activity of ADC. [Figure 9] This study demonstrates in vitro inhibition of topoisomerase I by exatecan delivery via ADC. [Figure 10] This shows the in vivo PK evaluation of ADCs and non-conjugate antibodies. [Figure 11] The UV chromatogram obtained by LC / MS measurement of P5(PEG12)-COOH is shown. [Figure 12] The UV chromatogram obtained by LC / MS measurement of P5(PEG24)-OSu is shown. [Figure 13] The UV chromatogram obtained by LC / MS measurement of NH2-VC-PAB-exatecan TFA salt is shown. [Figure 14] The UV chromatogram obtained by LC / MS measurement of NH2-VA-PAB-exatecan TFA salt is shown. [Figure 15]The UV chromatogram obtained by LC / MS measurement of P5(PEG2)-VC-PAB-exatecan is shown. [Figure 16] The UV chromatogram obtained by LC / MS measurement of P5(PEG12)-VC-PAB-exatecan is shown. [Figure 17] The UV chromatogram obtained by LC / MS measurement of P5(PEG24)-VC-PAB-exatecan is shown. [Figure 18] The UV chromatogram obtained by LC / MS measurement of P5(PEG12)-VA-PAB-exatecan is shown. [Figure 19] The UV chromatogram obtained by LC / MS measurement of P5(PEG12)-exatecan is shown. [Figure 20] This paper presents analytical characterization of DAR8-ADCs derived from synthesized mAbs and P5(PEG24)-VC-PAB-exatecan. [Figure 21] The analytical raw data for AV25 is shown. (A-D) Analytical characterization of one of the mAbs described. The antibody was expressed in Expi-CHO cells as previously described and purified by protein A chromatography. The mAbs were analyzed by HLPC-SEC (A), LC-MS (B), HLPC-HIC (C), and reduced SDS-PAGE (D). [Figure 22] The LC / MS analysis of the parent antibody is shown. [Figure 23] The LC / MS analysis of AV15 is shown. [Figure 24] The LC / MS analysis of AV18 is shown. [Figure 25] The LC / MS analysis of AV21 is shown. [Figure 26] The LC / MS analysis of AV25 is shown. [Figure 27] The LC / MS analysis of AV29 is shown. [Figure 28]The analytical raw data for AV25-P5(PEG24)-VC-PAB-exatecan DAR8 is shown. (A-D) Analytical characterization of one of the ADCs synthesized and purified as described above. A) Analytical size exclusion chromatography, B) LC-MS of the ADC preparation, C) Analytical hydrophobic interaction chromatography after conjugation. The data shows that the ADC was completely conjugated to DAR8 and that only a small amount of aggregates were present after purification. [Figure 29] The LC / MS analysis of parent P5(PEG24)-VC-PAB-exatecan DAR8 is shown. [Figure 30] The LC / MS analysis of AV15-P5(PEG24)-VC-PAB-exatecan (DAR8), an exemplary ADC of the present invention, is shown. [Figure 31] The LC / MS analysis of AV18-P5(PEG24)-VC-PAB-exatecan (DAR8), an exemplary ADC of the present invention, is shown. [Figure 32] The LC / MS analysis of AV21-P5(PEG24)-VC-PAB-exatecan (DAR8), an exemplary ADC of the present invention, is shown. [Figure 33] The LC / MS analysis of AV25-P5(PEG24)-VC-PAB-exatecan (DAR8), an exemplary ADC of the present invention, is shown. [Figure 34] The LC / MS analysis of AV29-P5(PEG24)-VC-PAB-exatecan (DAR8), an exemplary ADC of the present invention, is shown. [Figure 35] The results of ex vivo serum stability analysis of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8), a representative ADC of the present invention, are shown. [Figure 36] The results of in vivo efficacy analysis of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8), a representative ADC of the present invention, in a cell line-derived xenograft model (CDX) are shown. [Figure 37-1]The results of an in vivo efficacy analysis of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8), a representative ADC of the present invention, in a patient-derived xenograft model (PDX) are shown. [Figure 37-2] See the explanation in Figure 37-1. [Figure 37-3] See the explanation in Figure 37-1. [Figure 37-4] See the explanation in Figure 37-1. [Figure 38] The results of in vivo toxicity analysis of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8), a representative ADC of the present invention, in cynomolgus monkeys are shown. [Figure 39] MS analysis of anti-Napi2b comparative ADC synthesized by the method described above is shown. The signal is annotated with the measured mass (in Daltons) and absolute intensity. Exemplary spectra derived from the conjugation reaction are shown. The drug-to-antibody ratio of the final conjugate was estimated to be 3.5–4.0 from the MS signal. LC: light chain of anti-Napi2b comparative antibody, HC: heavy chain of anti-Napi2b comparative antibody. [Figure 40] The binding of unmodified AV25 mAb and AV25-P5(PEG24)-VC-PAB-exatecan DAR8 ADC to HEK293 cells transfected with Napi2a (left), Napi2b (center), and Napi2c (right) is shown as the MFI ratio. AV25 mAb and AV25-P5(PEG24)-VC-PAB-exatecan DAR8 ADC specifically bind to NaPi2b. The graph shows n=2±SD (note that the error bars are too small to display). [Figure 41] This report shows the cytotoxic dose-response of AV25-P5(PEG24)-VC-PAB-exatecan DAR 8 compared to an anti-Napi2b comparative ADC in three different cell lines. The mean and standard deviation of two measurements, as well as dose-response fitting, are shown. Fluorescence (in %) compared to the medium control is equal to the percentage of viable cells (%). [Figure 42]This study demonstrates dose-dependent induction of signs of DNA damage and apoptosis in response to treatment with increasing concentrations of AV25-P5(PEG24)-VC-PAB-exatecan DAR8. Corresponding non-targeting isotype control conjugates were included as negative controls. HCC-78 cells (A, high NaPi2b) and OVCAR-3 cells (B, high NaPi2b) were treated with increasing concentrations (0.05–12 μg / ml) of AV25-P5(PEG24)-VC-PAB-exatecan DAR8 or isotype ADC (isotype-P5(PEG24)-VC-PAB-exatecan DAR8) for 72 hours. Cells were stained for cleaved PARP (left), caspase 3 (center), and pH2AX (right) and analyzed by flow cytometry. Graphs show mean (n=2) ± SEM. [Figure 43] This graph shows the binding of the Fc region of AV25 HC-LALA and AV25 HC-wt antibodies to recombinant hexameric C1q complement protein, measured using a human C1q binding assay based on HTRF (homogenous time-resolved fluorescence) (HTRF Human C1q Binding Kit, Cisbio), as described by the manufacturer. In short, serial dilutions from 280 nM to 11.6 nM were measured for all antibodies tested (AV25 HC-wt, AV25 HC-LALA, α-MHC-I positive, IgG1 kit standard). The HTRF ratio was calculated by dividing the acceptor emission signal at 665 nm by the donor emission signal at 620 nm and multiplying by 10000. The graph shows the HTRF ratio at a concentration of 70 nM, after subtracting the background (dilution only) HTRF ratio. The graph shows the mean (n=2) ± SD. [Figure 44]This shows dose-dependent binding of the Fc region of AV25-HC-LALA antibody, AV25-P5(PEG24)-VC-PAB-exatecan DAR8 ADC, and AV25-HC-wt antibody to recombinant human FcRn and FcγR. This was measured using the Lumit® FcγR-binding immunoassay (FcγRn, FcγRI, FcγRIIa / CD32 R131 / H131 polymorphism, FcγRIIIa / CD16 V158 / F158 polymorphism, Promega) according to the manufacturer's instructions. Serial dilutions of AV25 HC-wt, AV25 HC-LALA, and AV25-P5(PEG24)-VC-PAB-exatecan DAR8, standard materials, and trastuzumab as a positive control were incubated with Tracer-LgBiT and FcγR-SmBiT. In the absence of an antibody analyte, or if no interaction occurs between the antibody under test and FcγR, Tracer-LgBiT binds to the FcγR-SmBiT target, resulting in a maximum luminescence signal. If successful interaction with FcγR occurs, the antibody / ADC under test competes with Tracer-LgBiT for binding to the FcγR target, resulting in a concentration-dependent decrease in the luminescence signal. Luminescence was measured using a microplate reader Infinite M200 Pro (Tecan). The graph shows n=1. [Figure 45] This describes an antibody-dependent cell-mediated cytotoxicity (ADCC) assay based on calcein release. Co-cultures of healthy donor (HD) NK cells and calcein-stained target-positive tumor cells (OVCAR-3 and HCC-78) in a 4:1 ratio were incubated with 15 μg / ml of a specified antibody or ADC (anti-MHC-I antibody served as a positive control). Specific cell death rate (%) was calculated by dividing the calcein released by antibody-mediated cell death by the calcein released by cells permeabilized with Triton X (maximum death). The graph shows the mean (n=2) ± SEM. [Figure 46]The results of in vivo efficacy analysis of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8), a representative ADC of the present invention, in a patient-derived non-small cell lung cancer (NSCLC) xenograft model (PDX, Lu7700) are shown. The left side shows the time course of tumor volume after a single treatment on day 0 for various dose levels of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8) (1 mg ADC / kg body weight, 3 mg ADC / kg body weight, and 5 mg ADC / kg body weight) and an isotype control with the same linker payload (5 mg / kg), compared to the untreated (vehicle) group. The right side shows the body weight of animals treated with various dose levels of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8) and an isotype control with the same linker payload, compared to the untreated (vehicle) group. All results are shown as average values ​​and SEM values ​​for 4 animals per group. [Figure 47]Figure 47A shows the results of an in vivo efficacy analysis of AV25-P5(PEG24)-VC-PAB-exatecan DAR 8, a representative ADC of the present invention, in a patient-derived ovarian cancer xenograft model (PDX, Ov6668). The left side shows the time course of tumor volume after a single treatment on day 0 with various dose levels of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8) (1 mg ADC / kg body weight, 3 mg ADC / kg body weight, and 5 mg ADC / kg body weight), compared to the untreated (vehicle) group. The right side shows the body weight of animals treated with various dose levels of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8) compared to the untreated (vehicle) group. All results are shown as mean values ​​and SEMs for 9 animals per group. Figure 47B shows the in vivo PK evaluation of total and intact ADCs at three dose levels in the dose-response efficacy study, as the mean and standard deviation of three measurements per time point obtained from mice treated once on day 0 with various dose levels of AV25-P5(PEG24)-VC-PAB-exatecan (1 mg ADC / kg body weight, 3 mg ADC / kg body weight, and 5 mg ADC / kg body weight). PK evaluation was performed as described in Example 1. [Figure 48] The results of in vivo efficacy analysis of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8), a representative ADC of the present invention, in a patient-derived ovarian cancer xenograft model are shown. The top section shows the time course of tumor volume after a single treatment on day 0 with 10 mg ADC / kg body weight of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8) or isotype-P5(PEG24)-VC-PAB-exatecan (DAR 8), compared to the untreated (vehicle) group. The bottom section shows the NaPi2B low-expression BRCA mutation model (bottom left) and the NaPi2B high-expression BRCA mutation model (bottom right). All results are shown as mean values ​​and SEM values ​​from 3 animals per group. [Figure 49]The dose-dependent binding of escalating upifitamab or AV25 to two different NaPi2b-positive cell lines (left: OVCAR-3, right: HCC78) is shown normalized to a nonspecific binding control and expressed as the MFI ratio. The mean and standard deviation of the two measurements, as well as the dose-response fitting, are shown. [Figure 50] The cytotoxic dose-response of AV25-P5(PEG24)-VC-PAB-exatecan DAR 8 in two different cell lines is shown, compared to upifitamab-P5(PEG24)-VC-PAB-exatecan DAR 8 and its isotype control, isotype-P5(PEG24)-VC-PAB-exatecan DAR 8. The mean and standard deviation of the two measurements, as well as the dose-response fitting, are shown. Fluorescence (in %) compared to the culture medium control is equal to the percentage of viable cells (%). [Modes for carrying out the invention]

[0022] Detailed description of the invention The present invention is described in detail below and is also illustrated by the accompanying embodiments and drawings.

[0023] The inventors have produced and characterized a novel specific anti-NaPi2b antibody to specifically target the extracellular domain of NaPi2b. This is particularly advantageous because it relates to a new therapeutic method for treating cancer (for example, preferably the cancer is a solid tumor and / or metastatic cancer, and more preferably the cancer is selected from the group consisting of lung cancer, ovarian cancer, thyroid cancer, non-squamous non-small cell lung cancer, non-mucinous ovarian cancer, papillary thyroid cancer, kidney cancer, endometrial cancer, uterine cancer, ureteral cancer, bladder cancer, and fallopian tube cancer).

[0024] The present invention provides a novel anti-NaPi2b antibody that exhibits reduced tendency for antibody modification by post-translational modification (e.g., deamide), improved on-target binding, improved target-dependent internal migration rate, limited tendency for aggregation and HMWS formation, and cross-reactivity with rat NaPi2b and cynomolgus monkey NaPi2b having similar binding ability to human NaPi2b (thus enabling better toxicity analysis and intercomparison); a novel NaPi2b-targeted antibody-drug conjugate (ADC) based thereon, generated by applying P5 conjugation technology (e.g., WO2018 / 041985) (based on modification of interchain cysteine ​​residues with an unsaturated phosphoamidate reagent); and therapeutic methods and their use, particularly in relation to cancer treatment.

[0025] The novel antibodies of the present invention optionally contain Fc silencing mutations such as leucine (L) to alanine (A) substitutions at positions 234 and 235 (LALA mutations), bind to human Napi2b (e.g., SEQ ID NO: 1) and / or rat Napi2b (e.g., SEQ ID NO: 2), exhibit cross-reactivity with rat Napi2b and cynomolgus monkey Napi2b, and optionally contain heavy chain variable region (V H ) does not have a dipeptide deamide moiety within CDR2, preferably the absent dipeptide deamide moiety is V H It is a humanized anti-NaPi2b monoclonal antibody (mAb) within CDR2, specifically NG (Asn-Gly).

[0026] The antibody-drug conjugate (ADC) of the present invention comprises an antibody against the anti-NaPi2b antibody of the present invention (NPT2B_human sodium-dependent phosphate transport protein 2B (e.g., having UniProt accession number: O95436 or SEQ ID NO: 1) and / or an antibody against NPT2B_rat sodium-dependent phosphate transport protein 2B (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO: 2)), wherein (a) the NaPi2b antibody is capable of binding to human Napi2b (e.g., SEQ ID NO: 1) and / or rat Napi2b (e.g., SEQ ID NO: 2), preferably, the binding to the human Napi2b and the rat Napi2b has substantially the same K D and more preferably, the antibody is selected from the group consisting of AV-25 antibody, AV-15 antibody, AV-18 antibody, AV-21 antibody, and AV-29 antibody, and most preferably, the substantially the same K D has a difference of up to 50% (e.g., a difference of up to 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 7%, 5%, 4%, 3%, 2%, or 1%); (b) the NaPi2b antibody is capable of cross-reactivity with rat Napi2b (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO: 2); (c) the NaPi2b antibody is capable of cross-reactivity with cynomolgus monkey (e.g., Macaca fascicularis) Napi2b (e.g., having UniProtKB accession number: A0A2K5UHY1 or SEQ ID NO: 3); (d) the NaPi2b antibody is preferably capable of internalization, preferably by internalization of the antibody through the antigen; (e) the NaPi2b antibody optionally may not have a dipeptide deamidation site within CDR2 of the heavy chain variable region (V H ), preferably, the absent dipeptide deamidation site is V HThe anti-NaPi2b antibody is NG (Asn-Gly) within CDR2; where the anti-NaPi2b antibody is one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most Preferably eight cytotoxic moieties (for example, cytotoxic payloads, e.g., tubulin inhibitors, e.g., topoisomerase I inhibitors, e.g., auristatin or camptothecin, e.g., MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), e.g., exatecan) are conjugated preferably via one or more linkers, and more preferably via one or more phosphoamidate linkers.

[0027] definition antibody When used herein, “antibody” may mean a protein comprising one or more polypeptides (including one or more binding domains and / or antigen-binding moieties, preferably antigen-binding domains) substantially or partially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. The term “immunoglobulin” (Ig) is used herein synonymously with “antibody.” Recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as numerous immunoglobulin variable region genes. In particular, “antibody” as used herein is typically a tetrameric glycosylated protein consisting of two light (L) chains, each about 25 kDa, and two heavy (H) chains, each about 50 kDa. Two types of light chains, called lambda and kappa, may be found in antibodies. Depending on the amino acid sequence of the constant domain of the heavy chain, immunoglobulins can be assigned to five major classes: A, D, E, G, and M, some of which can be further classified into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, but IgG is preferred in this invention. Antibodies of this invention having an IgE constant domain or a portion thereof that is bound by the Fc epsilon receptor I are also envisioned. IgM antibodies consist of five basic heterotetrameric units and an additional polypeptide called a J chain, containing 10 antigen-binding sites, while IgA antibodies consist of 2 to 5 basic quad-chain units, which can polymerize to form a multivalent aggregate combined with the J chain. In the case of IgG, the quad-chain unit is typically about 150,000 daltons. Each light chain contains a variable (V) domain (VL) and a constant (C) domain (CL) at the N-terminus. Each heavy chain contains an N-terminal V domain (VH), three or four C domains (CH), and a hinge region. While the constant domains are not directly involved in antibody-antigen binding, they can exhibit various effector functions, such as involvement in antibody-dependent cell-mediated cytotoxicity (ADCC).If the antibody needs to exhibit ADCC, it is preferable that it be an IgG1 subtype, as the IgG4 subtype will likely not have the ability to exhibit ADCC.

[0028] The term “antibody” also includes, but is not limited to, monoclonal antibodies, single-specific antibodies; polyspecific or multispecific antibodies such as bispecific antibodies; humanized antibodies, camelized antibodies, human antibodies, single-chain antibodies, chimeric antibodies, synthetic antibodies, recombinant antibodies, hybrid antibodies, mutant antibodies, grafted antibodies, and antibodies produced in vitro, with chimeric or humanized antibodies preferred. The term “humanized antibody” is generally defined as an antibody in which a CDR encoding HC and LC specificity is grafted onto a suitable human variable framework ("CDR grafting"). The term “antibody” also includes scFv, single-chain antibodies, diabodies or tetrabodies, domain antibodies (dAb), and nanobodies. From the viewpoint of the present invention, the term “antibody” also includes dimers, trimers, multimers, or bifunctional, trifunctional, or multifunctional antibodies having several antigen-binding sites.

[0029] Furthermore, when used in the present invention, the term “antibody” also refers to derivatives of antibodies (including fragments) as described herein. “Derivatives” of antibodies include amino acid sequences modified by the introduction of substitutions, deletions, or additions of amino acid residues. Furthermore, derivatives also include antibodies modified by covalently bonding any type of molecule to the antibody or protein. Examples of such molecules include, but are not limited to, sugars, PEGs, hydroxyl groups, ethoxy groups, carboxyl groups, or amine groups. In fact, covalent modifications of antibodies result in, but are not limited to, glycosylation, pegylation, acetylation, phosphorylation, and amidation.

[0030] The antibodies of the present invention are preferably “isolated” antibodies. “Isolated,” as used to describe the antibodies disclosed herein, means antibodies identified, separated, and / or recovered from the components of their production environment. Preferably, isolated antibodies are free from all other components originating from their production environment. Contaminating components originating from the production environment, such as those resulting from recombinant transfected cells, are substances that would typically interfere with the diagnostic or therapeutic use of the polypeptide and may include enzymes, hormones, and other protein or non-protein solutes. In a preferred embodiment, the antibody is purified to the extent that (1) at least 15 residues of the N-terminal or internal amino acid sequence can be obtained using a spinning cup sequencer, or (2) to the extent that it is considered homogeneous by SDS-PAGE under non-reducing or reducing conditions, using Coomassie blue staining or preferably silver staining. However, typically, isolated antibodies are prepared by at least one purification step.

[0031] The antibodies described herein can be used for diagnostic purposes, including sample testing and in vivo imaging, for which antibodies (or their conjugated fragments) can be conjugated to appropriate detectable substances to form immunoconjugates. For diagnostic purposes, appropriate substances are detectable labels, including radioisotopes for whole-body imaging, and radioisotopes, enzymes, fluorescent labels, and other appropriate antibody tags for sample testing. Detectable labels can be any of the various types currently used in the field of in vitro diagnostics, including particulate labels containing metal sols such as colloidal gold; isotopes; chromophores including fluorescent markers, biotin, luminescent markers, and phosphorescent markers; and enzymatic labels that convert a given substrate into a detectable marker; as well as polynucleotide tags that become apparent after amplification by polymerase chain reaction, etc. In this case, biotinylated antibodies would be detectable by avidin conjugation or streptavidin conjugation. Appropriate enzymatic labels include horseradish peroxidase and alkaline phosphatase, etc. For example, the label may be an alkaline phosphatase enzyme, or a suitable lanthanide chelate such as terbium(III) and europium(III), which is detected by measuring the presence or formation of chemiluminescence after conversion of a 1,2-dioxetane substrate (e.g., adamantylmethoxyphosphoryloxyphenyldioxetane (AMPPD), 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.1 3,7}decane}-4-yl)phenyl phosphate disodium (CSPD), and CDP and CDP-star®), or other luminescent substrates well known to those skilled in the art, such as terbium(III) and europium(III). The detection means will depend on the selected label. The appearance of the label or its reaction product can be observed visually if the label is particulate and accumulates at an appropriate level, or it can be observed using instruments such as a spectrophotometer, illuminometer, and fluorometer, in either case according to standard practices.

[0032] The "effector function" of an antibody refers to the biological activity that can be attributed to the antibody's Fc region (either the native sequence Fc region or the amino acid sequence variant Fc region), which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cell-mediated cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation. Antibodies, so to speak, mobilize effector cells to exert their effector function.

[0033] As used herein, the term “antigen-binding moiety” means a fragment of immunoglobulin (or intact antibody) and includes any polypeptide containing an antigen-binding fragment or antigen-binding domain. Preferably, the fragments are, for example, Fab, F(ab'), F(ab')2, Fv, scFv, Fd, disulfide-bonded Fv(sdFv), and other antibody fragments that retain the antigen-binding function described herein. Typically, such fragments are thought to contain an antigen-binding domain and have the same properties as the antibodies described herein. Therefore, the fragments may also preferably bind to the extracellular domain of NaPi2b.

[0034] As used herein, the term "specifically binding" means an antibody or fragment or derivative thereof that specifically binds to the NaPi2b protein but does not specifically bind to any other protein. The antibody or fragment or derivative thereof according to the present invention binds to the NAPI2B protein via the variable domain of the antibody.

[0035] The VH and VL domains, paired together, jointly form a single antigen-binding site. The CH domain closest to the VH is called CH1. Each L chain is linked to the H chain by a single disulfide covalent bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. The VH and VL domains consist of four regions with relatively conserved sequences called framework regions (FR1, FR2, FR3, and FR4), which form a scaffold for three regions with hypervariable sequences (complementarity-determining regions, CDRs). The CDRs contain most of the residues responsible for the specific interaction between the antibody and the antigen. The CDRs are called CDR1, CDR2, and CDR3. Therefore, the CDR components on the heavy chain are called H1 or H-CDR1 (or CRD-H1), H2 or H-CDR2 (or CDR-H2), and H3 or H-CDR3 (or CDR-H3), while the CDR components on the light chain are called L1 or L-CDR1 (or CRD-L1), L2 or L-CDR2 (or CDR-L2), and L3 or L-CDR3 (or CDR-L3).

[0036] The term "variable" refers to a portion of the immunoglobulin domain (i.e., the "variable domain") that exhibits sequence variability and is involved in determining the specificity and binding affinity of individual antibodies. Variability is not evenly distributed throughout the antibody's variable domain, but rather concentrated in the subdomains of the heavy chain and light chain variable regions. These subdomains are called "complementarity-determining regions" (CDRs).

[0037] The terms “CDR” and its plural form “CDRs” refer to complementarity-determining regions (CDRs), three of which form binding features for the light chain variable region (L1-CDR, L2-CDR, and L3-CDR), and three which form binding features for the heavy chain variable region (H1-CDR, H2-CDR, and H3-CDR). CDRs contribute to the functional activity of antibody molecules and are separated by amino acid sequences that constitute scaffolding or framework regions. The precise definitional boundaries and lengths of CDRs vary depending on the classification and numbering scheme. Therefore, CDRs may be referred to based on any other boundary definition, including Kabat, Chothia, contact definition, or any other numbering scheme described herein. Although the boundaries differ, each of these schemes has some overlap in the elements that constitute the so-called “hypervariable regions” within the variable sequence. Therefore, definitions of CDRs based on these schemes may differ in terms of length and the boundary regions with adjacent framework regions. However, numbering according to the so-called Kabat scheme is preferred.

[0038] The highly conserved (i.e., non-hypervariable) portions of the variable domain are called "framework" regions (FRMs). The variable domains of the natural heavy and light chains each contain four FRM regions, which primarily adopt a β-sheet configuration and are linked by three hypervariable regions. These hypervariable regions form loops, linking the β-sheet structure and, in some cases, forming part of it. The hypervariable regions of each chain are brought together very closely by the FRMs and, together with the hypervariable region of the other chain, contribute to the formation of the antigen-binding surface (see Kabat et al., above). The constant domains do not directly participate in antigen binding but exhibit various effector functions, such as antibody-dependent cell-mediated cytotoxicity and complement activation.

[0039] The term “binding domain” in relation to the present invention characterizes a domain of a polypeptide that specifically binds to / interacts with a given target epitope. “Epitope” is antigenic, and therefore the term epitope may also be referred to herein as “antigenic structure” or “antigenic determinant.” Thus, the binding domain is an “antigen interaction site.” According to the present invention, the term “antigen interaction site” defines a polypeptide motif that can specifically interact with a particular antigen or a particular group of antigens, such as the same antigen in different species. It is also understood that this binding / interaction constitutes “specific recognition.”

[0040] The terms “antigen-binding domain,” “antigen-binding portion,” “antigen-binding fragment,” and “antibody-binding region,” as used herein, refer to a portion of an antibody molecule containing amino acids responsible for the specific binding of an antibody to an antigen. The portion of the antigen that is specifically recognized and bound by the antibody is referred to herein as an “epitope,” as previously stated. As previously stated, the antigen-binding domain may typically include the antibody light chain variable region (VL) and the antibody heavy chain variable region (VH).

[0041] The term “epitope” also means a site on an antigen (in the context of this invention, the antigen is a NaPi2b protein) to which an antibody molecule binds. Preferably, an epitope is a site on a molecule (in the context of this invention, the antigen is a NaPi2b protein) to which an antibody or its antigen-binding portion, preferably an antibody, is produced and / or which the antibody binds. For example, an epitope can be recognized by an antibody or its antigen-binding portion. A “linear epitope” is an epitope whose recognized epitope is composed of a primary amino acid sequence. Typically, a linear epitope contains at least three, and more commonly at least five, for example, about eight to about ten amino acids, in a unique sequence.

[0042] The term "cross-reactivity" can refer to the ability of an antibody to react with similar antigenic sites on different proteins.

[0043] In this context, the term "specific" may mean that an antibody or its antigen-binding moiety binds to the target NaPi2b protein but not to another protein. The term "another protein" includes any protein in which the antibody or its antigen-binding moiety is closely related to or homologous to the target NAPI2B protein. However, the term "another protein" does not include cross-reactivity of an antibody or its antigen-binding moiety with a NaPi2b protein originating from a different species than the one in which the antibody or its antigen-binding moiety was produced.

[0044] Therefore, species-cross-reactive specific antibodies targeting the NaPi2b protein or their antigen-binding moieties are preferably intended by the present invention.

[0045] "K D The term "k" refers to the equilibrium dissociation constant, i.e., k, between an antibody and its antigen, or between the variable regions of one heavy chain and one light chain of an antibody or its fragment or derivative and their antigens. off / k onThis can mean a ratio; where the antigen is, for example, Napi2b, for example, full-length Napi2b and / or one or more fragments thereof, preferably, the one or more fragments comprising at least one extracellular domain (ECD) of Napi2b (e.g., the ECD comprising amino acids 122-135 and / or amino acids 235-361 and / or amino acids 429-485 and / or amino acids 547-552 of human Napi2b having SEQ ID NO: 1) and / or one or more fragments of the ECD (e.g., having a length of about 15-30 amino acids); for example, the full-length Napi2b and / or one or more fragments thereof may be fused to or unfused to one or more protein tags (e.g., 6×His tag, FLAG, HA, V5, Fc fusion, MBP, SUMO, TEV, GFP, TST); K D This is measured in vitro. D The affinity is inversely proportional to the affinity.

[0046] As used herein, the term "affinity" may mean the binding strength between a variable region of one heavy chain and one light chain of an antibody or a fragment or derivative thereof and their antigen; where the antigen is, for example, Napi2b, for example, full-length Napi2b and / or one or more fragments thereof, preferably the one or more fragments having at least one extracellular domain (ECD) of Napi2b (for example, the ECD is SEQ ID NO: The ECD comprises amino acids 122-135 and / or 235-361 and / or 429-485 and / or 547-552 of human Napi2b having 1, and / or one or more fragments of the ECD (e.g., having a length of about 15-30 amino acids); for example, the full-length Napi2b and / or one or more fragments thereof may be fused to or unfused to one or more protein tags (e.g., 6×His tag, FLAG, HA, V5, Fc fusion, MBP, SUMO, TEV, GFP, TST); affinity is measured in vitro. Affinity reveals the strength of the interaction between the epitope and the antigen-binding site of the antibody. Affinity can be calculated using the following formula. KA = [AB - AG] / [AB] × [AG] = k on / k off During the ceremony, KA = affinity constant [AB] = Molar concentration of vacant binding sites on the antibody [AG] = Molar concentration of vacant binding sites on the antigen [AB-AG] = Molar concentration of antibody-antigen complex

[0047] The terms “amino acid” or “amino acid residue” typically mean an amino acid selected from the group consisting of amino acids having the definitions accepted in the art, such as alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (Gln or Q); glutamic acid (Glu or E); glycine (Gly or G); histidine (His or H); isoleucine (He or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified amino acids, synthetic amino acids, or rare amino acids may also be used as desired. Generally, amino acids can be grouped into those having nonpolar side chains (e.g., Ala, Cys, He, Leu, Met, Phe, Pro, Val); charged side chains (e.g., Asp, Glu); positively charged side chains (e.g., Arg, His, Lys); or uncharged side chains (e.g., Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0048] The term “polypeptide” is used herein as equivalent to the term “protein.” Proteins (including their fragments, preferably biologically active fragments, and peptides, which typically have fewer than 30 amino acids) contain one or more amino acids linked to one another via covalent peptide bonds (which give rise to chains of amino acids). When used herein, the term “polypeptide” refers, for example, to a group of molecules consisting of more than 30 amino acids. Polypeptides may also form multimers, i.e., multiple polypeptide molecules, such as dimers, trimers, and higher-order oligomers. The polypeptide molecules forming such dimers, trimers, etc., may be identical or non-identical. As a result, the corresponding higher-order structures of such multimers are called homodimers or heterodimers, homotrimers or heterotrimers, etc. An example of a heteromultimer is the antibody molecule, which in its natural form consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms “polypeptide” and “protein” also refer to naturally modified polypeptides / proteins that are modified by post-translational modifications such as glycosylation, acetylation, and phosphorylation, for example. Such modifications are well known in the art.

[0049] The term "immune cells" refers to cells capable of producing antibodies. Immune cells of particular interest in this specification are, for example, lymphoid cells derived from the spleen, peripheral blood lymphocytes (PBLs), lymph nodes, inguinal lymph nodes, Peyer's patches, tonsils, bone marrow, umbilical cord blood, pleural fluid, and tumor-infiltrating lymphocytes (TILs).

[0050] One type of antibody variant encompassed by the present invention is an amino acid substitution variant. In these variants, at least one, two, three, four, five, six, seven, eight, nine, or ten amino acid residues in the Napi2b antibody molecule are substituted with other residues. The sites of greatest interest for substitutional mutagenesis include the CDR of the heavy chain and / or light chain, specifically the hypervariable region, but alterations of the FR of the heavy chain and / or light chain are also intended.

[0051] For example, if a CDR sequence contains 6 amino acids, it is expected that one, two, or three of these amino acids may be substituted. Similarly, if a CDR sequence contains 15 amino acids, it is expected that one, two, three, four, five, or six of these amino acids may be substituted.

[0052] Generally, when amino acids are substituted in one, more, or all of the heavy chain and / or light chain CDRs, it is preferable that the resulting "substituted" sequence is at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to the "original" CDR sequence, more preferably 65%, even more preferably 70%, particularly preferably 75%, and even more preferably 80%. This means that the degree to which the CDR is identical to the "substituted" sequence depends on the length of the CDR. For example, a CDR having five amino acids is preferably 80% identical to its substituted sequence in order that at least one amino acid is substituted. Therefore, the CDRs of Napi2b antibodies may have varying degrees of identity with respect to their substituted sequences; for example, CDRL1 may have 80% identity, while CDRL3 may have 90% identity.

[0053] A preferred substitution (or replacement) is a conservative substitution. However, any substitution (including non-conservative substitutions or one or more of the “exemplary substitutions” listed in Table I herein) is considered, as long as the antibody retains its ability to specifically bind to the Napi2b protein and / or its CDR has identity to the substituted sequence (at least 60% (e.g., at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), more preferably 65%, even more preferably 70%, particularly preferably 75%, and even more preferably 80% identity to the “original” CDR sequence).

[0054] Conservative substitutions are listed under the “Preferred Substitutions” section in Table I. If such substitutions alter biological activity, more substantial changes may be introduced, referred to as “Exemplary Substitutions” in Table I or further described later with respect to amino acid classes, and the products may be screened for desired characteristics.

[0055] chemical modification The antibody or its antigen-binding variant or fragment used in accordance with the present invention may be modified. Typical modifications that may be considered in the context of the present invention include, for example, the chemical modifications described below.

[0056] Possible chemical modifications to antibodies or their antigen-binding variants or fragments include acylation or acetylation of the amino terminus, or amidation or esterification of the carboxyl terminus, or such modifications at both ends. Modifications may also affect the amino group of the lysine side chain or the hydroxyl group of threonine. Other suitable modifications include, for example, extension of the amino group by polypeptide chains of varying lengths (e.g., XTEN technology or PASylation®), N-glycosylation, O-glycosylation, and chemical bonding of carbohydrates, e.g., hydroxyethyl starch (e.g., HESylation®) or polysialic acid (e.g., PolyXen® technology). Chemical modifications such as alkylation (e.g., methylation, propylation, butylation), arylation, and etherification may also be possible and are also envisioned.

[0057] Antibody-drug conjugates (ADCs) The term antibody-drug conjugate (or ADC), as used herein, may mean any antibody according to the present invention conjugated to one or more drug moieties (e.g., cytotoxic payloads). Preferably, the antibody-drug conjugate (ADC) of the present invention comprises an anti-Napi2b antibody of the present invention (e.g., a humanized monoclonal Napi2b-specific IgG1 antibody) conjugated to one or more cytotoxic payloads, where (a) the cytotoxic payload is camptothecin, mytansinoid, calicheamycin, duocalmycin, tubulisin, amatoxin, drastatin, and auristatin, e.g., monomethyl auristatin E (MMAE), pyro (b) The cytotoxic payload is selected from the group consisting of lobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and their analogues or prodrugs; and / or (b) the cytotoxic payload is exatecan (e.g., CAS number: 171335-80-1), DXD, SN38, camptothecin, topotecan, iri (c) The cytotoxic payload is a camptothecin moiety C selected from the group consisting of notecan, berothecan, lulutotecan, rubitecan, silatecan, cositecan, and gimatecan; and / or (c) the cytotoxic payload is conjugated via a cleavable linker (L), preferably the linker L is cleavable by a protease, glucuronidase, sulfatase, phosphatase, esterase, or disulfide reduction, and more preferably the linker is cleavable by a protease, preferably a cathepsin such as cathepsin B; and / or (d) the linker (L) comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; and / or (e) the cytotoxic payload is an exatecan conjugated via a chemical valine-citrulline-PAB releasing unit or a valine-alanine-PAB releasing unit, the releasing unit being cleavable by a protease.As used herein, “linker” (L) may mean any chemical moiety capable of linking the antibody of the present invention to one or more drug moieties (e.g., cytotoxic moieties (e.g., cytotoxic payloads, e.g., tubulin inhibitors, e.g., topoisomerase-I inhibitors, e.g., auristatin or camptothecin, e.g., MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), e.g., exatecan)). Preferably, L is a phosphoamidate linker; more preferably, linker L comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; most preferably, linker L is cleavable (e.g., readily cleaved by enzymes).

[0058] Sequence identity The terms "identity percentage (%)" or "sequence identity percentage (%)", as used in this invention, may mean the percentage of identical pairs of residues relative to the number of residues in the longer of the two sequences, after the polypeptide sequence of the invention has been (homologically) aligned with the sequence in question. The identity percentage is obtained by dividing the number of identical residues by the total number of residues and multiplying the result by 100.

[0059] The percentage of sequence homology or sequence identity can be determined herein, for example, using BLASTP, version blastp 2.2.5 (November 16, 2002; Altschul, SF et al. (1997) Nucl. Acids Res. 25, 3389-3402). In this embodiment, the percentage of homology is preferably based on the alignment of the entire polypeptide sequence including the propeptide sequence (matrix: BLOSUM 62; gap cost: 11.1) using the wild-type protein backbone as a reference in pairwise comparison. This is calculated as the percentage obtained by dividing the number of "positive" (homologous amino acids) shown as a result in the BLASTP program output by the total number of amino acids selected by the program for alignment.

[0060] The term "Napi2b" refers to sodium-dependent phosphate transport protein 2B and typically includes all known isoforms. Preferably, this sodium-dependent phosphate transport protein 2B has SEQ ID NO:1 or UniProtKB accession number O95436.

[0061] vector The nucleic acids of the present invention may also be in the form of a vector, present within a vector, and / or be part of a vector.

[0062] The term "vector" refers to a nucleic acid molecule used as a carrier for transferring (foreign) genetic material into a host cell, and non-limitingly includes plasmids, viruses, cosmids, and artificial chromosomes, such as bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). Typically, a designed vector includes an origin of replication, a multicloning site, and a selection marker. Typically, the vector itself is a nucleotide sequence, usually a DNA sequence, containing an insertion fragment (transgene) and a larger sequence that serves as the vector's "backbone." In addition to the transgene insertion fragment and backbone, a vector may include additional elements, such as gene regulatory elements, gene markers, antibiotic resistance, reporter genes, targeting sequences, or protein purification tags. Specifically envisioned in the context of this invention are expression vectors (expression constructs) for the expression of a transgene in a host cell, which typically include a gene regulatory sequence in addition to the transgene.

[0063] Generally, an expression vector is a vector capable of expressing the antibody of the present invention in vitro and / or in vivo (i.e., in a suitable host cell, host organism, and / or expression system). Those skilled in the art will readily understand that the selection of a particular vector is determined by, for example, the host cell, the intended copy number of the vector, and whether transient or stable expression of the antibody of the present invention is envisioned.

[0064] "Transient expression" occurs when nucleic acids (e.g., linear or nonlinear DNA or RNA molecules) or vectors that are incapable of autonomous replication are introduced into recipient host cells. The expression of the introduced gene occurs through the transient expression of the introduced sequence.

[0065] However, the “stable expression” of the nucleic acid sequences described herein is often preferred, which can be achieved by stably incorporating the nucleic acid sequences into the genome of a host cell, or by introducing a vector containing the nucleic acid sequences of the present invention that can autonomously replicate into a host cell.

[0066] The vectors provided herein are particularly intended to include gene regulatory elements functionally linked to the DNA sequence encoding the antibody of the present invention.

[0067] The term “gene regulatory element” refers to DNA sequences required for the expression of functionally linked coding sequences within a particular host organism. The term “gene regulatory element” includes other elements that can control gene expression, including controllable transcription promoters, operators, enhancers, silencers, transcription terminators, 5' and 3' untranslated regions, and start and end codons, which interact with host cellular proteins to perform transcription and translation. The exact nature of the regulatory regions required for gene expression can vary from organism to organism. Prokaryotic gene regulatory elements include, for example, promoters, optionally operator sequences, and ribosome-binding sites (RBS), while eukaryotic gene regulatory elements include promoters, polyadenylation (poly-A) signals, and enhancers.

[0068] Gene regulatory elements are assumed to be "functionally linked" to the gene they are intended to express, that is, positioned to functionally relate to that gene. For example, a promoter or enhancer is "functionally linked" to a coding nucleic acid sequence if it affects the transcription of that sequence. Functionally linked DNA sequences may or may not be adjacent. Typically, linking is achieved by ligation at a convenient restriction site or by synthetic oligonucleotide adapters or linkers.

[0069] host cell Furthermore, host cells containing the vectors described herein (e.g., recombinant host cells and / or isolated host cells) are also provided herein.

[0070] Various host cells can be used to express nucleic acid sequences encoding the antibodies described herein. Host cells can be prepared using genetic engineering methods known in the art. The process of introducing a vector into recipient host cells is also referred to below as "transformation" or "transfection." These terms are used synonymously herein.

[0071] Host cell transformation typically involves creating transient pores or "holes" in the cell wall and / or cell membrane to allow uptake of substances. Exemplary transformation protocols include the use of calcium phosphate, electroporation, cell compression, dendrimers, liposomes, cationic polymers such as DEAE-dextran or polyethyleneimine, sonoporation, optical transfection, impalefection, nanoparticles (gene guns), magnetofection, microparticle guns, alkaline cations (cesium, lithium), enzymatic digestion, agitation with glass beads, or viral vectors. The choice of method usually depends on the type of cell being transformed, the vector introduced into the cell, and the conditions under which the transformation takes place.

[0072] As used herein, the term “host cell” may mean any cell or cell culture that serves as the recipient of an antibody (Ab) encoding vector or isolated nucleic acid sequence as described herein. Suitable host cells include, but are not limited to, prokaryotic or eukaryotic cells, as well as bacteria, yeast cells, fungal cells, plant cells, and animal cells, such as insect cells and mammalian cells, such as mouse, rat, macaque, or human cells.

[0073] For example, antibodies (Ab) can be produced within bacteria. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are also suitable hosts for cloning or expression of the NAPI2B antibodies of the present invention. Exemplary examples include hosts of the genus Saccharomyces cerevisiae, Schizosaccharomyces pombe, and Kluyveromyces, such as K. lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC 56500), K. drosophilarum (ATCC 36906), K. thermotolerance, and K. marxianus; and the genus Yarrowia (EP 402). This includes 226); Pichia pastoris (EP 183 070); Candida; Trichoderma reesia (EP 244 234); Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces occidentalis; and hosts of filamentous fungi, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus, e.g., A. nidulans and A. niger.

[0074] Host cells suitable for the expression of the glycosylated antibody constructs of the present invention may also be derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Numerous baculovirus strains and variants, as well as corresponding permissible insect host cells derived from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori (silkworm), have been identified. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of silkworm NPV, are publicly available.

[0075] Plant cell cultures of cotton, maize, potato, soybean, petunia, tomato, Arabidopsis, and tobacco can also be used as hosts. Cloning vectors and expression vectors useful for protein production in plant cell cultures are known to those skilled in the art.

[0076] Examples of useful mammalian host cell lines include: SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); human fetal kidney cell line (293 or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (CHO), mouse Sertoli cells (TM4); monkey kidney cells (CVI ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL 1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, 1413 8065); mouse mammary tumor cells (MMT 060562, ATCC These include CCL51 cells, TRI cells, MRC 5 cells, FS4 cells, and human hepatocellular carcinoma cells (Hep G2).

[0077] patient Where used herein, the terms “patient” or “subject” mean human or non-human animals, usually mammals. Specifically, mammals are assumed to be, for example, rabbits, mice, rats, guinea pigs, hamsters, dogs, cats, pigs, cows, goats, sheep, horses, monkeys, apes, or preferably humans. Thus, the methods, uses, and compounds described in this document are generally applicable to both human and veterinary diseases.

[0078] treatment The term “treatment” in all its grammatical forms includes therapeutic or preventive treatments. “Therapeutic or preventive treatments” include preventive treatments aimed at complete prevention of clinical and / or pathological symptoms, or therapeutic treatments aimed at improvement or remission of clinical and / or pathological symptoms of a disease. Therefore, the term “treatment” also includes improvement or prevention of cancer.

[0079] In the present invention, the term “therapeutic effect” usually means a desirable or beneficial effect of treatment, e.g., improvement or remission of disease symptoms. The term “symptoms” of a disease is used herein to describe its perceptible manifestation, and this term includes both clinical symptoms (defined below as signs of the disease that may be detected during a physical examination and / or perceived by the patient (i.e., subjective symptoms)) and pathological symptoms, meaning the manifestation of the disease at the cellular and molecular levels. The therapeutic effect of treatment with NaPi2b-ADC of the present invention can be evaluated using routine methods in the art, for example, by measuring leukemia burden by blood / bone marrow analysis (cytomorphology, flow cytometry, genetics), clinical chemistry, or radiological techniques (e.g., CT). Alternatively, the overall condition of each patient (e.g., health status, well-being) can also be evaluated, which also helps a skilled physician to assess whether a therapeutic effect has been elicited. Those skilled in the art know of numerous other methods suitable for observing the therapeutic effect of the compounds of the present invention.

[0080] dose Preferably, a therapeutically effective dose of the compound described herein is administered. “Therapeutically effective dose” means the amount of the compound described herein that elicits a therapeutic effect. The precise dose of the Ab-NaPi2b-ADC of the present invention depends on the purpose of treatment (e.g., induction or maintenance of remission) and can be determined by those skilled in the art using known techniques. Adjustments may be necessary depending on the route of administration, age, weight, overall health, sex, diet, administration time, drug interactions, and severity of the condition, which can be determined by those skilled in the art using routine experimental methods.

[0081] Administration Various routes, including but not limited to oral, topical, transdermal, subcutaneous, intravenous, intraperitoneal, intramuscular, or intraocular, preferably subcutaneous and / or intravenous, are applicable to the administration of the compounds according to the present invention. However, if desired, those skilled in the art can readily select any other route.

[0082] composition The NAPI2B antibody and / or ADC of the present invention are intended to be administered in the form of a pharmaceutical composition.

[0083] The term "pharmaceutical composition" specifically means a composition suitable for administration to humans, i.e., a composition that is preferably sterile and / or contains pharmaceutically acceptable components. However, compositions suitable for administration to non-human animals are also assumed herein. Preferably, a pharmaceutical composition comprises the Ab-NaPi2b-ADC of the present invention together with one or more pharmaceutically acceptable excipients. The term "excipients" includes bulking agents, binders, disintegrants, coating agents, adsorbents, anti-adhesion agents, flow enhancers, preservatives, antioxidants, flavoring agents, colorants, sweeteners, solvents, auxiliary solvents, buffers, chelating agents, viscosity modifiers, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers, or osmotic pressure modifiers. The pharmaceutical compositions of the present invention can be formulated in various forms, for example, as solids, liquids, gases, or lyophilized forms; in particular, they may be in the form of ointments, creams, transdermal patches, gels, powders, tablets, solutions, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, liquids, elixirs, extracts, tinctures, or fluid extracts, or in forms particularly suitable for a desired method of administration.

[0084] The pharmaceutical composition of the present invention may further contain one or more additional active ingredients. Preferably, these active ingredients are therapeutically effective for treating the diseases described herein and are present in the composition in therapeutically effective amounts.

[0085] Accordingly, in view of the foregoing, the present invention also provides a pharmaceutical composition comprising one or more NaPi2b antibodies and / or ADCs of the present invention. This pharmaceutical composition is particularly intended for use in methods of therapeutic and / or prophylactic treatment of cancer.

[0086] kit Kits are also provided herein. A kit may consist of two or more parts and may contain, preferably, a therapeutically effective amount and in a pharmaceutically acceptable form, the NaPi2b antibody and / or ADC of the present invention. The components of the kit may be contained in containers or vials. The kit is envisioned to contain additional active ingredients useful for the treatment of cancer.

[0087] In some aspects / aspects, the present invention relates to anti-NaPi2b antibodies (e.g., antibodies against NPT2B_human sodium-dependent phosphate transporter protein 2B (e.g., having UniProt accession number: O95436 or SEQ ID NO: 1) and / or antibodies against NPT2B_rat sodium-dependent phosphate transporter protein 2B (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO: 2)), wherein (a) the NaPi2b antibody is capable of binding to human Napi2b (e.g., SEQ ID NO: 1) and / or rat Napi2b (e.g., SEQ ID NO: 2), and preferably, the binding to human Napi2b and rat Napi2b is substantially the same K D The antibody is selected from the group consisting of AV-25 antibody, AV-15 antibody, AV-18 antibody, AV-21 antibody, and AV-29 antibody, and most preferably the same K D(b) The NaPi2b antibody has a difference of up to 50% (e.g., a difference of up to 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 7%, 5%, 4%, 3%, 2%, or 1%); (c) The NaPi2b antibody is capable of cross-reactivity with rat Napi2b (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO: 2); (d) The NaPi2b antibody is capable of cross-reactivity with cynomolgus monkey (e.g., macaque fascicularis) Napi2b (e.g., having UniProtKB accession number: A0A2K5UHY1 or SEQ ID NO: 3); (e) The NaPi2b antibody is capable of internal transfer, preferably by antigen-mediated internal antibody transfer; (e) The NaPi2b antibody optionally has a heavy chain variable region (V H It is possible for the CDR2 of ) to not have a dipeptide deamide moiety, and preferably, the absent dipeptide deamide moiety is V H This is an NG (Asn-Gly) within CDR2.

[0088] In some aspects / embodiments, the present invention relates to monoclonal human or humanized IgG1 anti-NaPi2b antibodies, preferably comprising a kappa (κ) light chain.

[0089] In some aspects / applications, the present invention relates to a hybridoma that produces the antibody of the present invention.

[0090] In some aspects / applications, the present invention relates to nucleic acids encoding the antibodies of the present invention.

[0091] In some aspects / modes, the present invention relates to an expression vector comprising at least one nucleic acid molecule of the present invention.

[0092] The present invention further relates. In some aspects / portrayals, the present invention relates to isolated host cells (e.g., isolated recombinant host cells) comprising the vector and / or nucleic acids of the present invention.

[0093] In some aspects / applications, the present invention relates to antibody-drug conjugates (ADCs) comprising the anti-NaPi2b antibody of the present invention.

[0094] In some aspects / applications, the present invention relates to an antibody-drug conjugate (ADC) of the present invention comprising an anti-NaPi2b antibody of the present invention (e.g., a humanized monoclonal NaPi2b-specific IgG1 antibody) conjugated to a cytotoxic payload, wherein (a) the cytotoxic payload is selected from the group consisting of camptothecin, mytansinoids, calicheamycin, duochamycin, tubulisin, amatoxin, drastatin, and auristatin, e.g., monomethyl auristatin E (MMAE), pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and analogs or prodrugs thereof; and / or (b) the cytotoxic payload is exatecan, DXD, SN38, camptothecin, topotecan, irinotecan, vero (c) The cytotoxic payload is a camptothecin moiety C selected from the group consisting of tecan, lulutotecan, rubitecan, silatecan, cositecan, and gimatecan; and / or (c) the cytotoxic payload is conjugated via a cleavable linker (L), preferably the linker L is cleavable by a protease, glucuronidase, sulfatase, phosphatase, esterase, or disulfide reduction, more preferably the linker is cleavable by a protease, preferably a cathepsin such as cathepsin B; and / or (d) the linker (L) comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; and / or (e) the cytotoxic payload is exatecan conjugated via a chemical valine-citrulline-PAB releasing unit or a valine-alanine-PAB releasing unit, the releasing unit being cleavable by a protease.

[0095] In some aspects / encompassing ways, the present invention relates to cancer. Cancer can be any cancer. Preferably, the cancer is a solid tumor and / or metastatic cancer, and more preferably, the cancer is selected from the group consisting of lung cancer, ovarian cancer, thyroid cancer, non-squamous non-small cell lung cancer, non-mucinous ovarian cancer, papillary thyroid cancer, kidney cancer, endometrial cancer, uterine cancer, ureteral cancer, bladder cancer, and fallopian tube cancer.

[0096] In some aspects / applications, the present invention relates to compositions or kits comprising anti-NaPi2b, antibody-drug conjugates (ADCs), hybridomas, nucleic acids, expression vectors, and / or host cells.

[0097] In some aspects / applications, the present invention relates to a method for synthesizing antibody-drug conjugates (ADCs) of the present invention.

[0098] In some aspects / encompassing, the present invention relates to methods of treatment (e.g., of a patient), as well as the use of the antibodies, antibody-drug conjugates (ADCs), nucleic acids, expression vectors, host cells, compositions, and / or kits of the present invention.

[0099] In some aspects of the present invention, the antibody of the present invention is expressed in CHO cells as Fc-silencing (LALA mutation) IgG1 and purified by protein A chromatography.

[0100] The present invention further relates to the following items: 1. Anti-NaPi2b antibodies (e.g., antibodies against NPT2B_human sodium-dependent phosphate transporter 2B (e.g., having UniProt accession number: O95436 or SEQ ID NO: 1) and / or antibodies against NPT2B_rat sodium-dependent phosphate transporter 2B (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO: 2)): (a) The NaPi2b antibody is capable of binding to human Napi2b (e.g., SEQ ID NO: 1) and / or rat Napi2b (e.g., SEQ ID NO: 2), and preferably, the binding to human Napi2b and rat Napi2b is substantially the same K D The antibody is selected from the group consisting of AV-25 antibody, AV-15 antibody, AV-18 antibody, AV-21 antibody, and AV-29 antibody, and most preferably the same K D This has a difference of up to 50% (for example, a difference of up to 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 7%, 5%, 4%, 3%, 2%, or 1%); (b) The NaPi2b antibody is capable of cross-reactivity with rat Napi2b (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO: 2), preferably the cross-reactivity is improved (e.g., at least 10%) compared to the corresponding cross-reactivity of the parent antibody (e.g., including SEQ ID NO: 54 and SEQ ID NO: 55, shown, for example, in Figure 1) with rat Napi2b (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO: 2), more preferably the cross-reactivity with rat Napi2b is measured using endogenous rat Napi2b, most preferably the endogenous Napi2b is located on the cell surface; (c) The NaPi2b antibody is capable of cross-reactivity with cynomolgus monkey (e.g., macaque fascicularis) Napi2b (e.g., having UniProtKB accession number: A0A2K5UHY1 and SEQ ID NO: 3); (d) The NaPi2b antibody is capable of internal migration, preferably via antigen-mediated internal migration, and preferably such internal migration is improved (e.g., by at least 10%, e.g., 15%) compared to the corresponding internal migration of the parent antibody (e.g., including SEQ ID NO: 54 and SEQ ID NO: 55, shown, e.g., in Figure 1); (e) The NaPi2b antibody may optionally have a heavy chain variable region (VH It is possible for the CDR2 of ) to not have a dipeptide deamide moiety, and preferably, the absent dipeptide deamide moiety is V H This is an NG (Asn-Gly) within CDR2. 2. Anti-NaPi2b antibody affects the heavy chain variable region (V H ) does not have a dipeptide deamide moiety within CDR2, preferably the absence of such dipeptide deamide moiety is V H It is NG (Asn-Gly) within CDR2, and more preferably, the absence of the deamide moiety means that the corresponding V H An anti-NaPi2b antibody of any one of the above items, wherein post-translational modification of CDR2 is reduced (which, for example, improves the homogeneity of the antibody and / or simplifies the production process). 3. An anti-NaPi2b antibody that has cross-reactivity with rat Napi2b (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO: 2); preferably, the cross-reactivity is improved (e.g., at least 10%, e.g., 15%) compared to the corresponding cross-reactivity of parent antibodies (e.g., including SEQ ID NO: 54 and SEQ ID NO: 55, shown, e.g., in Figure 1) with rat Napi2b (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO: 2); more preferably, the cross-reactivity with rat Napi2b is measured using endogenous rat Napi2b, most preferably, the endogenous Napi2b is located on the cell surface; an anti-NaPi2b antibody of any one of the above items. 4. An anti-NaPi2b antibody having the ability to intermigrate internally, preferably by antigen-mediated internal transfer; more preferably, the internal transfer is improved (e.g., by at least 10%, e.g., 15%) compared to the corresponding internal transfer of a parent antibody (e.g., including SEQ ID NO: 54 and SEQ ID NO: 55, shown, e.g., in Figure 1), wherein the anti-NaPi2b antibody of any one of the above items. 5. The corresponding K of the parent antibodies (e.g., including SEQ ID NO: 54 and SEQ ID NO: 55, shown, for example, in Figure 1),D Compared to that, K is improved (for example, at least 10%, for example, 15%). D An anti-NaPi2b antibody that binds to any one of the above items, specifically to human Napi2b (e.g., SEQ ID NO: 1) and / or rat Napi2b (e.g., SEQ ID NO: 2). 6. An anti-NaPi2b antibody that can bind (e.g., specifically bind) to any one of the above items, to the extracellular domain of NaPi2b (e.g., including amino acids 122-135 and / or amino acids 235-361 and / or amino acids 429-485 and / or amino acids 547-552 of human NaPi2b having SEQ ID NO: 1). 7. The following characteristics: (a) (For example, in OVCAR-3 cells endogenously expressing Napi2b (e.g., HTB-161, ATCC)), having a KD of about 0.01 to about 10 nmol / L, preferably in the range of about 1 to about 10 nmol / L, more preferably about 1 to about 7 nmol / L, and even more preferably about 1 to about 4 nmol / L, relative to endogenously expressed human Napi2b. (More preferably, the KD is measured by the FACS assay method.) More preferably, having a KD in the range of about 2.661 to about 6.644 nmol / L; and / or (b) Optionally, having a KD in the range of about 0.01 to about 10 nmol / L for immobilized exogenous full-length Napi2b and / or one or more fragments thereof. (Preferably, the one or more fragments include at least one extracellular domain (ECD) of Napi2b (e.g., the ECD includes amino acids 122-135 and / or 235-361 and / or 429-485 and / or 547-552 of human Napi2b having SEQ ID NO: 1) and / or one or more fragments of the ECD (e.g., having a length of about 15-30 amino acids); the full-length Napi2b and / or one or more fragments thereof are fused to or unequivocally fused to one or more protein tags (e.g., 6×His tag, FLAG, HA, V5, Fc fusion, MBP, SUMO, TEV, GFP, TST); preferably, the KD is measured by an ELISA assay; and preferably, the KD is about 0.05-0.2 nmol / L). More preferably, the KD is in the range of about 0.071 to about 0.147 nmol / L. An anti-NaPi2b antibody having one or more of the above items. 8. (a) The anti-NaPi2b antibody has a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 4, and SEQ ID NO: An antibody comprising a light chain variable region having an amino acid sequence having at least 80% identity to 5 (for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%); preferably, the anti-NaPi2b antibody is an AV-25 antibody comprising a light chain containing SEQ ID NO: 6 and a heavy chain containing SEQ ID NO: 7; (b) The anti-NaPi2b antibody has a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 8, and SEQ ID NO: An antibody comprising a light chain variable region having an amino acid sequence having at least 80% identity to 9 (for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%); preferably, the anti-NaPi2b antibody is an AV-15 antibody comprising a light chain containing SEQ ID NO: 10 and a heavy chain containing SEQ ID NO: 11; (c) The anti-NaPi2b antibody has a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 12, and SEQ ID NO: An antibody comprising a light chain variable region having an amino acid sequence having at least 80% identity to 13 (for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%); preferably, the anti-NaPi2b antibody is an AV-18 antibody comprising a light chain containing SEQ ID NO: 14 and a heavy chain containing SEQ ID NO: 15; (d) The anti-NaPi2b antibody has a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 16, and SEQ ID NO: An antibody comprising a light chain variable region having an amino acid sequence having at least 80% identity to 17 (for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%); preferably, the anti-NaPi2b antibody is an AV-21 antibody comprising a light chain containing SEQ ID NO: 18 and a heavy chain containing SEQ ID NO: 19; (e) The anti-NaPi2b antibody has a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 20, and SEQ ID NO: An antibody comprising a light chain variable region having an amino acid sequence having at least 80% identity to 21 (for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%); preferably, the anti-NaPi2b antibody is an AV-29 antibody comprising a light chain containing SEQ ID NO: 22 and a heavy chain containing SEQ ID NO: 23. One of the anti-NaPi2b antibodies listed above. 9. (a) An antibody comprising a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 24, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 25, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 26, and a light chain variable region including a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 27, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 28, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 29; (b) An antibody comprising a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 30, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 32, and a light chain variable region including a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 33, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 34, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 35; (c) An antibody comprising a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 36, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 37, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 38, and a light chain variable region including a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 39, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 40, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 41; (d) An antibody comprising a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 42, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 43, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 44, and a light chain variable region including a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 45, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 46, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 47; (e) An antibody comprising a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 48, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 49, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 50, and a light chain variable region including a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 51, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 52, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 53. An anti-NaPi2b antibody, which is one of the above items. 10. The following characteristics: (a) Monoclonal antibody; (b) Chimeric antibodies and / or humanized antibodies; (c) Specific recognition of Napi2b, which is overexpressed in cancer cells; (d) Human IgG antibody, preferably human IgG1 antibody; (e) Contains kappa (κ) light chain; (f) Contains a lambda (λ) light chain; (g) Includes Fc silencing mutations such as leucine (L) to alanine (A) substitutions (LALA mutations) at positions 234 and 235. (Here, the LALA mutation can reduce the effector function of immune cells.) (h) It can be internally transported by target cells expressing Napi2b (e.g., cancer cells). (Preferably, the internally transported antibody is directed towards the lysosome); (i) tumor-selective antibodies (Preferably, the tumor is a liquid tumor and / or a solid tumor); (j) Antibodies selective for malignant cells; (k) Glycosylation-dependent binding to human Napi2b and / or rat Napi2b. (Here, the antibody binds to the glycosylated Napi2b protein.) (l) (For example, in OVCAR-3 cells that endogenously express Napi2b (e.g., HTB-161, ATCC)), a K concentration of about 0.01 to about 10 nmol / L, preferably in the range of about 1 to about 10 nmol / L, more preferably in the range of about 1 to about 7 nmol / L, and even more preferably in the range of about 1 to about 4 nmol / L relative to endogenously expressed human Napi2b. D Having (More preferably, the K D (This is measured by the FACS assay method.) More preferably, K in the range of about 2.661 to about 6.644 nmol / L D Having; (m) Optionally, a K2 solution ranging from approximately 0.01 to approximately 10 nmol / L for immobilized exogenous full-length Napi2b and / or one or more fragments thereof. D Having (Preferably, the one or more fragments include at least one extracellular domain (ECD) of Napi2b (e.g., the ECD includes amino acids 122-135 and / or amino acids 235-361 and / or amino acids 429-485 and / or amino acids 547-552 of human Napi2b having SEQ ID NO: 1) and / or one or more fragments of the ECD (e.g., having a length of about 15-30 amino acids), and the full-length Napi2b and / or one or more fragments thereof are fused to or not fused to one or more protein tags (e.g., 6×His tag, FLAG, HA, V5, Fc fusion, MBP, SUMO, TEV, GFP, TST); preferably, preferably the K D This is measured by an ELISA assay and is preferably about 0.05 to about 0.2 nmol / L. More preferably, K in the range of about 0.071 to about 0.147 nmol / L D Having An anti-NaPi2b antibody having one or more of the above items. 11. A monoclonal anti-NaPi2b antibody, one of the above items. 12. An anti-NaPi2b antibody from any of the above items, which is a chimeric antibody and / or a humanized antibody. 13. An anti-NaPi2b antibody capable of specifically recognizing Napi2b, which is overexpressed in cancer cells, according to any one of the above items. 14. An anti-NaPi2b antibody, preferably a human IgG antibody, and more preferably a human IgG1 antibody, which is one of the above items. 15. An anti-NaPi2b antibody containing a kappa (κ) light chain, one of the above items. 16. An anti-NaPi2b antibody containing a lambda (λ) light chain, one of the above items. 17. The anti-NaPi2b antibody contains Fc silencing mutations such as leucine (L) to alanine (A) substitutions (LALA mutations) at positions 234 and 235 (e.g., in the Fc region of the antibody). The LALA mutation can reduce the effector function of immune cells (for example, the antibody includes a mutated Fc region (or crystalline fragment region) (for example, according to any one of the above items), for example, a tail region of the antibody that interacts with cell surface Fc receptors in the unmutated state (for example, where the Fc region of the immunoglobulin molecule consists of a constant region of the heavy chain, which can bind to antibody receptors (Fc receptors) and complement C1q components on cells in the unmutated state)), One of the anti-NaPi2b antibodies listed above. 18. An anti-NaPi2b antibody can be internally translocated by target cells expressing Napi2b (e.g., cancer cells); preferably, the internally translocated antibody is directed towards lysosomes, one of the anti-NaPi2b antibodies of the above items. 19. An anti-NaPi2b antibody which is tumor-selective, preferably the tumor is a liquid tumor and / or a solid tumor, any one of the above items. 20. An anti-NaPi2b antibody, which is selective for malignant cells, and is one of the anti-NaPi2b antibodies listed above. 21. An anti-NaPi2b antibody of any one of the above items that can bind to human Napi2b and / or rat Napi2b in a glycosylation-dependent manner and binds to the glycosylated form of Napi2b protein. 22. When an anti-NaPi2b antibody is detected (for example, in OVCAR-3 cells that endogenously express Napi2b (e.g., HTB-161, ATCC)), it reacts with endogenously expressed human Napi2b at a K level in the range of approximately 0.01 to approximately 10 nmol / L, preferably in the range of approximately 1 to approximately 10 nmol / L, more preferably in the range of approximately 1 to approximately 7 nmol / L, and even more preferably in the range of approximately 1 to approximately 4 nmol / L. D Having, and more preferably the K D However, it is measured by the FACS assay method, and more preferably, K in the range of about 2.661 to about 6.644 nmol / L. D An anti-NaPi2b antibody having any one of the above items. 23. Anti-NaPi2b antibodies react with immobilized exogenous full-length Napi2b and / or one or more fragments thereof in a K2 concentration ranging from approximately 0.01 to approximately 10 nmol / L. D The fragment has, preferably, one or more fragments comprising at least one extracellular domain (ECD) of Napi2b (e.g., the ECD comprises amino acids 122-135 and / or amino acids 235-361 and / or amino acids 429-485 and / or amino acids 547-552 of human Napi2b having SEQ ID NO: 1) and / or one or more fragments of the ECD (e.g., having a length of about 15-30 amino acids), and the full-length Napi2b and / or one or more fragments thereof are fused to or not fused to one or more protein tags (e.g., 6×His tag, FLAG, HA, V5, Fc fusion, MBP, SUMO, TEV, GFP, TST), preferably, preferably the K D The K2 is measured by an ELISA assay and is preferably about 0.05 to about 0.2 nmol / L, and more preferably in the range of about 0.071 to about 0.147 nmol / L. D Having, One of the anti-NaPi2b antibodies listed above. 24. An anti-NaPi2b antibody from any of the above items, wherein the antibody is bound to a labeling group. 25. An anti-NaPi2b antibody from any of the above items, which can be obtained using a hybridoma (for example, the antibody is a recombinant antibody). 26. An anti-NaPi2b antibody of any one of the above items, obtained according to Example 1 or 2 of this Specification and / or having the features described in Example 1 or 2 of this Specification (e.g., Figures 1-33, in particular Figures 3, 4, 5, 6, 7, 8, 9, 19, and / or 20). 27. Preferably an anti-NaPi2b antibody selected from the group consisting of SEQ ID NO: 4 to 53, comprising one or more (e.g., two) CDRs, heavy chain variable regions, light chain variable regions, heavy chains, light chains, and / or signal sequences of any of the items. 28. An anti-NaPi2b antibody of any one of the items, preferably comprising at least one (e.g., two) heavy chains and light chains of any one of the items. 29. Consists of one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions (or mutations), which are preferably: CDR (e.g., CDR1, CDR2, CDR3, e.g., CDR-H1, CDR-H2, CDR-H3, CDR-H1, CDR-L1, CDR-L2, and / or CDR-L3, e.g., those listed in any one of the above items, e.g., the sequence listings disclosed herein), V H (Variable region heavy chain), V L (Variable region light chain), C H (Steady region heavy chain), or C L An anti-NaPi2b antibody of any one of the items, located within one or more regions selected from the group consisting of (constant region light chain), F(ab), and / or Fc region (for example, as defined in Figure 1 herein). 30. Consists of one or more (e.g., two, three, four, five, six, seven, eight, nine, or ten) highly conserved, conserved, or equivalent amino acid substitutions (or mutations), for example, “conserved or equivalent substitutions” means the substitutions listed as “exemplary substitutions” in Table I below, and “highly conserved substitutions” means, as used herein, the substitutions shown under the heading “preferred substitutions” in Table I below. Preferably, one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) highly conserved, conserved, or equivalent amino acid substitutions (or mutations) are: CDR (e.g., CDR1, CDR2, CDR3, e.g., CDR-H1, CDR-H2, CDR-H3, CDR-H1, CDR-L1, CDR-L2, and / or CDR-L3, e.g., those listed in any one of the above items, e.g., the sequence listings disclosed herein), V H (Variable region heavy chain), V L (Variable region light chain), C H (Steady region heavy chain), or C L An anti-NaPi2b antibody of any one of the items, located within one or more regions selected from the group consisting of (constant region light chain), F(ab), and / or Fc region (for example, as defined in Figure 1 herein). 31. Any one of the above items, in which the percentage of HMWS (high molecular weight species) of the antibody is less than 5% (as shown in Figure 20, for example). 32. A hybridoma that produces any one of the monoclonal antibodies listed above. 33. A nucleic acid that encodes one of the antibodies listed above. 34. An expression vector comprising at least one nucleic acid molecule from any of the items described above. 35. An isolated host cell (e.g., an isolated recombinant host cell) containing any one of the vectors and / or nucleic acids described above. 36. An antibody-drug conjugate (ADC) containing one of the anti-NaPi2b antibodies listed above. 37. The anti-NaPi2b antibody has one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) cytotoxic moieties (e.g., cytotoxic An antibody-drug conjugate (ADC) of any one of the above items is conjugated to a payload, for example, a tubulin inhibitor, for example, a topoisomerase I inhibitor, for example, auristatin or camptothecin, for example, MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), for example, exatecan), preferably via one or more linkers, more preferably via one or more phosphoamidate linkers. 38. An antibody-drug conjugate (ADC) of any one of the above items, wherein an anti-NaPi2b antibody is conjugated to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) camptothecin (e.g., exatecan) cytotoxic moieties, preferably via one or more linkers, more preferably via one or more phosphoamidate linkers. 39. An antibody-drug conjugate (ADC) of any one of the above items, wherein an anti-NaPi2b antibody is conjugated to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) exatecan cytotoxic moieties via one or more linkers, preferably via one or more phosphoamidate linkers. 40. The antibody-drug conjugate (ADC) contains a humanized monoclonal NaPi2b-specific IgG1 antibody conjugated to a cytotoxic payload. Here, (a) The cytotoxic payload is selected from the group consisting of camptothecin, mytansinoids, calicheamycin, duocalmycin, tubulisin, amatoxin, drastatin, and auristatin, e.g., monomethyl auristatin E (MMAE), pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and their analogues or prodrugs; and / or (b) The cytotoxic payload is a camptothecin moiety C selected from the group consisting of exatecan, DXD, SN38, camptothecin, topotecan, irinotecan, berotecan, lulutotecan, rubitecan, silatecan, cocitecan, and gimatecan; and / or (c) The cytotoxic payload is conjugated via a cleavable linker (L), preferably the linker L is cleavable by a protease, glucuronidase, sulfatase, phosphatase, esterase, or disulfide reduction, and more preferably the linker is cleavable by a protease, preferably a cathepsin such as cathepsin B; and / or (d) The linker L comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; and / or (e) The cytotoxic payload is an exatecan conjugated via a chemical valine-citrulline-PAB releasing unit or a valine-alanine-PAB releasing unit, the releasing unit being cleavable by a protease. An antibody-drug conjugate (ADC) from any of the above items. 41. One or more anti-NaPi2b antibodies inflict camptothecin (exatecan) cell damage (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cells, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8 cells). The harmful portion is conjugated via conjugation with an ethinylphosphoneamidate linker (for example, to all eight interchain cysteine ​​residues), preferably each phosphoneamidate linker having at least one PEG24 portion (for example, to prevent aggregation of the ADC), and more preferably the ADC having up to eight such linker payload portions and eight PEG24 portions, an antibody-drug conjugate (ADC) of any one of the above items. 42. An antibody-drug conjugate (ADC) having a formula selected from the group consisting of the following items, which is any one of the above items: (a) Formula I: TIFF2026513097000002.tif61148(b) Formula II: In formula TIFF2026513097000003.tif62148, n is in the range of 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8); (c) Formula III: In formula TIFF2026513097000004.tif60148, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); (d) Formula IV: In formula TIFF2026513097000005.tif61148, n is in the range of 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8); (e) Formula V: In formula TIFF2026513097000006.tif61148, n is in the range of 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8); (f) Equation VI: In formula TIFF2026513097000007.tif61149, n is in the range of 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8); (g) Formula VII: In formula TIFF2026513097000008.tif61148, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); (h) Formula VIII: In formula TIFF2026513097000009.tif60128, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); (i) Formula IX: In formula TIFF2026513097000010.tif60128, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); (j) Formula X: In formula TIFF2026513097000011.tif60128, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); (k) Formula XI: In formula TIFF2026513097000012.tif55154, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); (l) Formula XII: In formula TIFF2026513097000013.tif55154, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); (m)Formula XIII: In formula TIFF2026513097000014.tif55154, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); (n) Formula XIV: In formula TIFF2026513097000015.tif55154, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); (o)Formula XV: In formula TIFF2026513097000016.tif55154, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); (p)Formula XVI: In formula TIFF2026513097000017.tif55154, n is in the range of 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8). 43. (a) Anti-NaPi2b monoclonal antibodies can specifically recognize human Napi2b (e.g., SEQ ID NO: 1) and / or rat Napi2b (e.g., SEQ ID NO: 2) overexpressed on cancer cells; and (b) When ADC binds to Napi2b overexpressed on cancer cells, the ADC can be internalized by the cell and transported into the lysosomal compartment, where preferably a lysosomal protease (e.g., cathepsin B) can release a cytotoxic payload from the ADC. An antibody-drug conjugate (ADC) from any of the above items. 44. Any one of the above items, an antibody-drug conjugate (ADC) in which the proportion (%) of HMWS (high molecular weight species) of the antibody is less than 5% (as shown in Figure 20, for example). 45. A method for producing antibody-drug conjugates (ADCs), comprising the following steps: (a) One or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, most preferably 6 to 10, most preferably 7 to 10, most preferably 4 or 8, most preferably 8) cytotoxic moieties (e.g.) For example, the process involves conjugating a cytotoxic payload, such as a tubulin inhibitor, such as a topoisomerase I inhibitor, such as auristatin or camptothecin, such as MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), such as exatecan (e.g., CAS number: 171335-80-1), preferably via one or more linkers, and more preferably via one or more phosphoamidate linkers. 46. ​​A method for producing an antibody-drug conjugate (ADC) of any one of the above items, wherein an anti-Napi2b antibody is conjugated to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) camptothecin (e.g., exatecan (e.g., CAS number: 171335-80-1)) cytotoxic moieties, preferably via one or more linkers, more preferably via one or more phosphoamidate linkers. 47. A method for producing an antibody-drug conjugate (ADC) of any one of the above items, wherein an anti-Napi2b antibody is conjugated to one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) exatecan cytotoxic moieties via one or more linkers, preferably one or more phosphoamidate linkers. 48. Anti-Napi2b antibody is present in one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 cells, preferably 1 to 10, more preferably 2 to 10, most preferably 4 to 10, even more preferably 6 to 10, even more preferably 7 to 10, even more preferably 4 or 8, most preferably 8) camptothecin (exatecan (e.g., CAS number: 171335-80-1)) cells. A method for producing an antibody-drug conjugate (ADC) of any one of the above items, wherein the toxic portion is conjugated via conjugation with an ethinylphosphoneamidate linker (for example, to all eight interchain cysteine ​​residues), preferably each phosphoneamidate linker having at least one PEG portion having up to 24 PEG units (for example, to prevent aggregation of the ADC), and more preferably the ADC having up to eight linker payload portions and eight PEG24 portions. 49. The ADC contains a humanized monoclonal Napi2b-specific IgG1 antibody conjugated to a cytotoxic payload. (a) The cytotoxic payload is selected from the group consisting of camptothecin, mytansinoids, calicheamycin, duocalmycin, tubulisin, amatoxin, drastatin, and auristatin, e.g., monomethyl auristatin E (MMAE), pyrrolobenzodiazepine dimers, indolino-benzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and their analogues or prodrugs; and / or (b) The cytotoxic payload is a camptothecin moiety C selected from the group consisting of exatecan (e.g., CAS number: 171335-80-1), DXD, SN38, camptothecin, topotecan, irinotecan, berotecan, lulutotecan, rubitecan, silatecan, cocitecan, and gimatecan; and / or (c) The cytotoxic payload is conjugated via a cleavable linker (L), preferably the linker L is cleavable by a protease, glucuronidase, sulfatase, phosphatase, esterase, or disulfide reduction, and more preferably the linker is cleavable by a protease, preferably a cathepsin such as cathepsin B; and / or (d) The linker L comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; and / or (e) The cytotoxic payload is exatecan (e.g., CAS number: 171335-80-1) conjugated via a chemical valine-citrulline-PAB releasing unit or a valine-alanine-PAB releasing unit, wherein the releasing unit is cleavable by a protease. A method for producing one of the antibody-drug conjugates (ADCs) described above. 50. An antibody-drug conjugate (ADC) produced by any one of the methods described above. 51. An antibody-drug conjugate (ADC) having an HMWS (high molecular weight species) ratio (%) of less than 5%, preferably thereby reducing the aggregation of the ADC and / or reducing the toxicity of the ADC to normal (e.g., non-cancerous) tissue. 52. Any one of the above-mentioned antibody-drug conjugates (ADCs) having a drug-to-antibody ratio (DAR) in the range of 0 to 20, preferably in the range of 1 to 20, more preferably in the range of 2 to 12, most preferably in the range of 4 to 10, and most preferably in the range of 4 to 8. 53. An antibody-drug conjugate (ADC) of any one of the above items, wherein the DAR of the ADC is 4 or 8, preferably 8. 54. A composition or kit comprising any one of the above items: anti-Napi2b, antibody-drug conjugate (ADC), hybridoma, nucleic acid, expression vector, and / or host cell. 55. Any one of the above items, which is a pharmaceutical and / or diagnostic composition. 56. Any one of the above compositions or kits, wherein the ratio (DAR) of a drug (e.g., cytotoxic moiety (e.g., cytotoxic payload, e.g., tubulin inhibitor, e.g., topoisomerase I inhibitor, e.g., auristatin or camptothecin, e.g., MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), e.g., exatecan) to an antibody is in the range of 0 to 20, preferably in the range of 1 to 20, more preferably in the range of 2 to 12, most preferably in the range of 4 to 10, and most preferably in the range of 4 to 8. 57. A composition or kit comprising any one of the above items: an anti-NaPi2b antibody, an antibody-drug conjugate (ADC), a hybridoma, a nucleic acid, an expression vector, and / or a host cell. 58. A method for treating, improving, preventing and / or diagnosing cancer, preferably the cancer being a solid tumor and / or metastatic cancer, and more preferably the cancer being selected from the group consisting of lung cancer, ovarian cancer, thyroid cancer, non-squamous non-small cell lung cancer, non-mucinous ovarian cancer, papillary thyroid cancer, kidney cancer, endometrial cancer, uterine cancer, ureteral cancer, bladder cancer, and fallopian tube cancer, wherein the method comprises the step of administering a therapeutically effective or prophylactically effective amount of any one of the aforementioned antibodies, antibody-drug conjugates (ADCs), nucleic acids, expression vectors, host cells, compositions, or kits. 59. Any antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition, or kit for use as a pharmaceutical and / or therapeutic agent. 60. Any one of the above items for use in one or more of the following methods: an antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition, or kit: (a) A method for treating, improving, preventing and / or diagnosing cancer, preferably the cancer being a solid tumor and / or metastatic cancer, and more preferably the cancer being selected from the group consisting of lung cancer, ovarian cancer, thyroid cancer, non-squamous non-small cell lung cancer, non-mucinous ovarian cancer, papillary thyroid cancer, kidney cancer, endometrial cancer, uterine cancer, ureteral cancer, bladder cancer, and fallopian tube cancer; (b) Methods for monitoring the progression of cancer and / or for evaluating the effectiveness of cancer treatments; (c) Methods for screening candidate compounds for anticancer activity; (d) Methods for altering the resistance of cancer cells to chemotherapy; (e) Methods for making cancer cells sensitive to chemotherapy; (f) Methods for inhibiting the proliferation of cancer cells expressing NaPi2b; (g) Methods for the production or preparation of antibodies; (h) Methods for immunizing non-human animals; (i) Methods for preparing hybridomas; (j) Any one of the methods described above; (k) One of the methods (a) to (j) which is in vivo, in vitro, or ex vivo. 61. Use of any one of the above items for one or more of the following: an antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition, or kit: (a) Treatment, improvement, prevention, and / or diagnosis of cancer (preferably, the cancer is a solid tumor and / or metastatic cancer, and more preferably, the cancer is selected from the group consisting of lung cancer, ovarian cancer, thyroid cancer, non-squamous non-small cell lung cancer, non-mucinous ovarian cancer, papillary thyroid cancer, kidney cancer, endometrial cancer, uterine cancer, ureteral cancer, bladder cancer, and fallopian tube cancer); (b) Monitoring the progression of cancer and / or evaluating the effectiveness of cancer treatments; (c) Screening candidate compounds for their anticancer activity; (d) Altering the resistance of cancer cells to chemotherapy; (e) Making cancer cells sensitive to chemotherapy; (f) Inhibiting the proliferation of cancer cells that express NaPi2b; (g) Production or preparation of antibodies; (h) Immunizing non-human animals; (i) Preparation of hybridomas; (j) in any one of the above items; (k) Any one of (a) through (j) is an in vivo, in vitro, or ex vivo use.

[0101] Where used herein, the singular forms “a,” “an,” and “the” refer to multiple objects unless otherwise indicated in the context. For example, “a reagent” refers to one or more of such reagents, and “the method” refers to equivalent steps and methods known to those skilled in the art, which may be modified or used in place of the method described herein.

[0102] Unless otherwise specified, the term “at least” preceding a series of elements should be understood to refer to all elements in that series. Those skilled in the art will recognize, or can verify through experiments not exceeding the scope of routine experimentation, numerous equivalents of the specific embodiments of the present invention described herein. Such equivalents are intended to be encompassed by the present invention.

[0103] Whenever the terms “and / or” are used herein, they include the meanings of “and,” “or,” and “all or any other combination of the elements linked by the terms.”

[0104] When used herein, the terms "about" or "approximately" mean within 20%, preferably within 10%, and more preferably within 5% of a given value or range.

[0105] The terms "less than" or "more than" do not include that specific number.

[0106] For example, "less than 20" means less than the specified number. Similarly, "more than" or "greater than" means more than or greater than the specified number; for example, "more than 80%" means more than or greater than the specified number, which is 80%.

[0107] Throughout this specification and the subsequent claims, unless otherwise indicated in the context, the word “comprise,” and variations such as “comprises” and “comprising,” shall be understood to mean encompassing the integer or process, or group of integers or processes, described herein, but not excluding any other integers and processes, or groups of integers and processes. Where used herein, the term “comprising” may be replaced by the terms “containing” or “including,” or sometimes, where used herein, the term “having.” Where used herein, “consisting of” excludes any element, process, or component not specified.

[0108] The term "includes" means "includes, but is not limited to." "Includes" and "includes, but is not limited to" are used synonymously.

[0109] It should be understood that the present invention is not limited to, and therefore may vary, the specific methodologies, protocols, materials, reagents, and substances described herein. The technical terms used herein are for the sole purpose of describing specific embodiments and are not intended to limit the scope of the present invention as defined solely by the claims.

[0110] All publications cited throughout this Specified, including all patents, patent applications, scientific publications, and instructions, whether preceding or following, are incorporated herein by reference in their entirety. Nothing herein should be construed as admitting that the present invention has no prior rights to such disclosures based on prior art. In the event that any material incorporated by reference conflicts with or is inconsistent with this Specified, this Specified shall prevail over any such material.

[0111] The contents of all documents and patent documents cited herein are incorporated in their entirety by reference. [Examples]

[0112] Examples of the Invention A deeper understanding of the present invention and its advantages will be evident from the following examples, which are provided for illustrative purposes only. These examples do not limit the scope of the present invention in any way.

[0113] Example 1: General information Chemicals, solvents, and antibodies Chemicals and solvents were purchased from Merck (Merck group, Germany), TCI (Tokyo Chemical Industry Co., Ltd., Japan), Iris Biotech (Iris Biotech GmbH, Germany), MCE (MedChemExpress, USA), and Carl Roth (Carl Roth GmbH + Co. KG, Germany), and used without further purification. Dry solvents were purchased from Merck (Merck group, Germany). PEG24 was purchased from BiochemPEG (Pure Chemistry Scientific Inc., United States).

[0114] Preparative HPLC For small-scale preparative HPLC, a VP 250 / 10 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH & Co. Kg, Germany) was used in a BUCHI Pure C-850 Flash-Prep system (BUCHI Labortechnik AG, Switzerland). The following gradient was used: Method C: (A=H2O+0.1%TFA (trifluoroacetic acid), B=MeCN (acetonitrile)+0.1%TFA, flow rate 6 ml / min, 30%B 0-5 min, 30-70%B 5-35 min, 99%B 35-45 min). For larger scales, a VP 250 / 21 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH & Co. Kg, Germany) was used, with the following gradient: Method D: (A=H2O+0.1%TFA (trifluoroacetic acid), B=MeCN (acetonitrile)+0.1%TFA, flow rate 14 ml / min, 30%B 0-5 min, 30-70%B 5-35 min, 99%B 35-45 min).

[0115] LC-MS Low molecular weight molecules, linker payloads, antibodies, and ADCs were analyzed using Waters H-class instruments equipped with a 4-solvent control system, Waters sample control system-FTN, Waters PDA detector, and Waters column control system, using an Acquity UPLC protein BEH C4 column (300 Å, 1.7 μm, 2.1 mm × 50 mm) for antibodies and ADCs. In this case, samples were eluted at a column temperature of 80°C. The following gradients were used: A: 0.1% formic acid dissolved in H2O, B: 0.1% formic acid dissolved in MeCN. Mass spectrometry was performed using a Waters XEVO G2-XS QTof analyzer. Proteins were ionized in positive ion mode by applying a 40kV cone voltage. Raw data were analyzed using MaxEnt 1. Small molecules and linker payloads were analyzed using an Acquity UPLC-BEH C18 column (300Å, 1.7μm, 2.1mm×50mm). In this case, the sample was eluted at a column temperature of 45°C and a flow rate of 0.4 mL / min. The following gradient was used: A: 0.1% formic acid dissolved in H2O, B: 0.1% formic acid dissolved in MeCN. 2%B 0-1 min, 2-98%B 1-5 min, 98%B 5-5.5 min, 98-2%B 5.5-6 min, 2%B 6-7 min.

[0116] CDR mutagenesis and antibody expression For efforts to induce CDR mutations in parental antibodies, 22 × 10 8 A phage display library containing 100 sequences (66% functional) was prepared. This preparation involved homology modeling of the antibody Fv region, selection of surface CDR residues potentially involved in antigen binding (18 positions identified for VH and 16 for VK), and NGS data (approximately 2 × 10⁶). 6The analysis was performed based on the amino acid usage at each position in the IgG sequence (Figure 1). Panning of the CDR mutant phage library to select antibodies with improved affinity was performed by applying stepwise antigen restriction four times to antigen / Napi2b expressing cells, achieved by increasing cell dilution. From each panning, 384 clones (1536 clones in total) were selected and produced as soluble scFv antibodies in Escherichia coli (E. coli). These were screened for binding to human Napi2b expressing HEK293 cells and parental HEK293 cells using flow cytometry. A signal-to-noise ratio (N / S) greater than 12 between the MFI of positive cell lines and the MFI of negative cell lines was selected as the hit selection criterion. According to the above selection criterion, 375 clones were identified and their DNA sequences were determined. From these results, 255 different antibody sequences were identified, and the offrate of these clones was further analyzed using BLI technology. In detail, biotinylated Napi2b antigen was immobilized on a streptavidin sensor, and the binding and dissociation of scFv were measured. Off-rate dynamics were modeled, and clones that met the following criteria—namely, a significant binding response and high modeling accuracy—were ranked. For further detailed analysis, the following five antibody clones / sequences were selected: AV-15, AV-18, AV-21, AV-25, and AV-29 (Figure 1).

[0117] Next, antibodies were transiently expressed in Expi-CHO-S cells (Thermo Fisher) by co-transfecting cells with pcDNA3.4 expression plasmids (Thermo Fisher) encoding the heavy and light chains of each sequence in a 1:1 ratio using the Expi-CHO transfection system (Thermo Fisher). Cells were collected by centrifugation at 300 g, 4°C for 5 minutes. To remove fine particles from the supernatant, the supernatant was centrifuged at 4000-5000 g, 4°C for 30 minutes. For further clarification, the supernatant was passed through a 0.22 μm filter. Antibodies were purified from the clarified and filtered supernatant by protein A chromatography and analyzed by HPLC-SEC, HPLC-HIC, LC-MS, and SDS-PAGE.

[0118] Preparative size exclusion chromatography Protein purification by size exclusion chromatography was performed using an AKTA Pure FPLC system (GE Healthcare, United States) equipped with an F9-C fraction collector.

[0119] Measurement of ADC concentration ADC concentrations were measured in 96-well plates using the Pierce® Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific, USA) and Bradford Reagent B6916 (Merck, Germany) along with a pre-diluted bovine gamma globulin (Thermo Fisher Scientific, USA) protein assay standard. The results from both assay methods were arithmetically averaged.

[0120] Sample preparation of ADCs and antibodies for MS To deglycosylate the antibody and ADC, 0.5 μl of PNGase-F solution (Pomega, Germany, recombinant, cloned from Elizabethkingia miricola, 10 u / μl) and 5 μl of 100 mM DTT aqueous solution were added to 50 μl of PBS containing 0.2 mg / mL antibody or ADC, and the solution was incubated at 37°C for at least 2 hours. The glycosylated mAb and ADC were incubated with 10 mM DTT at a concentration of 0.2 mg / mL for 1 hour. 2 μl of each sample was injected and the samples were subjected to LC / MS.

[0121] Analytical size exclusion chromatography ADC analysis size exclusion chromatography (A-SEC) was performed on a Vanquish Flex UHPLC system equipped with a DAD detector, split sampler FT (4°C), column compartment H (25°C), and binary pump F (Thermo Fisher Scientific, USA), using a MAbPac SEC-1 300 Å, 4 × 300 mm column (Thermo Fisher Scientific, USA) at a flow rate of 0.15 mL / min. Separation of various ADC / mAb populations was achieved by a constant composition gradient over 30 minutes using pH 7 phosphate buffer (20 mM Na₂HPO₄ / NaH₂PO₄, 300 mM NaCl, 5% v / v isopropyl alcohol) as the mobile phase. 8 μg of ADC / mAb was loaded onto the column for A-SEC analysis. UV chromatograms were recorded at 220 nm and 280 nm.

[0122] Analytical Hydrophobic Interactions Chromatography Measurements were performed on a Vanquish Flex UHPLC system (2.9) using a MabPac HIC butyl 4.6 × 100 mm column (Thermo Fischer Scientific, USA). Separation of various ADCs / antibodies was achieved using the following gradient: A: 1M (NH4)2SO4, 500 mM NaCl, 100 mM NaH2PO4 pH 7.4, B: 20 ​​mM NaH2PO4, 20% (v / v) isopropyl alcohol, pH 7.4. 0%B: 0-1 min, 0-95%B: 1-15 min, 95%B: 15-20 min, 95-0%B: 20-23 min, 0%B: 23-25 ​​min, flow rate 700 μL / min. For each analysis, 15 μg of sample was loaded onto the column. UV chromatograms were recorded at 220 nm and 280 nm.

[0123] Charge Variant Analysis Measurements were performed using a ProPac Elite WCX 5μm 4×150mm column (Thermo Fischer Scientific) on a Vanquish Flex UHPLC system (2.9). Antibody charge variant separation was performed with the following gradient: A: 1×CX-1 buffer pH 5.6, B: 1×CX-1 buffer pH 10.2 (Thermo Fisher Scientific). 0~100% B: 0~60 minutes, flow rate 1 ml / min. 24 μg of sample was loaded onto the column for each analysis. UV chromatograms were recorded at 280 nm. Charge variant analysis was performed as described above. Changes after 7 days of incubation at 40°C compared to day 0 are shown (percentage decrease in the main peak (%) and percentage increase in acidic and basic species (%)) (Figure 2).

[0124] SDS-PAGE Samples were prepared for SDS-PAGE by incubation (95°C, 5 minutes) in SDS sample buffer (BioRad) supplemented with 25 mM DTT, separated using a 4-20% polyacrylamide gel (BioRad 4-20% Mini Protean TGX), and subsequently stained with Coomassi stain (Thermo Fisher Scientific Imperial protein stain).

[0125] ADC synthesis: A common method for conjugating a P5-based linker-payload construct to an antibody to obtain DAR8. 50 μl of each anti-NaPi2b antibody (parent, AV15, AV18, AV21, AV25, AV29) in a 10.0 mg / ml solution dissolved in P5-conjugation buffer (50 mM Tris, 1 mM EDTA, 100 mM NaCl, pH 8.3, room temperature) was mixed with 3.33 μl of 10 mM TCEP solution in P5-conjugation buffer. Immediately afterward, 1.67 μl of a 40 mM solution of P5-exatecan construct dissolved in DMSO was added. This mixture was shaken at 350 rpm, 25°C for 16 hours. The reaction mixture was purified by preparative size exclusion chromatography using 25 ml of Superdex® 200 Increase 10 / 300GL (Cytiva, Sweden) and eluted with sterile PBS (Merck, Germany) at a flow rate of 0.8 ml / min. The antibody-containing fractions were combined and concentrated by spin filtration (Amicon® Ultra-2mL MWCO:30kDa, Merck, Germany).

[0126] Binding to human and rat NaPi2B, as evaluated by flow cytometry. HEK293 cells stably overexpressing human Napi2b and rat Napi2b were generated by stably incorporating a human or rat full-length Napi2b-mCherry-(GGGS)3x-mCherry expression construct under the control of the human EF1 promoter and a puromycin selection cassette under the control of the CMV promoter into the parental HEK293 cell line. Briefly, cells were transfected with linear plasmids using Lipfectamine 2000 (Thermo Fisher) and selected with the antibiotic puromycin. Subsequently, cells were cloned as single cells in 96-well plates by serial dilution under continuous antibiotic selection. Clones were screened for target expression by flow cytometry and for mCherry fluorescence by flow cytometry and fluorescence microscopy. Clones positive for the target and mCherry were grown and used for further experiments. Equilibrium binding constant (K) D To determine the optimal antibody-positive cell type (mCherry), HEK293 cells stably expressing full-length NaPi2b-mCherry from humans (A and C), cynomolgus monkeys (B), or rats (D) were incubated with antibodies ranging from 0.002 to 200 nM. These cells were stained with Alexa-labeled anti-human IgG H+L secondary antibody (Thermo Fisher Scientific) and analyzed by flow cytometry. Antibody binding was investigated in mCherry-positive cells. The mean fluorescence intensity (MFI) ratio was normalized relative to the secondary antibody control. The assay was performed in pairs, and data points were analyzed by nonlinear regression using a single-site specific binding model, using Prism 9 software. D The values ​​were obtained. The graph shows the mean (n=2) ± SEM (Figure 3).

[0127] Binding to immobilized human NaPi2B, as evaluated by ELISA. The binding of parental mAb clones and AV mAb clones to purified, immobilized recombinant human Napi2b antigen at increasing concentrations was investigated using an ELISA setup.

[0128] To measure the binding of parental mAb clones and AV mAb clones to the Napi2b antigen, surface-treated flat-bottomed 96-well plates (Nunclon, Thermo Fisher Scientific) were coated with 1 μg / mL of purified recombinant human Napi2b antigen consisting of Napi2b extracellular loop 2 expressed as an Fc-fusion-6His tagged protein in Expi-HEK293 cells. After blocking with 2% bovine serum albumin (Carl Roth) in 1×PBS-Tween20 (0.05%), the antibody concentration was increased (0.00015 μg / ml, 0.00046 μg / ml, 0.00137 μg / ml, 0.00412 μg / ml, 0.01235 μg / ml, 0.03704 μg / ml, 0.11111 μg / ml, 0.33333 μg / ml, 1.00000 μg / ml, 3.00000 μg / ml, 9.00000 μg / ml) and conjugated at room temperature for 2 hours. The conjugated antibodies were detected by incubation with HRP-conjugated goat anti-human kappa light chain secondary antibody (diluted 1:10000 in blocking solution) at room temperature for 1 hour. Ultra-TMB (Thermo Fisher, 34028) substrate was added and incubated at room temperature for 15-30 minutes, after which 100 μl / well of 1M sulfuric acid was added. Within 10 minutes of adding the acid, the absorbance at 450 nm was measured using a microplate reader Infinite M1000 Pro (Tecan). The apparent dissociation constant (K) was then determined. D ) was calculated using nonlinear regression with a single-site specific binding model with Prism 9 software. D The values ​​(in μg / ml) are 0.02209 (parent mAb), 0.01581 (AV15), 0.01494 (AV18), 0.01248 (AV21), 0.01060 (AV25), and 0.01238 (AV29). The graph shows the mean (n=2) ± SEM (Figure 4).

[0129] Internal migration evaluated by flow cytometry To investigate internal migration using pHrodo, a goat anti-human IgG Fcγ fragment-specific secondary antibody (Jackson ImmunoResearch) was labeled using the pHrodo® Deep Red antibody labeling kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Napi2b-positive OVCAR-3 cells were incubated with 5 μg / ml parental mAb clones and AV mAb clones in the presence of equimolar amounts of pHrodo Deep Red-labeled secondary antibody at 37°C for 1 hour, 5 hours, and 24 hours. An increase in MFI indicates the presence of AV antibodies in the late endosomal and lysosomal compartments. The MFI ratio was determined by dividing the MFI of cells incubated with pHrodo by the MFI of unstained cells (Figure 5).

[0130] Melting curves evaluated by NanoDSF The thermal stability of the protein was measured using nano-differential scanning fluorescence (nanoDSF), which measures the temperature-dependent changes in the internal fluorescence of tryptophan and tyrosine residues (Tycho NT.6, NanoTemper Technologies). For this purpose, 1 μM of protein mixed in PBS was absorbed into a capillary tube and then placed into a reader. The internal fluorescence of the protein was then measured at 330 nM and 350 nM while incubating at increasing temperature. Changes in the fluorescence signal indicate transitions in the protein's folding state, and the temperature at which the transition occurs is the inflection temperature (T). i ) or melting temperature (T m It is also known as (Haffke, M. et al., Label-free Thermal Unfolding Assay of G Protein-Coupled Receptors for Compound Screening and Buffer Composition Optimization. 2016) (Figure 6).

[0131] In vitro cytotoxicity assessed by the resazurin assay. To investigate the direct cytotoxicity of ADC, each cell was seeded in a 96-well plate (flat-bottom, 5000 cells / well, suspended in 100 μl of culture medium) and incubated with increasing concentrations of ADC in the medium (0-3 μg / ml) for 7 days to create a dose-response curve. Before viability analysis, the supernatant on adherent cells was removed and replaced with fresh medium. Subsequently, cell death was analyzed using resazurin (Sigma-Aldrich) at a final concentration of 55 μM as a cell viability discriminant dye. Fluorescence emission at 590 nM was measured using a microplate reader Infinite M1000 Pro (Tecan). Cell viability was determined by dividing the fluorescence of ADC-treated cells by the fluorescence of control cells similarly treated with culture medium alone. The graph shows the mean (n=2) ± SEM (Figure 7).

[0132] Bystander dies To analyze the bystander activity of ADC against target-negative cells, 20,000 NaPi2B-positive cells (OVCAR-3) were incubated with gradually increasing concentrations of ADC (0-3 μg / ml). After 5 days, half of the cell culture supernatant was transferred to 5,000 NaPi2B-negative cells (SW-620), and incubated for another 5 days. Cell death was analyzed by measuring viability using resazurin as described above (Figure 8).

[0133] In vitro inhibition of topoisomerase I by delivery of exatecan via ADC. Inhibition of topoisomerase I by exatecan delivery via TUB-040 ADC induces DNA damage markers. OVCAR-3 cells were treated with 5 μg / mL TUB-040 or 5 nM free exatecan for 72 hours. Cells were stained with Live / Dead stains, as well as for the DNA damage markers active caspase-3, cleaved PARP, and phosphorylated H2A.X (Ser-139), and analyzed by flow cytometry. Graphs show mean (n=2) ± SEM (Figure 9).

[0134] In vivo PK evaluation for one ADC In vivo pharmacokinetic (PK) experiments were conducted using AV25 and AV25-P5(PEG24)-VC-PAB-exatecan. Female Sprague Dolly rats were treated with 10 mg / kg of unconjugated AV25 antibody or ADC. Blood samples were collected at various time points, and the amount of ADC was quantified using total antibody ELISA and intact ADC ELISA assays.

[0135] To evaluate the pharmacokinetic activity (PK) of ADC in vivo, total antibody concentrations in the serum of ADC-treated SD rats were measured at various time points. All humanized anti-NaPi2B antibodies were analyzed in rat serum in the range of 2000–15.6 ng / ml. Nunc 96-well plates (100 μl / well) were coated with NaPi2B diluted in PBS (required concentration: 0.25 μg / ml) and sealed with PCR foil. The plates were incubated in a refrigerator, maintaining a temperature of 2–8°C overnight. The coated plates were washed three times with 300 μl of PBST. 200 μl / well of blocking solution (2% albumin mixed with PBST) was added, the plates were sealed, and incubated at room temperature for 1 hour. The coated plates were washed three times with 300 μl of PBST. Prepared standards (2000–15.6 ng / ml each of ADC, QC, and test samples) were added at a rate of 100 μl per well, the plate was sealed, and incubated at room temperature for 1 hour. The plate was washed three times with 300 μl of PBST. 100 μl / well of anti-human IgG (γ-chain specific)-peroxidase antibody (diluted 1:60000 in PBS) was added and incubated at room temperature for 1 hour. The plate was washed three times with 300 μl of PBST. 50 μl / well of TMB was added, the plate was sealed, and incubated at room temperature for 15 minutes. 50 μl / well of 1 M sulfuric acid was added. Absorbance at a wavelength of 450 nm was measured using a Tecan plate reader.

[0136] To evaluate the in vivo stability of ADC, the concentration of intact ADC in the serum of ADC-treated SD rats was measured at various time points. Intact ADC was analyzed in rat serum in the range of 2000–15.6 ng / ml. Nunc 96-well plates (100 μl / well) were coated with rabbit anti-exatecan mAb (required concentration: 1 μg / ml) diluted in PBS and sealed with PCR foil. The plates were incubated in a refrigerator, maintaining a temperature of 2–8°C overnight. The coated plates were washed three times with 300 μl of PBST. 200 μl / well of blocking solution (2% albumin mixed with PBST) was added, the plates were sealed, and incubated at room temperature for 1 hour. The coated plates were washed three times with 300 μl of PBST. Prepared standards (2000–15.6 ng / ml each for ADC, QC, and test samples) were added at a rate of 100 μl per well, the plate was sealed, and incubated at room temperature for 1 hour. The plate was washed three times with 300 μl of PBST. 100 μl / well of goat anti-human IgG (H+L) (adsorbed) (diluted 1:25000 in PBS) was added and incubated at room temperature for 1 hour. The plate was washed three times with 300 μl of PBST. 100 μl / well of TMB was added, the plate was sealed, and incubated at room temperature for 10 minutes. 100 μl / well of 1 M sulfuric acid was added. Absorbance at a wavelength of 450 nm was measured using a Tecan plate reader (Figure 10).

[0137] Linker-payload synthesis General method 2 for synthesizing PEGylated P5 structural units by Staudinger phosphonite reaction TIFF2026513097000018.tif58149

[0138] In a 25 ml Schlenk flask, under an argon atmosphere, 267 mg of bis(diisopropylamino)chlorophosphine (1.00 mmol, 1.00 equivalent) was added, cooled to 0°C, and 2.20 mL of ethinyl magnesium bromide solution (0.5 M in THF, 1.10 mmol, 1.10 equivalent) was added dropwise. The yellowish solution was raised to room temperature and stirred for a further 30 minutes. 3.00 mmol (3.0 equivalent) of the desired PEG alcohol, dissolved in 5.56 mL of 1H tetrazole solution (0.45 M in MeCN, 2.50 mmol, 2.50 equivalent), was added, and the white suspension was stirred overnight at room temperature. To form the desired phosphonite, 31 The reaction was monitored by P-NMR. 1.0 mmol (1.0 equivalent) of the desired azide dissolved in 2 mL of DMF, THF, or MeCN was added, and the suspension was stirred at room temperature for 24 hours. The crude reaction mixture was purified by preparative HPLC.

[0139] P5(PEG12)-OSu TIFF2026513097000019.tif27128

[0140] The title compound was synthesized according to General Method 2 from 19.5 mg of bis(diisopropylamino)chlorophosphine (73 μmol, 1.00 equivalent), 146 μL of ethynylmagnesium bromide solution (0.5 M, 73 μmol, 1.00 equivalent in THF), 100 mg of dodecaethylene glycol (183 μmol, 2.50 equivalent), 400 μL of 1H-tetrazole solution (0.45 M, 183 μmol in MeCN), and 19 mg of 4-azidobenzoic acid-N-hydroxysuccinimide (73 μmol, 1.00 equivalent). After preparative HPLC (Method D) and lyophilization, the product was obtained as a colorless oil (42.5 mg, 50 μmol, 68%). TIFF2026513097000020.tif45165

[0141] P5(PEG12)-COOH TIFF2026513097000021.tif24128

[0142] The title compound was synthesized from 40 mg of bis(diisopropylamino)chlorophosphine (150 μmol, 1.00 equivalent), 360 μL of ethynylmagnesium bromide solution (0.5 M in THF, 180 μmol, 1.2 equivalents), 245 mg of PEG12 (450 μmol, 3.0 equivalents), 0.83 mL of 1H-tetrazole solution (0.45 M in MeCN, 450 μmol, 2.5 equivalents), and 39 mg of 4-azidobenzoic acid (150 μmol, 1.00 equivalent) according to General Method 2. After preparative HPLC (Method D) and lyophilization, the product was obtained as a colorless oil. (25 mg, 34 μmol, 23%). Results of HR-MS: C 33 H 57 NO 16 P + [M+H] + Calculated value 754.3410, measured value 754.3398 (UV trace in Figure 11).

[0143] P5(PEG24)-OSu TIFF2026513097000022.tif27128

[0144] The title compound was synthesized from 41 mg of bis(diisopropylamino)chlorophosphine (159 μmol, 1.00 equivalent), 370 μL of ethynylmagnesium bromide solution (0.5 M in THF, 185 μmol, 1.2 equivalents), 450 mg of PEG24 (388 μmol, 2.50 equivalents), 1.02 mL of 1H-tetrazole solution (0.45 M in MeCN, 466 μmol, 3.0 equivalents), and 40 mg of 4-azidobenzoic acid N-hydroxysuccinimide ester (155 μmol, 1.00 equivalent) according to General Method 2. After preparative HPLC (Method D) and lyophilization, the product was obtained as a colorless oil. (79 mg, 57 μmol, 37%). Results of MS: C 61 H 109 N2O 30 P 2+ [M+2H] 2+ Calculated value 690.3396, measured value 690.81. (UV trace in Figure 12).

[0145] NH2-VC-PAB-exatecan TFA salt TIFF2026513097000023.tif54158

[0146] 34.3 mg of exatecan mesylate (0.0645 mmol, 1.0 equivalent) was placed in a screw-cap vial and suspended in 645 μL of dry DMSO. 241 μL (0.0967 mmol, 1.5 equivalents) of 0.4 mol / L Fmoc-VC-PAB-PNP solution in dry DMSO, 64.5 μL (0.0645 mmol, 1.0 equivalent) of 1 mol / L HOBt hydrate solution in dry DMSO, and 113 μL (0.645 mmol, 10.0 equivalents) of DIPEA were added. The yellow solution was stirred at 50°C for 2 hours. Then, 425 μL of 50% (w / w) diethanolamine solution in dry DMSO was added, and the reaction mixture was stirred at room temperature for a further 30 minutes. 1.5 ml of MeCN and 2.5 mL of H2O were added, and the resulting yellow solution was directly purified by preparative HPLC using Method D. After lyophilization, 47.3 mg (76.7%, 0.0495 mmol) of a yellowish solid was obtained as TFA salt. HR-MS result: C 43 H 50 FN8O9 + [M+H] + Theoretical value: 841.3680, measured value: 841.3696 (UV trace in Figure 13).

[0147] NH2-VA-PAB-exatecan TFA salt TIFF2026513097000024.tif54157

[0148] 1.23 mg of exatecan mesylate (0.00232 mmol, 1.0 equivalent) was placed in a screw-cap vial and suspended in 23 μL of dry DMSO. 8.7 μL (0.00348 mmol, 1.5 equivalents) of 0.4 mol / L Fmoc-VA-PAB-PNP solution in dry DMSO, 2.3 μL (0.00232 mmol, 1.0 equivalent) of 1 mol / L HOBt hydrate solution in dry DMSO, and 4 μL (0.0232 mmol, 10.0 equivalents) of DIPEA were added. The yellow solution was stirred overnight at room temperature. Then, 15 μL of 50% (w / w) diethanolamine solution in dry DMSO was added, and the reaction mixture was stirred for a further 30 minutes at room temperature. 1.5 ml of MeCN and 2.5 mL of H2O were added, and the yellow solution was directly purified by preparative HPLC using Method C. After freeze-drying, a yellowish solid was obtained containing 1.01 mg (50.0%, 0.00116 mmol) of TFA salt. HR-MS result: C 40 H 44 FN6O8 + [M+H] + Theoretical value: 755.3200, measured value: 755.3201 (UV trace in Figure 14).

[0149] P5(PEG2)-VC-PAB-Exatecan TIFF2026513097000025.tif54157

[0150] 23.4 μL (0.00468 mmol, 1.0 equivalent) of 200 mM NH2-VC-PAB-exatecan TFA salt solution in dry DMSO, 46.8 μL (P5(PEG2)-COOSu, 0.00936 mmol, 2.0 equivalent) of 200 mM 2-(2-hydroxyethoxy)ethyl-N-(4-benzoic acid-N-hydroxysuccinimide)-P-ethynylphosphoneamidate solution, and 4.08 μL (0.0234 mmol, 5.0 equivalent) of DIPEA were placed in a screw-cap vial. The solution was shaken at 50°C for 5 hours, allowed to cool to room temperature, and 1.5 ml of MeCN and 2.5 mL of H2O were added. This solution was directly purified by preparative HPLC using Method C. After freeze-drying, 1.33 mg (25.0%, 0.00117 mmol) of a yellowish solid was obtained. HR-MS result: C 56 H 64 FN9O 14 P + [M+H] + Theoretical value: 1136.4289, measured value: 1136.4306. (UV trace in Figure 15).

[0151] P5(PEG12)-VC-PAB-Exatecan TIFF2026513097000026.tif52156

[0152] Into a screw-cap vial, 51 μL (0.0102 mmol, 1.0 equivalent) of a 200 mM solution of NH2-VC-PAB-exatecan TFA salt in dry DMSO, 102 μL (P5(PEG12)-COOH, 0.0204 mmol, 2.0 equivalents) of a 200 mM solution of PEG12-N-(4-benzoic acid)-P-ethynylphosphonamidate in dry DMSO, 102 μL (0.0255 mmol, 2.5 equivalents) of a 250 mM solution of Pybop in dry DMSO, and 8.89 μL (0.051 mmol, 5.0 equivalents) of DIPEA were added. The solution was shaken at room temperature for 2 hours, 1.5 ml of MeCN and 2.5 mL of H2O were added, and this solution was directly purified by preparative HPLC using Method D. After lyophilization, 15.91 mg (99.0%, 0.0101 mmol) of a yellowish solid was obtained. Results of HR-MS: C 76 H 105 FN9O 24 P 2+ [M+H] 2+ Theoretical value 788.8492, measured value 788.8485. (UV trace in Figure 16).

[0153] P5(PEG24)-VC-PAB-exatecan TIFF2026513097000027.tif54157

[0154] Into a screw-cap vial, 102 μL (0.0204 mmol, 1.0 equivalent) of a 200 mM solution of NH2-VC-PAB-exatecan TFA salt in dry DMSO, 204 μL (P5(PEG24)-OSu, 0.0408 mmol, 2.0 equivalents) of a 200 mM solution, and 17.78 μL (0.102 mmol, 5.0 equivalents) of DIPEA were added. The solution was shaken at room temperature overnight, 1.5 ml of MeCN and 2.5 mL of H2O were added, and this solution was directly purified by preparative HPLC using Method D. After lyophilization, 25.76 mg (60.0%, 0.01224 mmol) of a yellowish solid was obtained. Results of HR-MS: C 100 H 153 FN9O 36 P2+ [M+H] 2+ Theoretical value 1053.5081, measured value 1053.50833. (UV trace in Figure 17).

[0155] P5(PEG12)-VA-PAB-Exatecan TIFF2026513097000028.tif54158

[0156] 11.6 μL (0.00116 mmol, 1.0 equivalent) of a 100 mM NH2-VA-PAB-Exatecan TFA salt solution in dry DMSO, 8.7 μL (P5(PEG12)-COOH, 0.00174 mmol, 1.5 equivalents) of a 200 mM PEG12-N-(4-benzoic acid)-P-ethynylphosphonamidate solution in dry DMSO, 11.6 μL (0.00232 mmol, 2.0 equivalents) of a 200 mM Pybop solution in dry DMSO, and 2.02 μL (0.0116 mmol, 10.0 equivalents) of DIPEA were placed in a screw-cap vial. The solution was shaken at room temperature for 2 hours, 1.5 ml of MeCN and 2.5 mL of H2O were added, and this solution was directly purified by preparative HPLC using Method C. After lyophilization, 0.56 mg (32.2%, 0.000375 mmol) of a yellowish solid was obtained. Results of HR-MS: C 73 H 99 FN7O 23 P 2+ [M+H] 2+ Theoretical value 745.8252, measured value 745.8255. (UV trace in Figure 18).

[0157] P5(PEG12)-Exatecan TIFF2026513097000029.tif54134

[0158] 50 μL (0.005 mmol, 1.0 equivalent) of a 100 mM exatecan mesylate suspension in dry DMSO, 20 μL (P5(PEG12)-COOH, 0.005 mmol, 1.0 equivalent) of a 250 mM PEG12-N-(4-benzoic acid)-P-ethynylphosphoneamidate solution in dry DMSO, 20 μL (0.006 mmol, 1.2 equivalents) of a 300 mM Pybop solution in dry DMSO, and 4.33 μL (0.025 mmol, 5.0 equivalents) of DIPEA were placed in a screw-cap vial. The solution was shaken at room temperature for 2 hours, and 1.5 ml of MeCN and 2.5 mL of H2O were added. This solution was directly purified by preparative HPLC using Method C. After freeze-drying, 2.63 mg (45.0%, 0.0023 mmol) of a yellowish solid was obtained. HR-MS result: C 57 H 77 FN4O 19 P + [M+H] + Theoretical value: 1171.4899, measured value: 1171.4852. (UV trace in Figure 19).

[0159] Characterization of mAb and ADC Summary of analysis of ADCs derived from synthesized mAbs and P5(PEG24)-VC-PAB-exatecan (Figure 20) The ratio (%) of HMWS was measured by size exclusion chromatography for analysis as described above. This ratio (%) indicates the area of peaks with shorter retention times compared to the monomeric species. Charge variant analysis was carried out as described above. It shows the changes after incubation at 40 °C for 7 days compared to day 0. The retention time of the ADC was measured by hydrophobic interaction chromatography as described above. Ti was measured by nanoDSF as described above and determined from the melting curve. The binding affinity was measured as described above, using flow cytometry (FACS) for OVCAR-3 cells or ELISA for the isolated protein. The cytotoxicity was evaluated by the resazurin assay method as described above. The internalization is shown as the ratio (%) of the MFI ratio of pHrodo at 5 - 6 hours after compared to time point 0 as described above.

[0160] Analytical raw data (Figure 21) of exemplary monoclonal antibodies. (A - D) Analytical characterization of one of the mAbs described. The antibody was expressed in Expi-CHO cells and purified by protein A chromatography as described above. The mAb was analyzed by HLPC-SEC (A), LC-MS (B), HLPC-HIC (C), and reducing SDS-PAGE (D).

[0161] LC / MS analysis of antibody clones Parent (Figure 22) Theoretical value LC: 23473, measured value: 23472; Theoretical value HC: 48705, measured value: 48706. AV15 (Figure 23) Theoretical value LC: 23459, measured value: 23458; Theoretical value HC: 48705, measured value: 48706. AV18 (Figure 24) Theoretical value LC: 23473, measured value: 23472; Theoretical value HC: 48632, measured value: 48634. AV21 (Figure 25) Theoretical value LC: 23473, measured value: 23472; Theoretical value HC: 48564, measured value: 48566. [[ID=2⑧]]AV25 (Figure 26) Theoretical value LC: 23443, measured value: 23442; Theoretical value HC: 48632, measured value: 48634. AV29 (Figure 27) Theoretical value LC: 23443, measured value: 23442; Theoretical value HC: 48634, measured value: 48636.

[0162] Example of ADC analysis raw data (Figure 28) Figure 28. (A-D) Analytical characterization of AV25-P5(PEG24)-VC-PAB-exatecan (DAR8) synthesized and purified as described above. A) Analytical size exclusion chromatography, B) LC-MS of ADC preparation, C) Analytical hydrophobic interaction chromatography after conjugation. The data show that the ADC was completely conjugated to DAR8 and that only a small amount of aggregates were present after purification.

[0163] LC / MS analysis of ADC Parent-P5(PEG24)-VC-PAB-exatecanDAR8 (Figure 29) Theoretical value LC: 25579, measured value: 25577; Theoretical value HC: 55023, measured value: 55021. AV15-P5(PEG24)-VC-PAB-Exatecan DAR8 (Figure 30) Theoretical value LC: 25565, measured value: 25563; Theoretical value HC: 55023, measured value: 55022. AV18-P5(PEG24)-VC-PAB-Exatecan DAR8 (Figure 31) Theoretical value LC: 25579, measured value: 25577; Theoretical value HC: 54950, measured value: 54947. AV21-P5(PEG24)-VC-PAB-Exatecan DAR8 (Figure 32) Theoretical value LC: 25579, measured value: 25577; Theoretical value HC: 54882, measured value: 54881. AV25-P5(PEG24)-VC-PAB-Exatecan DAR8 (Figure 33) Theoretical value LC: 25549, measured value: 25547; Theoretical value HC: 54950, measured value: 54950. AV29-P5(PEG24)-VC-PAB-Exatecan DAR8 (Figure 34) Theoretical value LC: 25549, measured value: 25547; Theoretical value HC: 54952, measured value: 54950.

[0164] Example 2: Ex vivo serum stability, in vivo efficacy in cell line-derived xenograft models (CDX), and in vivo efficacy in patient-derived xenograft models (PDX) of an exemplary ADC of the present invention: Ex vivo serum stability: Serum samples from each species were spiked with AV25-P5(PEG24)-VC-PAB-exatecan DAR 8 at a concentration of 0.2 mg / ml, mixed with at least 80% serum. The samples were sterile filtered using a UFC30GV0S centrifugal filter unit (Merck, Germany) and incubated at 37°C for 1, 2, 3, 5, and 7 days. Samples on day 0 were processed further without modification.

[0165] Recombinant NaPi2B antigen was conjugated to Thermo NHS magnetic beads according to the manufacturer's instructions. The bead storage solution was removed from 40 μl of NaPi2B conjugated bead suspension. These beads were incubated with 100 μl of serum-ADC mixture pre-mixed with 200 μl of PBS for 2 hours at room temperature. The supernatant was then removed, and the resin was washed twice with 1 mL of PBS-T. Subsequently, the beads were incubated with 10 μl of 100 mM glycine buffer (pH 2.5) at room temperature for 15 minutes. This solution was rebuffered in PBS using a 75 μL Zeba® spin desalting column (Thermo Fisher Scientific, USA) at 7K MWCO. The samples were further processed for MS analysis as described above. The drug-to-antibody ratio (DAR) was calculated from the MS intensity of light chain adducts conjugated to 0 or 1 molecule of P5(PEG24)-VC-PAB-exatecan and the MS intensity of heavy chain adducts conjugated to 0 to 3 molecules of P5(PEG24)-VC-PAB-exatecan.

[0166] These results clearly demonstrate that the linker between the AV25 antibody and the exatecan drug molecule is highly stable in different species of serum, and that the amount of payload does not significantly decrease (no change in drug-to-antibody ratio (DAR)) even after incubation of the ADC for several days (Figure 35).

[0167] In vivo efficacy of ADCs in cell line-derived xenotransplantation models (CDX): All animal experiments were conducted in accordance with German animal welfare laws and approved by local authorities. In short, 1 x 10 7 100 μL + 100 μL Matrigel were subcutaneously injected into CB17-Scid mice. The tumor size was 0.1-0.15 cm on day 15 after transplantation. 3 Treatment was initiated when the mean tumor volume reached [a certain value]. After randomly dividing animals into treatment and control groups, five animals in each group were treated once on day 0 by intravenous injection of either AV25-P5(PEG24)-VC-PAB-exatecan DAR 8, isotype-P5(PEG24)-VC-PAB-exatecan DAR 8, or vehicle at a dose of 1 mg / kg, 3 mg / kg, or 5 mg / kg. Tumor volume, body weight, and overall health status were recorded throughout the study.

[0168] In the case of targeted AV25-P5(PEG24)-VC-PAB-exatecan DAR 8, complete tumor remission was observed with a single injection at all dose levels (1 mg / kg, 3 mg / kg, and 5 mg / kg). In contrast, with the isotype-P5(PEG24)-VC-PAB-exatecan DAR 8, a non-targeted isotype control (left), only a slight effect was observed even at the highest dose of 5 mg / kg (Figure 36). This clearly demonstrates the remarkable effect of NaPi2B targeting by AV25-P5(PEG24)-VC-PAB-exatecan DAR 8. In in vivo models derived from the same OVCAR3 cell line, AV25-P5(PEG24)-VC-PAB-exatecan DAR 8 was clearly superior at all dose levels tested compared to other NaPi2B-targeting ADCs known in the literature, such as rifastuzumab vedotin. AV25-P5(PEG24)-VC-PAB-exatecan DAR 8 showed no tumor recurrence at all up to the end of the observation period, even with a single low dose of 1 mg / kg, which is extremely unusual compared to other NaPi2B-targeting ADCs administered at higher doses and more frequently in the same OVCAR3 model.

[0169] In vivo efficacy of ADCs in patient-derived xenograft models (PDX): All animal experiments were conducted in accordance with German animal welfare laws and approved by local authorities. In short, 3x3 mm samples of tumor tissue derived from patients were subcutaneously transplanted into the flanks of immunodeficient female NMRI nu / nu mice. The tumors were 0.1–0.15 cm in size. 3 Treatment was initiated when the mean tumor volume reached [a certain value]. Animals in each group were treated once on day 0 by intravenous injection with either AV25-P5(PEG24)-VC-PAB-exatecan DAR 8 or vehicle at a dose of 10 mg / kg. Tumor volume, body weight, and overall health status were recorded throughout the study.

[0170] The data clearly demonstrate very high and long-lasting in vivo efficacy in patient-derived tumor models compared to vehicle controls in various tumor types, including lung cancer, endometrial cancer, and ovarian cancer (Figure 37).

[0171] In vivo toxicity in cynomolgus monkeys: A toxicological study was conducted in cynomolgus monkeys. Two animals from each group were intravenously administered 10 mg / kg and 20 mg / kg of AV25-P5(PEG24)-VC-PAB-exatecan on days 1 and 22, respectively, and final dissection was performed on day 43. Three different analytes, including total ADC, intact ADC, and free exatecan, were analyzed from animal plasma samples by ELISA and LC / MS-MS. Exposure and pharmacokinetic (PK) parameters were evaluated for each analyte. No significant changes in body weight, clinical chemistry, hematological tests, or histopathological tests were observed at these dose levels. In this study, the HNSTD (maximum dose without serious toxicity) was determined to be at least 20 mg / kg.

[0172] For PK analysis, both total ADCs and intact ADCs were measured by ELISA. The first ELISA setup was for the quantification of human antibodies (total antibody / total mAb).

[0173] The first ELISA setup is for the quantification of human antibodies (total antibody / total mAb). For this purpose, purified recombinant NaPi2B antigen is immobilized on a protein-binding plate. When the sample and standard dilution are added to the wells, AV25-P5(PEG24)-VC-PAB-exatecan is expected to be captured independently of DAR. A specific human detection antibody that recognizes Kappa LC is added to the wells to enable the quantification of the captured total antibody.

[0174] The second ELISA setup is designed to specifically measure intact ADCs (linker-payload conjugated). For this purpose, an anti-payload specific antibody is immobilized on the binding plate. Only AV25-P5(PEG24)-VC-PAB-exatecan conjugated with (at least one) payload molecule is detected. When the sample and standard dilution are added to the wells, it is expected that only AV25-P5(PEG24)-VC-PAB-exatecan conjugated with the payload will be captured. A specific human detection antibody that recognizes Kappa LC is added to the wells to enable quantification of the captured total antibody.

[0175] The amount of free exatecan in plasma samples from cynomolgus monkeys treated with AV25-P5(PEG24)-VC-PAB-exatecan was measured by liquid chromatography-mass spectrometry (LCMS). A quantitative method was established using a Xevo G2-XS qTOF instrument with multiple reaction monitoring (MRM) acquisition mode and mass transitions specific to exatecan. Deuterated exatecan-d5 was used as an internal standard in all measurements. To quantify the amount of free exatecan in serum samples from different treatment groups and time points, a liquid extraction procedure was performed to separate the contained proteins from lipophilic low-molecular-weight molecules. Pre-diluted exatecan and exatecan-d5 at concentrations of 100 nM and 10 nM, respectively, were prepared in 1:1 ACN:H2O to prepare calibration curves and control samples. This assay was performed according to the quantitative assay method previously described for DxD by Nagai et al., 2018.

[0176] PK analysis showed that no increase in in vivo clearance occurred in cross-reactive species, indicating a favorable exposure profile (Figure 38).

[0177] In this study, the HNSTD (maximum dose without serious toxicity) was determined to be at least 20 mg / kg. This dose is at least six times higher than the doses investigated in toxicological studies of other NaPi2B-targeting ADCs (Figure 38).

[0178] This significant improvement in ADC tolerability and superior PK profile may be due to improvements in the properties of the antibody clones described herein, for example, the reduced aggregation tendency of the aforementioned AV25 clone. Antibody and ADC aggregation has been shown to be one of the major off-target toxicity promoters of ADCs.

[0179] Example 3: Further Characterization of AV25 mAb and ADC Based Thereon For all data presented, AV25-P5(PEG24)-VC-PAB-exatecan and AV25 refer to the LALA variant of AV25 mab unless explicitly named AV25-wt.

[0180] Synthesis of anti-Napi2b comparative antibody Anti-NaPi2b comparative antibody Light chain TIFF2026513097000030.tif31170 and heavy chain DNA encoding TIFF2026513097000031.tif58165 was synthesized (Geneart, Thermo Fisher). The heavy chain signal sequence (SEQ ID NO: 58, MDWTWRILFLVAAATGAHS) and the light chain signal sequence (SEQ ID NO: 59, MLPSQLIGFLLLWVPASRG) were added. The light and heavy chain sequences were cloned into pcDNA3.4-TOPO (Thermo Fisher) expression plasmids. Next, antibodies were transiently expressed in Expi-CHO-S cells (Thermo Fisher) by co-transfecting cells with pcDNA3.4 expression plasmids (Thermo Fisher) encoding the heavy and light chains of each sequence in a 1:1 ratio using the Expi-CHO transfection system (Thermo Fisher). Cells were collected by centrifugation at 300g, 4°C for 5 minutes. To remove particulate matter from the supernatant, the supernatant was centrifuged at 4000-5000 g at 4°C for 30 minutes. For further clarification, the supernatant was passed through a 0.22 μm filter. Antibodies were purified from the clarified and filtered supernatant by protein A chromatography.

[0181] Synthesis of anti-Napi2b comparative ADCs 50 μl of a 10 mg / mL anti-Napi2b comparative antibody (66.67 μM) solution in Dulbecco's PBS (Merck KGaA) was mixed with 1.33 μl of TCEP solution (0.5 mM in buffer, Merck KGaA, diluted to 10 mM in PBS, 4 equivalents of TCEP relative to the antibody). After incubation at room temperature for 30 minutes, 1.66 μl of a 20 mM solution of maleimidocaproylvaline citruyline p-aminobenzylcarbamate-monomethyl auristatin E (MC-VC-PAB-MMAE) in DMSO (10.0 equivalents relative to the antibody) was added. The mixture was shaken at room temperature (25°C) for 1 hour (350 RPM). The reaction mixture was purified by preparative size exclusion chromatography using 25 ml of Superdex® 200 Increase 10 / 300GL (Cytiva, Sweden) and eluted with sterile PBS (Merck, Germany) at a flow rate of 0.8 ml / min. As described above, the antibody-containing fraction was combined and concentrated by spin filtration (Amicon® Ultra-2 mL MWCO: 30 kDa, Merck, Germany), and analyzed by MS. MS analysis was performed as described in Example 1.

[0182] Figure 39 shows the MS analysis of the anti-Napi2b comparative ADC synthesized by the method described above. The signal is annotated with the measured mass (in Daltons) and absolute intensity. Exemplary spectra derived from the conjugation reaction are shown. The drug-to-antibody ratio of the final conjugate was estimated to be 3.5–4.0 from the MS signal. LC: light chain of the anti-Napi2b comparative antibody, HC: heavy chain of the anti-Napi2b comparative antibody.

[0183] Binding to human NaPi2a, human NaPi2b, and human NaPi2c, as evaluated by flow cytometry. To measure concentration-dependent binding, HEK293 cells transiently or stably expressing human NaPi2a-mCherry, human Napi2b-mCherry, and human Napi2c-mCherry were incubated with antibody or ADC concentrations ranging from 0.002 to 200 nM, stained with Alexa-labeled anti-human IgG H+L secondary antibody (Thermo Fisher Scientific), and analyzed by flow cytometry. The mean fluorescence intensity (MFI) ratio was normalized relative to a non-specific binding control. The assay was performed in pairs, and data points were analyzed by nonlinear regression using a single-site specific binding model with Prism 9 software. Graphs show the mean (n=2) ± SD.

[0184] This experiment revealed the specificity of AV25 and AV25-P5(PEG24)-VC-PAB-exatecan to Napi2b. Notably, AV25 bound only to HEK293 cells that overexpress Napi2b (SLC34A2) but not to either Napi2a (SLC34A1) or Napi2c (SLC34A3). Both are members of the SLC34 family and have 45.5–50.9% sequence homology to Napi2b.

[0185] This experiment clearly demonstrates that AV25 exhibits the highest selectivity for target NaPi2b compared to other NaPi2 proteins. This highlights the key features of the antibodies disclosed herein for selective tumor targeting.

[0186] Figure 40 shows the binding of unmodified AV25 mAb and AV25-P5(PEG24)-VC-PAB-exatecan DAR8 ADC to HEK293 cells transfected with Napi2a (left), Napi2b (center), and Napi2c (right), expressed as MFI ratios. AV25 mAb and AV25-P5(PEG24)-VC-PAB-exatecan DAR8 ADC specifically bind to NaPi2b. The graph shows n=2±SD (note that the error bars are too small to display).

[0187] In vitro cytotoxicity assessed by the resazurin assay. Synthesis of anti-Napi2b comparative ADC and AV25-P5(PEG24)-VC-PAB-exatecan DAR8 To investigate the direct cytotoxicity of ADC, each cell was seeded in a 96-well plate (flat-bottom, 5000 cells / well, suspended in 100 μl of medium) and incubated with increasing concentrations of ADC in the medium (0–12 μg / ml) for 7 days to create a dose-response curve. Before viability analysis, the supernatant on adherent cells was removed and replaced with fresh medium. Subsequently, cell death was analyzed using resazurin (Sigma-Aldrich) at a final concentration of 55 μM as a cell viability discriminant dye. Fluorescence emission at 590 nM was measured using a microplate reader Infinite M200 Pro (Tecan). Cell viability was determined by dividing the fluorescence of ADC-treated cells by the fluorescence of control cells similarly treated with medium alone. The graph shows the mean (n=2) ± SEM.

[0188] In this experiment, Napi2b was compared to an anti-Napi2b comparative ADC that targets Napi2b. 高 The cytotoxicity of AV25-P5(PEG24)-VC-PAB-exatecan DAR 8 against expressing cell lines was investigated. In HCC-78 cells, AV25-P5(PEG24)-VC-PAB-exatecan had an IC50 concentration 1 / 15.5 times lower than that of anti-Napi2b comparative ADC, demonstrating a significant increase in selectivity. No nonspecific toxicity was observed in target-negative cells.

[0189] Figure 41 shows the cytotoxic dose-response of AV25-P5(PEG24)-VC-PAB-exatecan DAR 8 compared to an anti-NaPi2b comparative ADC in three different cell lines. The mean and standard deviation of the two measurements, as well as the dose-response fitting, are shown. Fluorescence (in %) compared to the medium control is equal to the percentage of viable cells (%).

[0190] In vitro inhibition of topoisomerase I by delivery of exatecan via ADC. Inhibition of topoisomerase I by delivery of exatecan via AV25-P5(PEG24)-VC-PAB-exatecan DAR8 ADC induces DNA damage, which is detected based on the accumulation of cleaved PARP, active caspase 3, and phosphorylated histone 2AX (pH2AX). OVCAR-3 cells and HCC-78 cells (both Napi2b) 高 Cells were treated for 72 hours with AV25-P5(PEG24)-VC-PAB-exatecan or isotype control (isotype-P5(PEG24)-VC-PAB-exatecan) at progressively increasing concentrations (0.05–12 μg / ml). Cells were stained with live / dead staining agents (Thermo Fisher Scientific), fixed and permeabilized using a fixation / permeabilization kit (BD Biosciences), and then stained for the DNA damage markers active caspase 3, cleaved PARP, and pH2AX (Ser-139) (all BD Biosciences), and analyzed by flow cytometry. The graph shows the mean (n=2) ± SEM.

[0191] AV25-P5(PEG24)-VC-PAB-exatecan DAR8 induces a concentration-dependent accumulation of DNA damage, as indicated by increased HCC-78(A) and OVCAR-3(B) cells positive for cleaved PARP, active caspase 3, and pH2AX, while the isotype control remains inactive.

[0192] This experiment clearly demonstrates that ADCs selectively deliver exatecan to target cells and induce cell death by inhibiting topoisomerase I.

[0193] Figure 42 shows the dose-dependent induction of signs of DNA damage and apoptosis in response to treatment with gradually increasing concentrations of AV25-P5(PEG24)-VC-PAB-exatecan DAR8. Corresponding non-targeting isotype control conjugates were included as negative controls. HCC-78 cells (A, NaPi2b) 高 ) and OVCAR-3 cells (B, NaPi2b 高 The cells were treated with AV25-P5(PEG24)-VC-PAB-exatecan DAR8 or isotype ADC (isotype-P5(PEG24)-VC-PAB-exatecan DAR8) at progressively increasing concentrations (0.05–12 μg / ml) for 72 hours. The cells were stained for cleaved PARP (left), caspase 3 (center), and pH2AX (right) and analyzed by flow cytometry. The graph shows the mean (n=2) ± SEM.

[0194] Interaction with complement factor C1q To reduce or even prevent interactions between the IgG1 backbone AV25 antibody and complement factors or Fc receptors (FcRs), which can lead to unwanted immune activation and / or intracellular uptake via FcRs, the Fc portion of the AV25 antibody and the induced ADC, AV25-P5(PEG24)-VC-PAB-exatecan DAR8, were silenced using two point mutations, L234A and L235A(LALA).

[0195] In this experiment, the LALA-silenced AV25 TIFF2026513097000032.tif58165 and Fc-wild-type AV25 The interaction between TIFF2026513097000033.tif58165 and complement factor C1q was analyzed. The C1q interaction investigation was performed using the HTRF Human C1q Binding Kit (Cisbio) according to the manufacturer's instructions. Briefly, AV25 HC-wt and HC-LALA, reference materials, and anti-MHC-I positive controls (Invivogen) known to interact with C1q were captured and collected by anti-human IgG Fab-biotin; this anti-human IgG Fab-biotin forms a complex with streptavidin and binds to streptavidin-labeled d2 (fluorescent acceptor). When the antibody binds to human C1q, the europium cryptotate (fluorescent donor)-labeled anti-C1q antibody can approach the fluorescent acceptor, triggering fluorescence resonance energy transfer (FRET). Fluorescence emission at 665 nm was measured using a microplate reader, Infinite M200 Pro (Tecan). The graph shows the mean (n=2) ± SD.

[0196] AV25 HC-wt binds well to C1q, similar to the standard and anti-MHC-I positive controls, but in the case of AV25 HC-LALA, the interaction with C1q is completely abolished. Reduced interaction with C1q is expected to decrease unwanted activation of the innate immune system.

[0197] Figure 43 shows the binding of the Fc region of AV25 HC-LALA and AV25 HC-wt antibodies to recombinant hexameric C1q complement protein, as measured using a human C1q binding assay based on HTRF (homogenous time-resolved fluorescence) (HTRF Human C1q Binding Kit, Cisbio), according to the manufacturer's instructions. Briefly, serial dilutions from 280 nM to 11.6 nM were measured for all antibodies tested (AV25 HC-wt, AV25 HC-LALA, α-MHC-I positive, IgG1 kit standard). The HTRF ratio was calculated by dividing the acceptor emission signal at 665 nm by the donor emission signal at 620 nm and multiplying by 10000. The graph shows the HTRF ratio at a concentration of 70 nM, subtracting the background (dilution only) HTRF ratio. The graph shows the mean (n=2) ± SD.

[0198] Interaction with FcR To reduce or even prevent interactions between the IgG1 backbone AV25 antibody and complement factors or Fc receptors (FcRs), which can lead to unwanted immune activation and / or intracellular uptake via FcRs, the Fc portion of the AV25 antibody and the induced ADC, AV25-P5(PEG24)-VC-PAB-exatecan DAR8, were silenced using two point mutations, L234A and L235A(LALA).

[0199] In this experiment, the LALA-silenced AV25 TIFF2026513097000034.tif58166 and AV25-P5(PEG24)-VC-PAB-exatecanDAR8 and Fc-wild-type AV25 The interaction between TIFF2026513097000035.tif58165 and the Fc gamma receptor (FcγR) was analyzed. Following the manufacturer's instructions, FcγR interactions were investigated using the Lumit® FcγR-binding immunoassay (FcγRn, FcγRI, FcγRIIa / CD32 R131 / H131 polymorphism, FcγRIIIa / CD16 V158 / F158 polymorphism, Promega), based on the principle of competition and luciferase detection. In short, AV25 HC-wt, HC-LALA, and AV25-P5(PEG24)-VC-PAB-exatecan DAR8, along with trastuzumab as a positive control, were incubated with Tracer-LgBiT and FcγR-SmBiT. In the absence of an antibody analyte, or if no interaction occurs between the antibody under test and FcγR, Tracer-LgBiT binds to the FcγR-SmBiT target, resulting in a maximum luminescence signal. If successful interaction with FcγR occurs, the antibody / ADC under test competes with Tracer-LgBiT for binding to the FcγR target, resulting in a concentration-dependent decrease in the luminescence signal. Luminescence is measured using a microplate reader, Infinite M200 Pro (Tecan). The graph shows n=1.

[0200] AV25 HC-wt binds well to all FcγRs, similar to the IgG1-positive control, but for AV25 HC-LALA and AV25-P5(PEG24)-VC-PAB-exatecan DAR8, the interaction with FcγRs is significantly reduced or almost completely absent. Interestingly, even trastuzumab, as a positive control, shows reduced interaction with all FcγRs compared to the aforementioned positive control. In FcγRn interaction experiments, mouse AV25 served as another negative control.

[0201] This experiment clearly demonstrated that incorporating the LALA mutation into the AV25 antibody reduced undesirable interactions with FcγRI(CD64), CD16, and CD32. Reduced interactions with these receptors are expected to decrease the undesirable uptake of AV25-related ADC conjugates into non-target cells, thereby mitigating undesirable toxicity. Interactions with FcRn were only slightly reduced.

[0202] Figure 44 shows the dose-dependent binding of the Fc region of the AV25-HC-LALA antibody, AV25-P5(PEG24)-VC-PAB-exatecan DAR8 ADC, and AV25-HC-wt antibody to recombinant human FcRn and FcγR. This was measured using the Lumit® FcγR-binding immunoassay (FcγRn, FcγRI, FcγRIIa / CD32 R131 / H131 polymorphism, FcγRIIIa / CD16 V158 / F158 polymorphism, Promega) according to the manufacturer's instructions. Serial dilutions of AV25 HC-wt, AV25 HC-LALA, and AV25-P5(PEG24)-VC-PAB-exatecan DAR8, standard materials, and trastuzumab as a positive control were incubated with Tracer-LgBiT and FcγR-SmBiT. In the absence of an antibody analyte, or if no interaction occurs between the antibody under test and FcγR, Tracer-LgBiT binds to the FcγR-SmBiT target, resulting in a maximum luminescence signal. If successful interaction with FcγR occurs, the antibody / ADC under test competes with Tracer-LgBiT for binding to the FcγR target, resulting in a concentration-dependent decrease in the luminescence signal. Luminescence was measured using a microplate reader Infinite M200 Pro (Tecan). The graph shows n=1.

[0203] ADCC To reduce or even prevent interactions between the IgG1 backbone AV25 antibody and complement factors or Fcγ receptors (FcγRs), which can lead to unwanted immune activation and / or intracellular uptake via FcRs, the Fc portion of the AV25 antibody and the induced ADC, AV25-P5(PEG24)-VC-PAB-exatecan DAR8, were silenced using two point mutations, L234A and L235A(LALA).

[0204] In this experiment, Fc-wild-type AV25 mAb TIFF2026513097000036.tif58165 and LALA Silenced AV25 mAb Antibody-dependent cell-mediated cytotoxicity (ADCC) induced after interaction with FcγRIIIa (CD16) of TIFF2026513097000037.tif58165 and AV25-P5(PEG24)-VC-PAB-exatecan DAR8, as well as isotypes and anti-MHC-I positive controls (Invivogen), were analyzed. In the calcein-release-based antibody-dependent cell-mediated cytotoxicity (ADCC) assay, peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor bufficoat (purchased from DONAS GmbH) using LeucoSep tubes (Greiner Bio-One) and a Histopaque®-1077 (density 1.077 g / ml, Merck) density gradient, according to a standard protocol. Subsequently, using a human NK cell isolation kit (Miltenyi Biotec) according to the manufacturer's instructions, natural killer (NK) cells were sorted by MACS (magnetic cell separation) using negative selection to obtain intact human primary NK cells. Napi2b-positive target cells OVCAR-3 and HCC-78 (both Napi2b 高The cells were stained with 16 μM calcein AM (Thermo Fisher Scientific). 40,000 NK cells and 10,000 calcein-stained target cells were incubated in a 4:1 ratio in the presence of 15 μg / ml antibody or ADC. Cells permeabilized with 2.5% Triton X 100 (Merck) were used as a positive control corresponding to maximum calcein release. After 4 hours, the supernatant was transferred to a black, flat, unbound 96-well plate (Greiner Bio-One), and fluorescence at 485 / 535 nM was measured using an Infinite M200 Pro reader (Tecan). Specific cell death rate (%) was calculated by subtracting background calcein release (NK+ target) from the calcein released by antibody-mediated death, and then dividing that by subtracting background calcein release (target only) from the calcein released by Triton X-permeabilized cells (maximum death). The graph shows the mean (n=2) ± SEM.

[0205] Anti-MHC-I positive controls and AV25 HC-wt induced high ADCC activity in human NK cells, while none of the LALA-silencing antibodies and ADCs (AV25 HC-LALA, AV25-P5(PEG24)-VC-PAB-exatecan DAR8, and isotype controls) induced ADCC, or only minimally. The reduction in ADCC is expected to decrease the undesirable toxicity of AV25-related antibodies.

[0206] Figure 45 shows an antibody-dependent cell-mediated cytotoxicity (ADCC) assay based on calcein release. Co-cultures of healthy donor (HD) NK cells and calcein-stained target-positive tumor cells (OVCAR-3 and HCC-78) in a 4:1 ratio were incubated with 15 μg / ml of a specified antibody or ADC (anti-MHC-I antibody served as a positive control). Specific cell death rate (%) was calculated by dividing the calcein released by antibody-mediated cell death by the calcein released by cells permeabilized with Triton X (maximum death). The graph shows the mean (n=2) ± SEM.

[0207] Investigation of dose-response and exposure PDX of AV25-P5(PEG24)-VC-PAB-exatecan DAR8 (Lu7700) All animal experiments were conducted in accordance with German animal welfare laws and approved by local authorities. In short, 3x3 mm samples of patient-derived tumor tissue (Lu7700 non-small cell lung cancer (NSCLC) model, EPO Experimentelle Pharmakologie & Onkologie Berlin-Buch GmbH) were subcutaneously transplanted into the flanks of immunodeficient female NMRI nu / nu mice. The tumors were 0.1–0.15 cm in size. 3 Treatment was initiated when the mean tumor volume reached 5 mg / kg. Animals in each group were treated once on day 0 by intravenous injection with AV25-P5(PEG24)-VC-PAB-exatecan DAR 8 at 5 mg / kg, 3 mg / kg, or 1 mg / kg, isotype-P5(PEG24)-VC-PAB-exatecan DAR 8 at 5 mg / kg, or vehicle. Tumor volume, body weight, and overall health status were recorded throughout the study.

[0208] The data demonstrate that in the in vivo NSCLC PDX model, all ADC dose levels tested exhibited superior and long-term efficacy compared to vehicle controls. This effect is highly specific to targeted anti-TPBG antibodies, as exemplified by the highest dose, non-targeted isotype-controlled ADC group.

[0209] Figure 46 shows the results of in vivo efficacy analysis of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8), a representative ADC of the present invention, in a patient-derived NSCLC xenograft model (PDX, Lu7700). The left side shows the time course of tumor volume after a single treatment on day 0 for various dose levels of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8) (1 mg ADC / kg body weight, 3 mg ADC / kg body weight, and 5 mg ADC / kg body weight) and an isotype control with the same linker payload (5 mg / kg), compared to the untreated (vehicle). The right side shows the body weight of animals treated with various dose levels of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8) and an isotype control with the same linker payload, compared to the untreated (vehicle). All results are shown as average values ​​and SEM values ​​for 4 animals per group.

[0210] Investigation of dose-response and exposure PDX of AV25-P5(PEG24)-VC-PAB-exatecan DAR8 (Ov6668) All animal experiments were conducted in accordance with German animal welfare laws and approved by local authorities. In short, 3x3 mm samples of patient-derived tumor tissue (Ov6668 ovarian cancer model, EPO Experimentelle Pharmakologie & Onkologie Berlin-Buch GmbH) were subcutaneously transplanted into the flanks of immunodeficient female NMRI nu / nu mice. The tumors were 0.1–0.15 cm in size. 3 Treatment was initiated when the mean tumor volume reached [a certain value]. Animals in each group were treated once on day 0 by intravenous injection with AV25-P5(PEG24)-VC-PAB-exatecan DAR 8 or vehicle at 5 mg / kg, 3 mg / kg, or 1 mg / kg. Tumor volume, body weight, and overall health status were recorded throughout the study.

[0211] The data demonstrate that, in the in vivo PDX model, all ADC dose levels tested showed the highest and longest efficacy compared to the vehicle control. Dose-proportionality of exposure was demonstrated in the PK data. PK analysis was performed as described in Example 1.

[0212] In the case of targeted AV25-P5(PEG24)-VC-PAB-exatecan DAR8, complete tumor remission was observed in ovarian cancer PDX with a single injection at dose levels of 5 mg / kg and 3 mg / kg. Significant tumor regression was also observed at 1 mg / kg in ovarian cancer models, followed by partial tumor regrowth after day 40. PK analysis demonstrated a dose-proportional exposure profile and high stability of AV25-P5(PEG24)-VC-PAB-exatecan DAR8 ADC at the tested dose levels of 5 mg / kg, 3 mg / kg, and 1 mg / kg.

[0213] The data demonstrate that AV25-P5(PEG24)-VC-PAB-exatecan DAR 8 ADC exhibits superior and long-term efficacy compared to the vehicle at all tested ADC dose levels in an in vivo PDX model. The absence of weight loss suggests the highest level of tolerability. PK analysis shows a dose-proportional exposure profile and high stability of AV25-P5(PEG24)-VC-PAB-exatecan DAR 8 ADC at tested dose levels of 5 mg / kg, 3 mg / kg, and 1 mg / kg.

[0214] Figure 47A shows the results of an in vivo efficacy analysis of AV25-P5(PEG24)-VC-PAB-exatecan DAR 8, a representative ADC of the present invention, in a patient-derived ovarian cancer xenograft model (PDX, Ov6668). The left side shows the time course of tumor volume after a single treatment on day 0 with various dose levels of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8) (1 mg ADC / kg body weight, 3 mg ADC / kg body weight, and 5 mg ADC / kg body weight), compared to the untreated (vehicle) group. The right side shows the body weight of animals treated with various dose levels of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8) compared to the untreated (vehicle) group. All results are shown as mean values ​​and SEMs for 9 animals per group. Figure B shows the in vivo PK evaluation of total and intact ADCs at three dose levels in the dose-response efficacy study, as the mean and standard deviation of three measurements per time point obtained from mice treated once on day 0 with various dose levels of AV25-P5(PEG24)-VC-PAB-exatecan (1 mg ADC / kg body weight, 3 mg ADC / kg body weight, and 5 mg ADC / kg body weight). PK analysis was performed as described in Example 1.

[0215] AV25-P5(PEG24)-VC-PAB-Exatecan DAR8 PDX study (10 mg / kg) The figure shows the results of in vivo efficacy analysis of a single 10 mg / kg dose of either AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8), a representative ADC of the present invention, or isotype AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8), in three patient-derived ovarian cancer xenograft (PDX) models with varying NaPi2b expression levels and BRCA mutation statuses. The PDX experiments were conducted as described in Example 2.

[0216] The data clearly demonstrate very high and long-lasting in vivo efficacy in patient-derived ovarian cancer tumor models compared to vehicle controls, even in BRCA mutation PDX models and models with low Napi2b target expression, which are very difficult to treat.

[0217] Figure 48 shows the results of in vivo efficacy analysis of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8), a representative ADC of the present invention, in a patient-derived ovarian cancer xenograft model. The top section shows the time course of tumor volume after a single treatment on day 0 with 10 mg ADC / kg body weight of AV25-P5(PEG24)-VC-PAB-exatecan (DAR 8) or isotype-P5(PEG24)-VC-PAB-exatecan (DAR 8), compared to the untreated (vehicle) model. The bottom section shows the NaPi2B low-expression BRCA mutation model (bottom left) and the NaPi2B high-expression BRCA mutation model (bottom right). All results are shown as mean values ​​and SEM values ​​from 3 animals per group.

[0218] Binding of upifitamab to NaPi2b-expressing cell lines OVCAR-3 and HCC-78, as evaluated by flow cytometry, versus AV25. Upifitamab is an antibody having the same sequence as the parent mAb of this disclosure. Upifitamab was purchased from MedChemExpressed to perform the following experiments.

[0219] To measure concentration-dependent binding, NaPi2b-positive OVCAR-3 and HCC-78 were incubated with antibody concentrations ranging from 0.00075 to 30 μg / ml, stained with Alexa-labeled anti-human IgG H+L secondary antibody (Thermo Fisher Scientific), and analyzed by flow cytometry. The mean fluorescence intensity (MFI) ratio was normalized relative to a non-specific binding control. The assay was performed in pairs, and data points were analyzed by nonlinear regression using a one-site specific binding model with Prism 9 software. The graph shows the mean (n=2) ± SD.

[0220] In this experiment, we compared the binding of AV25 to upifitamab in NaPi2b-positive cell lines, and K D The value was determined. It is worth noting that AV25 (K in OVCAR-3) D = 0.44 μg / ml, K in HCC-78 D =0.42μg / ml) Upifitamab (K in OVCAR-3) D = 1.11 μg / ml, K in HCC-78 D Compared to (=1.27 μg / ml), the binding was improved, and K D The value is getting lower.

[0221] This experiment clearly demonstrates that AV25 binding is superior to upifitamab.

[0222] Figure 49 shows dose-dependent binding of escalating upifitamab or AV25 to two different NaPi2b-positive cell lines (left: OVCAR-3, right: HCC78), normalized to a nonspecific binding control and expressed as the MFI ratio. The mean and standard deviation of the two measurements, as well as the dose-response fitting, are shown.

[0223] In vitro cytotoxicity of upifitamab-P5(PEG24)-VC-PAB-exatecan versus AV25-P5(PEG24)-VC-PAB-exatecan DAR8, as evaluated by the rezazurin assay. Upifitamab is an antibody having the same sequence as the parent mAb of this disclosure. Upifitamab was purchased from MedChemExpressed to perform the following experiments. Upifitamab was further conjugated to P5(PEG24)-VC-PAB-exatecan as described above in Example 1 and purified as a DAR8 conjugate.

[0224] To investigate the direct cytotoxicity of ADC, each cell was seeded in a 96-well plate (flat-bottom, 5000 cells / well, suspended in 100 μl of medium) and incubated with increasing concentrations of ADC in the medium (0–12 μg / ml) for 7 days to create a dose-response curve. Before viability analysis, the supernatant on adherent cells was removed and replaced with fresh medium. Subsequently, cell death was analyzed using resazurin (Sigma-Aldrich) at a final concentration of 55 μM as a cell viability discriminant dye. Fluorescence emission at 590 nM was measured using a microplate reader Infinite M200 Pro (Tecan). Cell viability was determined by dividing the fluorescence of ADC-treated cells by the fluorescence of control cells similarly treated with medium alone. The graph shows the mean (n=2) ± SEM.

[0225] In this experiment, Napi2b was directly compared to upifitamab-P5(PEG24)-VC-PAB-exatecan DAR 8. 高 The cytotoxicity of AV25-P5(PEG24)-VC-PAB-exatecan DAR 8 against expression cell lines (OVCAR-3 and HCC-78) was investigated. In both cell lines, AV25-P5(PEG24)-VC-PAB-exatecan exhibited increased selectivity, with an IC50 concentration 1 / 3.5 to 1 / 3.8 that of upifitamab-P5(PEG24)-VC-PAB-exatecan. No nonspecific toxicity was observed in non-targeted isotype controls.

[0226] This experiment clearly demonstrated that AV25-P5(PEG24)-VC-PAB-exatecan has superior ability to kill cancer cells compared to upifitamab-P5(PEG24)-VC-PAB-exatecan, suggesting an improved response in cancer patients to targeted therapy based on a targeted AV25 antibody compared to the parent mAb or upifitamab.

[0227] Figure 50 shows the cytotoxic dose-response of AV25-P5(PEG24)-VC-PAB-exatecan DAR 8 in two different cell lines, compared to upifitamab-P5(PEG24)-VC-PAB-exatecan DAR 8 and the isotype control, isotype-P5(PEG24)-VC-PAB-exatecan DAR 8. The mean and standard deviation of the two measurements, as well as the dose-response fitting, are shown. Fluorescence (in %) compared to the culture medium control is equal to the percentage of viable cells (%).

[0228] Those skilled in the art will readily understand that the present invention is well-suited to achieving its objectives and obtaining the results and benefits mentioned, as well as those inherent in the invention. Furthermore, it will be readily apparent to those skilled in the art that various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention. The compositions, methods, procedures, treatments, molecules, and specific compounds described herein are representative and illustrative of specific embodiments and are not intended to limit the scope of the invention. Modifications and other uses thereof that those skilled in the art may conceive are within the scope of the invention and are defined by the claims. The reference or discussion herein of previously published documents should not necessarily be construed as an acknowledgment that such documents are part of the current art or common general knowledge.

[0229] The invention as described herein by example can be adequately implemented in the absence of any one or more elements or limitations not specifically disclosed herein. Therefore, terms such as “comprising,” “including,” and “containing” should be interpreted comprehensively and non-restrictively. Furthermore, the terms and expressions used herein are intended to be descriptive rather than restrictive, and while there is no intention to exclude any equivalents of the exhibited and described features or any part thereof, it should be recognized that various modifications are possible within the scope of the claimed invention. Thus, while the invention is specifically disclosed by exemplary embodiments and any features, it should be understood that modifications and changes to the invention as embodied herein are possible for those skilled in the art, and that such modifications and changes are considered to be within the scope of the invention.

[0230] The present invention has been described extensively and generally in this specification. Each of the groups of subspecies and subgenera within the scope of the comprehensive disclosure also forms part of the present invention. This includes a general description of the present invention with any condition or negative limitation of excluding any subject matter from a genus, whether or not the subject matter to be excluded is specifically mentioned herein. All documents referenced herein, including patent applications and scientific publications, are incorporated herein by reference for all purposes.

[0231] Other embodiments are within the scope of the following claims. Furthermore, if any feature or aspect of the present invention is described in terms of the Markush group, it is likely that those skilled in the art will recognize that the present invention can also be described in terms of any individual member or subgroup of a member of the Markush group.

Claims

1. Anti-NaPi2b antibodies (for example, antibodies against NPT2B_human sodium-dependent phosphate transporter protein 2B (e.g., having UniProt accession number: O95436 or SEQ ID NO: 1) and / or antibodies against NPT2B_rat sodium-dependent phosphate transporter protein 2B (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO: 2)), (a) The NaPi2b antibody is capable of binding to human Napi2b (e.g., SEQ ID NO: 1) and / or rat Napi2b (e.g., SEQ ID NO: 2), and preferably, the binding to human Napi2b and rat Napi2b is substantially the same K D The antibody has, more preferably, the binding to the human Napi2b and the rat Napi2b is binding to endogenous Napi2b (e.g., located on the cell surface), most preferably the antibody is selected from the group consisting of AV-25 antibody, AV-15 antibody, AV-18 antibody, AV-21 antibody, and AV-29 antibody, and more preferably the substantially same K D It has a difference of up to 50% (for example, a difference of up to 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 7%, 5%, 4%, 3%, 2%, or 1%); (b) The NaPi2b antibody is preferably capable of cross-reactivity with rat Napi2b (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO: 2) by antigen-mediated internal transfer of the antibody, and more preferably, the internal transfer is improved (e.g., by at least 10%, e.g., 15%) compared to the corresponding internal transfer of the parent antibody (e.g., including SEQ ID NO: 54 and SEQ ID NO: 55, shown, e.g., in Figure 1); (c) The NaPi2b antibody is capable of cross-reactivity with cynomolgus monkey (e.g., Macaca fascicularis) Napi2b (e.g., having UniProtKB accession number: A0A2K5UHY1 or SEQ ID NO: 3); (d) The NaPi2b antibody is capable of internal migration, preferably by internal antibody migration via the antigen; and (e) The NaPi2b antibody has a heavy chain variable region (V H It is possible for the CDR2 of ) to not have a dipeptide deamide moiety, and preferably, the absent dipeptide deamide moiety is V H NG (Asn-Gly) in CDR2, Anti-NaPi2b antibody.

2. Anti-NaPi2b antibody, heavy chain variable region (V H ) does not have a dipeptide deamide moiety within CDR2, preferably the absence of such dipeptide deamide moiety is V H An anti-NaPi2b antibody according to any one of the claims, wherein NG (Asn-Gly) is present in CDR2.

3. (a) The anti-NaPi2b antibody has a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 4, and SEQ ID NO: An antibody comprising a light chain variable region having an amino acid sequence having at least 80% identity with respect to 5 (for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), Preferably, the anti-NaPi2b antibody is an AV25 antibody comprising a light chain containing SEQ ID NO: 6 and a heavy chain containing SEQ ID NO: 7; (b) The anti-NaPi2b antibody has a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 8, and SEQ ID NO: An antibody comprising a light chain variable region having an amino acid sequence that has at least 80% identity with respect to 9 (for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), Preferably, the anti-NaPi2b antibody is an AV-15 antibody comprising a light chain containing SEQ ID NO: 10 and a heavy chain containing SEQ ID NO: 11; (c) The anti-NaPi2b antibody has a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 12, and SEQ ID NO: An antibody comprising a light chain variable region having an amino acid sequence that has at least 80% identity with respect to 13 (for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), Preferably, the anti-NaPi2b antibody is an AV-18 antibody comprising a light chain containing SEQ ID NO: 14 and a heavy chain containing SEQ ID NO: 15; (d) The anti-NaPi2b antibody has a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 16, and SEQ ID NO: An antibody comprising a light chain variable region having an amino acid sequence that has at least 80% identity with respect to 17 (for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), Preferably, the anti-NaPi2b antibody is an AV-21 antibody comprising a light chain containing SEQ ID NO: 18 and a heavy chain containing SEQ ID NO: 19; (e) The anti-NaPi2b antibody has a heavy chain variable region having an amino acid sequence that is at least 80% (e.g., at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) identical to SEQ ID NO: 20, and SEQ ID NO: An antibody comprising a light chain variable region having an amino acid sequence that has at least 80% identity with respect to 21 (for example, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%), Preferably, the anti-NaPi2b antibody is an AV-29 antibody comprising a light chain containing SEQ ID NO: 22 and a heavy chain containing SEQ ID NO:

23. The anti-NaPi2b antibody according to any one of the above claims.

4. (a) An antibody comprising a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 24, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 25, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 26, and a light chain variable region including a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 27, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 28, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 29; (b) An antibody comprising a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 30, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 31, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 32, and a light chain variable region including a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 33, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 34, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 35; (c) An antibody comprising a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 36, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 37, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 38, and a light chain variable region including a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 39, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 40, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 41; (d) An antibody comprising a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 42, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 43, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 44, and a light chain variable region including a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 45, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 46, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO: 47; (e) An antibody comprising a heavy chain variable region including a heavy chain CDR1 having the amino acid sequence shown in SEQ ID NO: 48, a heavy chain CDR2 having the amino acid sequence shown in SEQ ID NO: 49, and a heavy chain CDR3 having the amino acid sequence shown in SEQ ID NO: 50, and a light chain variable region including a light chain CDR1 having the amino acid sequence shown in SEQ ID NO: 51, a light chain CDR2 having the amino acid sequence shown in SEQ ID NO: 52, and a light chain CDR3 having the amino acid sequence shown in SEQ ID NO:

53. The anti-NaPi2b antibody according to any one of the above claims.

5. The following features: (a) Monoclonal antibody; (b) Chimeric antibodies and / or humanized antibodies; (c) Specific recognition of Napi2b, which is overexpressed in cancer cells; (d) Human IgG antibody, preferably human IgG1 antibody; (e) Contains kappa (κ) light chain; (f) Contains a lambda (λ) light chain; (g) Includes Fc silencing mutations such as leucine (L) to alanine (A) substitutions (LALA mutations) at positions 234 and 235. (Here, the LALA mutation can reduce the effector function of immune cells); (h) It can be internally transported by target cells expressing Napi2b (e.g., cancer cells). (Preferably, the internally transported antibody is directed towards the lysosome); (i) tumor-selective antibodies (Preferably, the tumor is a liquid tumor and / or a solid tumor); (j) Antibodies selective for malignant cells; (k) Glycosylation-dependent binding to human Napi2b and / or rat Napi2b. (Here, the antibody binds to the glycosylated Napi2b protein); (l) (For example, in OVCAR-3 cells that endogenously express Napi2b (e.g., HTB-161, ATCC),) K in the range of about 0.01 to about 10 nmol / L, preferably in the range of about 1 to about 10 nmol / L, more preferably about 1 to about 7 nmol / L, and even more preferably about 1 to about 4 nmol / L relative to endogenously expressed human Napi2b D Having (More preferably, the K D (This is measured by the FACS assay method.) More preferably, it has K in the range of about 2.661 to about 6.644 nmol / L D ; (m) Optionally, a K2 solution ranging from approximately 0.01 to approximately 10 nmol / L for immobilized exogenous full-length Napi2b and / or one or more fragments thereof. D Having (Preferably, the one or more fragments include at least one extracellular domain (ECD) of Napi2b (e.g., the ECD includes amino acids 122-135 and / or amino acids 235-361 and / or amino acids 429-485 and / or amino acids 547-552 of human Napi2b having SEQ ID NO: 1) and / or one or more fragments of the ECD (e.g., having a length of about 15 to about 30 amino acids), and the full-length Napi2b and / or one or more fragments thereof are fused to or not fused to one or more protein tags (e.g., 6×His tag, FLAG, HA, V5, Fc fusion, MBP, SUMO, TEV, GFP, TST), preferably, preferably the K D This is measured by an ELISA assay and is preferably about 0.05 to about 0.2 nmol / L. More preferably, K in the range of about 0.071 to about 0.147 nmol / L D Having; (n) Cross-reactivity with rat Napi2b (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO: 2); and / or (o) Heavy chain variable region (V H ) does not have a dipeptide deamide moiety within CDR2. (Preferably, the dipeptide deamide site that is not present is V H This is NG (Asn-Gly) within CDR2. An anti-NaPi2b antibody according to any one of the above claims, comprising one or more of the above.

6. A hybridoma that produces the monoclonal antibody according to any one of the above claims.

7. A nucleic acid encoding the antibody according to any one of the above claims.

8. An expression vector comprising at least one nucleic acid molecule according to any one of the above claims.

9. An isolated host cell (e.g., an isolated recombinant host cell) comprising the vector and / or nucleic acid according to any one of the claims above.

10. An antibody-drug conjugate (ADC) comprising the anti-NaPi2b antibody according to any one of the above claims.

11. A composition or kit comprising an anti-NaPi2b antibody, an antibody-drug conjugate (ADC), a hybridoma, a nucleic acid, an expression vector, and / or host cells according to any one of the preceding claims.

12. A composition according to any one of the claims, which is a pharmaceutical and / or diagnostic composition.

13. Methods for treating, improving, preventing, and / or diagnosing cancer, Preferably, the cancer is a solid tumor and / or metastatic cancer, and more preferably, the cancer is selected from the group consisting of lung cancer, ovarian cancer, thyroid cancer, non-squamous non-small cell lung cancer, non-mucinous ovarian cancer, papillary thyroid cancer, kidney cancer, endometrial cancer, uterine cancer, ureteral cancer, bladder cancer, and fallopian tube cancer. The method comprises the step of administering a therapeutically effective or prophylactic amount of the antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition, or kit described in any one of the claims, The aforementioned method.

14. The process of conjugating one or more antibodies according to any one of the above claims to one or more cytotoxic moieties (e.g., cytotoxic payloads, e.g., tubulin inhibitors, e.g., topoisomerase I inhibitors, e.g., auristatin or camptothecin, e.g., MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), e.g., exatecan) preferably via one or more linkers, more preferably via one or more phosphoamide linkers. A method for producing antibody-drug conjugates (ADCs) containing [the specified substance].

15. An antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition, or kit according to any one of the claims, for use as a pharmaceutical and / or in a therapeutic setting.

16. The following method: (a) A method for treating, improving, preventing and / or diagnosing cancer, preferably the cancer being a solid tumor and / or metastatic cancer, and more preferably the cancer being selected from the group consisting of lung cancer, ovarian cancer, thyroid cancer, non-squamous non-small cell lung cancer, non-mucinous ovarian cancer, papillary thyroid cancer, kidney cancer, endometrial cancer, uterine cancer, ureteral cancer, bladder cancer, and fallopian tube cancer; (b) Methods for monitoring the progression of cancer and / or for evaluating the effectiveness of cancer treatments; (c) Methods for screening candidate compounds for anticancer activity; (d) Methods for altering the resistance of cancer cells to chemotherapy; (e) Methods for making cancer cells sensitive to chemotherapy; (f) Methods for inhibiting the proliferation of cancer cells expressing NaPi2b; (g) Methods for the production or preparation of antibodies; (h) Methods for immunizing non-human animals; (i) Methods for preparing hybridomas; (j) The method according to any one of the claims above; (k) any one of the methods (a) to (j) that is in vivo, in vitro, or ex vivo. An antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition, or kit according to any one of the claims, for use in one or more of the above.

17. below: (a) Treatment, improvement, prevention, and / or diagnosis of cancer (Preferably, the cancer is a solid tumor and / or metastatic cancer, and more preferably, the cancer is selected from the group consisting of lung cancer, ovarian cancer, thyroid cancer, non-squamous non-small cell lung cancer, non-mucinous ovarian cancer, papillary thyroid cancer, kidney cancer, endometrial cancer, uterine cancer, ureteral cancer, bladder cancer, and fallopian tube cancer); (b) Monitoring the progression of cancer and / or evaluating the effectiveness of cancer treatments; (c) Screening candidate compounds for their anticancer activity; (d) Altering the resistance of cancer cells to chemotherapy; (e) Making cancer cells sensitive to chemotherapy; (f) Inhibiting the proliferation of cancer cells that express NaPi2b; (g) Production or preparation of antibodies; (h) Immunizing non-human animals; (i) Preparation of hybridomas; (j) in the method according to any one of the claims; (k) In vivo, in vitro, or ex vivo use of any one of (a) through (j) Use of one or more of the antibodies, antibody-drug conjugates (ADCs), nucleic acids, expression vectors, host cells, compositions, or kits according to any one of the claims.

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