Novel Anti-napi2b antibodies and antibody-drug-conjugates based thereon, therapeutic methods

Abstract

The present invention relates to novel anti-NaPi2b antibodies, novel antibody-drug conjugates (ADCs) based thereon and methods of treatment and uses thereof, in particular methods of treatment and uses thereof associated with the treatment of cancer.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of priority of European Patent Application EP22202150, filed with the European Patent Office on October 18, 2022, the entire content of which is incorporated herein by reference for all purposes.

[0003] Sequence Listing

[0004] This application contains a sequence listing in computer-readable form, which is incorporated herein by reference. Technical Field

[0005] The present invention relates to novel anti-NaPi2B antibodies, novel antibody-drug conjugates (ADCs) based thereon, and their therapeutic methods and uses, particularly therapeutic methods and uses related to cancer treatment. Background Art

[0006] NaPi2b, encoded by the SLC34A2 gene, is a multi-pass transmembrane, sodium-dependent phosphate transporter that is expressed in human lung cancer, ovarian cancer, and thyroid cancer, as well as in the normal tissues that give rise to these tumors. As a member of the SLC34 solute carrier protein family, it is responsible for transcellular inorganic phosphate uptake and maintenance of phosphate homeostasis, and is associated with cell differentiation and tumorigenesis. It has been reported that Napi2b mRNA / protein is expressed in non-squamous non-small cell lung cancer, non-mucinous ovarian cancer, and papillary thyroid cancer; normal tissue expression has been reported in the lung, bronchus, and kidney (Lin et al., 2015). In normal lung tissue, NaPi2b plays a role in phosphate transport, and mutations in SLC34A2 are associated with pulmonary and testicular microlithiasis (Corut et al., 2006). Differential expression in tumors (relative to most normal tissues), prominent cell surface localization, and high endocytic rate make Napi2b a promising target for ADC therapy. Several first-generation antibody-drug conjugates have also been developed in the past, however, either they do not produce long-term survival benefits or are associated with dose-limiting toxicities.

[0007] Therefore, there is a need for novel antibodies with improved properties and ADCs based thereon with improved toxicity profiles and higher functionality. Accordingly, the technical problem of the present invention is to meet this need.

[0008] The present invention meets this need in particular by providing novel anti-NaPi2b antibodies, novel NaPi2b-targeting antibody-drug conjugates (ADCs) based on said anti-NaPi2b antibodies, and their therapeutic methods and uses (especially related to cancer treatment), wherein the novel anti-NaPi2b antibodies have a reduced tendency to be modified by post-translational or post-expression and purification modifications (such as deamidation), improved on-target binding, improved target-dependent internalization rate, restricted aggregation and tendency to form high molecular weight species (HMWS), and cross-reactivity with rat NaPi2b and cynomolgus monkey NaPi2b with a similar binding ability compared to human NaPi2b, which allows for better toxicity analysis and comparability. The novel NaPi2b-targeting antibody-drug conjugate (ADC) is produced by applying the P5 conjugation technology (such as WO2018 / 041985), which is based on modifying the interchain cysteine residues with unsaturated phosphonamidate reagents. Summary of the Invention

[0009] This technical problem is solved by the subject matter defined in the claims.

[0010] Accordingly, the present invention relates to anti-NaPi2b antibodies (such as antibodies against NPT2B_human sodium-dependent phosphate transporter 2B (such as having UniProt accession number: O95436 or SEQ ID NO:1), and / or antibodies against NPT2B_rat sodium-dependent phosphate transporter 2B (such as having UniProt accession number: Q9JJ09 or SEQ ID NO:2)), wherein the NaPi2b antibody is capable of: (a) binding to human NaPi2b (such as SEQ ID NO:1) and / or rat NaPi2b (such as SEQ ID NO:2), preferably the binding to said human NaPi2b and said rat NaPi2b has approximately the same K D d, and further preferably the antibody is selected from: AV-25, AV-15, AV-18, AV-21 and AV-29 antibodies), and even more preferably, the approximately the same K Dwith a difference of up to 50% (e.g., up to 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 7%, 5%, 4%, 3%, 2% or 1% difference); (b) cross-reacts with rat NaPi2b (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO: 2), (c) cross-reacts with cynomolgus monkey (e.g., Macaca fascicularis) NaPi2b (e.g., having UniProtKB accession number: A0A2K5UHY1 or SEQ ID NO: 3), (d) internalizes, preferably by antigen-mediated antibody internalization; (e) optionally, does not have a dipeptide deamidation site in the CDR2 of the heavy chain variable region (V H ), preferably the missing dipeptide deamidation site is NG (Asn-Gly) in V H CDR2.

[0011] The invention also relates to a monoclonal human or humanized IgG1 anti-Napi2b antibody, which preferably comprises a κ light chain.

[0012] The invention also relates to a hybridoma, wherein the hybridoma produces the antibody of the invention.

[0013] The invention also relates to a nucleic acid encoding the antibody of the invention.

[0014] The invention also relates to an expression vector comprising at least one of the nucleic acid molecules of the invention.

[0015] The invention also relates to an isolated host cell (e.g., an isolated recombinant host cell) comprising the vector and / or the nucleic acid of the invention.

[0016] The invention also relates to an antibody-drug conjugate (ADC) comprising the anti-Napi2b antibody of the invention.

[0017] The invention also relates to the antibody-drug conjugate (ADC) of the invention, which comprises the anti-NaPi2b antibody of the invention (e.g., a humanized monoclonal NaPi2b-specific IgG1 antibody) conjugated to a cytotoxic payload / drug: (a) wherein the cytotoxic payload is selected from camptothecin, maytansine alkaloids, calicheamicin, duocarmycin, tubulysins, amanitin, dolastatin and auristatins (such as monomethyl auristatin E (MMAE)), pyrrolobenzodiazepine dimers, indolobenzodiazepine Dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors and their analogs or prodrugs; and / or (b) wherein the cytotoxic payload is a camptothecin moiety C, selected from exatecan, DXD, SN38, camptothecin, topotecan, irinotecan, belotecan, letotecan, rubitecan, silatecan, cositecan, and gemitecan; and / or (c) wherein the cytotoxic payload is conjugated via a cleavable linker (L), preferably wherein the linker L is cleavable by a protease, glucuronidase, sulfatase, phosphatase, esterase or by reduction of a disulfide, more preferably wherein the linker is cleavable by a protease, preferably by a cathepsin such as cathepsin B; and / or (d) wherein the linker (L) comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; and / or (e) wherein the cytotoxic payload is exatecan, which is conjugated via a chemical valine-citrulline-PAB or valine-alanine-PAB release unit, wherein the release unit is cleavable by a protease. Preferably, the drug (e.g., cytotoxic moiety (e.g., cytotoxic payload, e.g., tubulin disruptor, 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 antibody ratio (DAR) is in the range of 0 to 20; more preferably, the DAR is in the range of 4 to 8, and most preferably the DAR is 4 or 8.

[0018] The present invention also relates to a composition or kit comprising the anti-NaPiab antibody, antibody-drug conjugate (ADC), hybridoma, nucleic acid, expression vector, and / or host cell described in the present invention.

[0019] The present invention also relates to a method for synthesizing the antibody-drug conjugate (ADC) described in the present invention.

[0020] The present invention also relates to a treatment method and use of the antibody, antibody-drug conjugate (ADC), composition, and / or kit described in the present invention.

[0021] Summary of the Sequence Listing

[0022] As described herein, unless otherwise specified, reference is made to UniProtKB accession numbers (https: / / www.uniprot.org / release-notes / 2022-08-03-release, as available in UniProt release 2022_03, published on August 3, 2022).

[0023] This application contains a sequence listing in computer-readable form, which is incorporated herein by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present invention will be better understood with reference to the detailed description when considered in conjunction with the non-limiting examples and the drawings. The drawings illustrate:

[0025] Figure 1 Exemplary sequences of the anti-NaPi2b antibodies of the present invention are shown.

[0026] Figure 2 Charge variant analysis of exemplary antibodies of the present invention is shown.

[0027] Figure 3 Binding to human and rat NaPi2B as evaluated by flow cytometry is shown. Mean ± SEM for n = 2 is shown in the figure.

[0028] Figure 4 Binding to immobilized human NaPi2B as evaluated by ELISA is shown. Mean ± SEM for n = 2 is shown in the figure.

[0029] Figure 5 Internalization as evaluated by flow cytometry is shown.

[0030] Figure 6 Melting curves as evaluated by NanoDSF are shown.

[0031] Figure 7 In vitro cytotoxicity evaluated by resazurin assay in NaPi2B-positive cells is shown. Mean ± SEM for n = 2 is shown in the figure.

[0032] Figure 8 Bystander activity of the ADC is shown.

[0033] Figure 9 In vitro inhibition of topoisomerase-I by delivery of irinotecan by the ADC is shown.

[0034] Figure 10 In vivo PK evaluation of the ADC and the uncoupled antibody is shown.

[0035] Figure 11 UV chromatogram of P5(PEG12)-COOH measured by LC / MS is shown.

[0036] Figure 12 UV chromatogram of P5(PEG24)-OSu measured by LC / MS is shown.

[0037] Figure 13Shows the UV chromatogram of NH2-VC-PAB-irinotecan TFA salt measured by LC / MS.

[0038] Figure 14 Shows the UV chromatogram of NH2-VA-PAB-irinotecan TFA salt measured by LC / MS.

[0039] Figure 15 Shows the UV chromatogram of P5(PEG2)-VC-PAB-irinotecan measured by LC / MS.

[0040] Figure 16 Shows the UV chromatogram of P5(PEG12)-VC-PAB-irinotecan measured by LC / MS.

[0041] Figure 17 Shows the UV chromatogram of P5(PEG24)-VC-PAB-irinotecan measured by LC / MS.

[0042] Figure 18 Shows the UV chromatogram of P5(PEG12)-VA-PAB-irinotecan measured by LC / MS.

[0043] Figure 19 Shows the UV chromatogram of P5(PEG12)-irinotecan measured by LC / MS.

[0044] Figure 20 Shows the analytical characterization of the synthesized mAb and the DAR 8-ADC synthesized from P5(PEG24)-VC-PAB-irinotecan.

[0045] Figure 21 Shows the analytical raw data of AV25. (A-D) in the figure are the analytical characterizations of one of the mAbs. As described above, the antibody was expressed in Expi-CHO cells and purified by protein A chromatography. The mAb was analyzed by HLPC-SEC (A), LC-MS (B), HLPC-HIC (C), and reducing SDS-PAGE (D).

[0046] Figure 22 Shows the LC / MS analysis of the parental antibody.

[0047] Figure 23 Shows the LC / MS analysis of AV15.

[0048] Figure 24 Shows the LC / MS analysis of AV18.

[0049] Figure 25 Shows the LC / MS analysis of AV21

[0050] Figure 26Shows the LC / MS analysis of AV25.

[0051] Figure 27 Shows the LC / MS analysis of AV29.

[0052] Figure 28 Shows the raw analytical data of AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8. (A-D) in the figure are the analytical characterizations of one of the ADCs synthesized and purified as described above. A) Analytical size exclusion chromatography, B) LC-MS of the ADC product, C) Analytical hydrophobic interaction chromatography after conjugation. The data indicate that the ADC is fully conjugated with DAR 8 and only a small amount of aggregates are present after purification.

[0053] Figure 29 Shows the LC / MS analysis of parental-P5(PEG24)-VC-PAB-irinotecan DAR 8.

[0054] Figure 30 Shows the LC / MS analysis of an exemplary ADC of the present invention, AV15-P5(PEG24)-VC-PAB-irinotecan (DAR 8).

[0055] Figure 31 Shows the LC / MS analysis of an exemplary ADC of the present invention, AV18-P5(PEG24)-VC-PAB-irinotecan (DAR 8).

[0056] Figure 32 Shows the LC / MS analysis of an exemplary ADC of the present invention, AV21-P5(PEG24)-VC-PAB-irinotecan (DAR 8).

[0057] Figure 33 Shows the LC / MS analysis of an exemplary ADC of the present invention, AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8).

[0058] Figure 34 Shows the LC / MS analysis of an exemplary ADC of the present invention, AV29-P5(PEG24)-VC-PAB-irinotecan (DAR 8).

[0059] Figure 35 Shows the results of in vitro serum stability analysis of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8), which is a representative ADC of the present invention.

[0060] Figure 36Shows the in vivo efficacy analysis results of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8), which is a representative ADC of the present invention, in a cell line-derived xenograft model (CDX).

[0061] Figure 37 Shows the in vivo efficacy analysis results of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8), which is a representative ADC of the present invention, in a patient-derived xenograft model (PDX).

[0062] Figure 38 Shows the in vivo toxicity analysis results of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8), which is a representative ADC of the present invention, in cynomolgus monkeys.

[0063] Figure 39 Shows the MS analysis of an anti-NaPi2b comparative ADC synthesized by the above method. The measured mass and absolute intensity in daltons are used to label the signals. An exemplary spectrum from the conjugation reaction is shown. The ratio of the finally conjugated drug to the antibody estimated from the MS signals is 3.5 to 4.0. LC: light chain of the anti-NaPi2b comparative antibody, HC: heavy chain of the anti-NaPi2b comparative antibody.

[0064] Figure 40 Shows the binding of unmodified AV25 mAb and AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 ADC, expressed as the MFI ratio, to HEK293 cells transfected with Napi2a (left), Napi2b (middle), and Napi2c (right). AV25 mAb and AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 ADC specifically bind to NaPi2b. n = 2 ± SD is shown in the figure (note that the error bars are too small to be visible).

[0065] Figure 41 Shows the cytotoxicity dose-response of AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 and an anti-NaPi2b comparative ADC on 3 different cell lines. The mean and SD of two measurements are shown, as well as the dose-response fit. The fluorescence % of the medium control is equal to the % of live cells.

[0066] Figure 42 Shows the dose-dependent induction of DNA-damage and apoptosis markers in response to increasing concentrations of AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 treatment. The corresponding non-target isotype control conjugate is included as a negative control. HCC-78 cells (A, NaPi2b高 and OVCAR-3 cells (B, NaPi2b 高 ) were treated with increasing concentrations (0.05–12 μg / ml) of AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 or isotype ADC (isotype-P5(PEG24)-VC-PAB-irinotecan DAR 8) for 72 h, cells were stained for cleaved PARP (left), Caspase 3 (middle) and pH2AX (right), and analyzed by flow cytometry. Shown are mean ± SEM for n = 2.

[0067] Figure 43 Binding of the AV25 HC-LALA antibody Fc region relative to the AV25 HC-wt antibody to recombinant hexameric C1q complement protein is shown, measured in a human C1q binding assay based on HTRF (Homogeneous Time-Resolved Fluorescence) (HTRF human C1q binding kit, Cisbio) according to the manufacturer's instructions. Briefly, serial dilutions from 280 nM - 11.6 nM of all test antibodies (AV25 HC-wt, AV25 HC-LALA, α-MHC-I positive, IgG1 kit standard) were measured. 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 10,000. Shown is the HTRF ratio at a concentration of 70 nM, where the background (diluent only) HTRF ratio was subtracted. Shown are mean ± SD for n = 2.

[0068] Figure 44 Dose-dependent binding of the AV25-HC-LALA antibody and AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 ADC Fc regions relative to the AV25-HC-wt antibody to recombinant human FcRn and FcγR is shown, using Lumit TMFcγR-binding immunoassays (FcγRn, FcγRI, FcγRIIa / CD32 R131 / H131 polymorphism, FcγRIIIa / CD16 V158 / F158 polymorphism; Promega) were measured according to the manufacturer's instructions. Serial dilutions of AV25 HC-wt, AV25 HC-LALA, and AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8, standards, and trastuzumab as a positive control were incubated with Tracer-LgBiT and FcγR-SmBiT. In the absence of the antibody analyte, or if no interaction of the tested antibody with FcγR occurred, Tracer-LgBiT bound to the FcγR-SmBiT target, generating a maximal luminescence signal. In the case of successful interaction with FcγR, the tested antibody / ADC competed with Tracer-LgBiT for binding to the FcγR target, resulting in a concentration-dependent decrease in the luminescence signal. Luminescence was measured on a microplate reader Infinite M200Pro (Tecan). n = 1 in the figure.

[0069] Figure 45 Antibody-dependent cell cytotoxicity (ADCC) assays based on calcein release were shown. Co-cultures of healthy donor (HD)-derived NK cells and calcein-stained target-positive tumor cells (OVCAR-3 and HCC-78) at a ratio of 4:1 were incubated with 15 μg / ml of the indicated antibody or ADC (anti-MHC-I antibody was used as a positive control). The percentage of specific killing was calculated by dividing the calcein released by antibody-mediated cell killing by the calcein released by Triton X-permeabilized cells (maximal killing). Mean ± SEM with n = 2 was shown in the figure.

[0070] Figure 46Shows the results of in vivo efficacy analysis of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8), which is a representative ADC of the present invention, in a patient-derived non-small cell lung cancer (NSCLC) xenograft model (PDX, Lu7700). Shown on the left is the change in tumor volume over time relative to untreated (vehicle) after a single administration of different dose levels of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8) (1, 3, and 5 mg ADC / kg body weight), an isotype control carrying the same amount of linker-payload (5 mg / kg) on day 0. Shown on the right is the body weight of animals treated with different dose levels of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8) relative to untreated (vehicle) animals. All results are presented as the mean and SEM of 4 animals per group.

[0071] Figure 47 Shows the results in Figure 47 Panel A shows the results of in vivo efficacy analysis of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8), which is a representative ADC of the present invention, in a patient-derived ovarian cancer xenograft model (PDX, Ov6668). Shown on the left is the change in tumor volume over time relative to untreated (vehicle) after a single administration of different dose levels of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8) (1, 3, and 5 mg ADC / kg body weight) on day 0. Shown on the right is the body weight of animals treated with different dose levels of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8) relative to untreated (vehicle) animals. All results are presented as the mean and SEM of 9 animals per group. Figure 47 Panel B shows the in vivo PK evaluation of total antibody and intact ADC at three dose levels in a dose-response efficacy study, and the results are given as the mean and SD of 3 measurements at each time point for mice, which were administered a single dose of different dose levels of AV25-P5(PEG24)-VC-PAB-irinotecan (1, 3, and 5 mg ADC / kg body weight) on day 0. The PK evaluation was performed as described in Example 1.

[0072] Figure 48Shows the in vivo efficacy analysis results of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8), which is a representative ADC of the present invention, in a patient-derived ovarian cancer xenograft model. Shown at the top is the change in tumor volume over time relative to untreated (vehicle) after a single administration of 10 mg ADC / kg body weight of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8) or isotype-P5(PEG24)-VC-PAB-irinotecan (DAR 8) on day 0. Shown at the bottom are BRCA-mutant models with low NaPi2B expression (lower left) and high NaPi2B expression (lower right). All results are presented as the mean and SEM of 3 animals per group.

[0073] Figure 49 Shows the dose-dependent binding of increasing concentrations of Upifitamab or AV25 normalized to a non-specific binding control on two different NaPi2b-positive cell lines (OVCAR-3, left, HCC78, right), expressed as the MFI ratio. The mean and SD of two measurements are shown, as well as the dose-response fit.

[0074] Figure 50 Shows the cytotoxicity dose response of AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 compared to Upifitamab-P5(PEG24)-VC-PAB-irinotecan DAR 8 and an isotype control isotype-P5(PEG24)-VC-PAB-irinotecan DAR 8 on two different cell lines. The mean and SD of two measurements are shown, as well as the dose-response fit. The % fluorescence of the media control is equal to the % of live cells. Detailed Description

[0075] The present invention is described in detail below and is also illustrated by the appended examples and figures.

[0076] The inventors prepared and characterized novel specific anti-NaPi2b antibodies for specifically targeting the extracellular domain of NaPi2b. This is particularly advantageous as it relates to a new treatment method for treating cancer (e.g., preferably, the cancer is solid cancer and / or metastatic cancer, more preferably, the cancer is selected from lung cancer, ovarian cancer, thyroid cancer, non-squamous non-small cell lung cancer, non-mucinous ovarian cancer, papillary thyroid cancer, renal cancer, endometrial cancer, uterine cancer, ureteral cancer, bladder cancer, and fallopian tube cancer).

[0077] The present invention provides novel anti-NaPi2b antibodies, novel NaPi2b-targeting antibody-drug conjugates (ADCs) based on the anti-NaPi2b antibodies, and methods of treatment and uses thereof (especially related to cancer treatment). The novel anti-NaPi2b antibodies have a reduced tendency to be modified by post-translational modifications (such as deamidation), improved on-target binding, improved target-dependent internalization rate, limited aggregation and tendency to form high molecular weight species (HMWS), and cross-reactivity with rat NaPi2b and cynomolgus monkey NaPi2b with a similar binding ability to human NaPi2b, which allows for better toxicity analysis and comparability. The novel NaPi2b-targeting antibody-drug conjugate (ADC) is produced by applying the P5 conjugation technology (such as WO2018 / 041985), which is based on modifying the interchain cysteine residues with unsaturated phosphonamidate reagents.

[0078] The novel antibody of the present invention is a humanized anti-NaPi2b monoclonal antibody (mAb), which optionally contains Fc silent mutations, such as the substitution of leucine (L) with alanine (A) at positions 234 and 235 (LALA mutation), binds to human NaPi2b (such as SEQ ID NO:1) and / or rat NaPi2b (such as SEQ ID NO:2), has cross-reactivity with rat and cynomolgus monkey NaPi2b, and optionally does not have a dipeptide deamidation site in the CDR2 of the heavy chain variable region (V H ), preferably the missing dipeptide deamidation site is NG (Asn-Gly) in V H CDR2.

[0079] The antibody-drug conjugate (ADC) of the present invention comprises an anti-NaPi2b antibody (such as an antibody against NPT2B_human sodium-dependent phosphate transporter 2B (such as having UniProt accession number: O95436 or SEQ ID NO:1), and / or an antibody against NPT2B_rat sodium-dependent phosphate transporter 2B (such as having UniProt accession number: Q9JJ09 or SEQ ID NO:2)), wherein the NaPi2b antibody is capable of: (a) binding to human NaPi2b (such as SEQ ID NO:1) and / or rat NaPi2b (such as SEQ ID NO:2), preferably the binding to the human NaPi2b and the rat NaPi2b has approximately the same K D , and further preferably the antibody is selected from: AV-25, AV-15, AV-18, AV-21 and AV-29 antibodies), and further most preferably, the approximately same K Dwith up to 50% difference (e.g., up to 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 7%, 5%, 4%, 3%, 2% or 1% difference); (b) cross-reacts with rat NaPi2b (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO:2), (c) cross-reacts with cynomolgus monkey (e.g., Macaca fascicularis) NaPi2b (e.g., having UniProtKB accession number: A0A2K5UHY1 or SEQ ID NO:3), (d) internalizes, preferably by antigen-mediated antibody internalization; (e) optionally, does not have a dipeptide deamidation site in the CDR2 of the heavy chain variable region (V H ), preferably the missing dipeptide deamidation site is NG (Asn-Gly) in V H CDR2; wherein the anti-NaPi2b antibody is preferably 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, further most preferably 6 to 10, further most preferably 7 to 10, further most preferably 4 or 8, most preferably 8) cytotoxic moieties (e.g., cytotoxic payloads, e.g., microtubule disrupting agents, e.g., topoisomerase-I inhibitors, e.g., auristatin or camptothecin, e.g., MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), e.g., irinotecan) via one or more linkers, further preferably via one or more phosphoramidate linkers.

[0080] Definitions

[0081] antibody

[0082] As used herein, "antibody" refers to a protein comprising one or more polypeptides substantially or partially encoded by immunoglobulin genes or immunoglobulin gene segments, which comprise one or more binding domains and / or antigen-binding portions, preferably antigen-binding domains. The term "immunoglobulin" (Ig) as used herein is used interchangeably with "antibody". Well-known immunoglobulin genes include the κ, λ, α, γ, δ, ε, and μ constant region genes, as well as the myriad immunoglobulin variable region genes. In particular, an "antibody" as used herein is typically a tetrameric glycosylated protein composed of two light (L) chains (each approximately 25 kDa) and two heavy (H) chains (each approximately 50 kDa). Two types of light chains, termed λ and κ, can be found in antibodies. Immunoglobulins can be classified into five main classes, A, D, E, G, and M, based on the amino acid sequence of the heavy chain constant domain, and several of these classes can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2, with IgG being preferred in the context of the present invention. Antibodies related to the present invention are also contemplated, which have an IgE constant domain or a portion thereof bound by Fcε receptor I. IgM antibodies are composed of 5 basic heterotetrameric units along with an additional polypeptide called the J chain and contain 10 antigen-binding sites, while IgA antibodies contain 2 - 5 basic 4-chain units that can polymerize with the J chain to form multivalent aggregates. In the case of IgG, the 4-chain unit is typically approximately 150,000 daltons. Each light chain includes an N-terminal variable (V) domain (VL) and a constant (C) domain (CL). Each heavy chain includes an N-terminal V domain (VH), three or four C domains (CH), and a hinge region. The constant domains do not directly participate in antibody binding to antigen but can exhibit a variety of effector functions, such as participating in antibody-dependent cell cytotoxicity (ADCC). If an antibody is to exert ADCC, it is preferably of the IgG1 subclass, while the IgG4 subclass does not have the ability to exert ADCC.

[0083] The term "antibody" also includes, but is not limited to, and encompasses monoclonal, monospecific, poly- or multi-specific antibodies such as bispecific antibodies, humanized, camelized, human, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, grafted, and in vitro-generated antibodies, with chimeric or humanized antibodies being preferred. The term "humanized antibody" is generally defined as an antibody in which the CDRs specifically encoding the HC and LC have been transferred to an appropriate human variable framework ("CDR grafting"). The term "antibody" also includes scFv, single-chain antibodies, diabodies or tetrabodies, domain antibodies (dAbs), and nanobodies. For the purposes of the present invention, the term "antibody" shall also encompass bis-, tris-, or multimeric antibodies or bis-, tris-, or multifunctional antibodies having several antigen-binding sites.

[0084] In addition, the term "antibody" as used herein also refers to derivatives of the antibodies (including fragments) described herein. "Derivatives" of an antibody include amino acid sequences that have been altered by the introduction of amino acid residue substitutions, deletions, or additions. In addition, derivatives encompass antibodies that have been modified by covalent attachment of any type of molecule to the antibody or protein. Examples of such molecules include sugars, PEG, hydroxyl, ethoxy, carboxyl, or amino, but are not limited to these. In fact, covalent modification of the antibody results in glycosylation, pegylation, acetylation, phosphorylation, amidation, and the like, but is not limited to these.

[0085] The antibodies of the present invention are preferably "isolated" antibodies. As used herein, "isolated" in describing the disclosed antibodies means an antibody that has been identified, separated, and / or recovered from the components of its production environment. Preferably, the isolated antibody is not associated with all of the other components from its production environment. Contaminant components of its production environment, such as those caused by recombinant transfected cells, are materials that typically interfere with the diagnostic or therapeutic use of the polypeptide and can include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In a preferred embodiment, the antibody will be purified (1) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequencer, or (2) to homogeneity by SDS-PAGE using Coomassie blue or preferably silver stain, under non-reducing or reducing conditions. However, typically an isolated antibody will be prepared by at least one purification step.

[0086] The antibodies described herein can be used for diagnostic purposes, including sample testing and in vivo imaging, and for this purpose, the antibody (or its binding fragment) can be conjugated to a suitable detectable reagent to form an immunoconjugate. For diagnostic purposes, a suitable reagent is a detectable label, which includes radioisotopes for whole body imaging, as well as radioisotopes, enzymes, fluorescent labels, and other suitable antibody labels for sample testing. The detectable label can be any of the various types currently used in the field of in vitro diagnostics, including particulate labels comprising metal sols (such as colloidal gold), isotopes, chromophores (including fluorescent labels, biotin, luminescent labels, phosphorescent labels, etc.), and enzyme labels that convert a given substrate into a detectable label and polynucleotide tags that are revealed after amplification (e.g., by polymerase chain reaction). Biotinylated antibodies can subsequently be detected by avidin or streptavidin binding. Suitable enzyme labels include horseradish peroxidase, alkaline phosphatase, and the like. For example, the label can be the enzyme alkaline phosphatase, which is in a 1,2-dioxetane substrate (such as adamantylmethoxyphosphoryloxy phenyl dioxetane (AMPPD), 3-(4-(methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo{3.3.1.13,7}dec}-4-yl)phenyl disodium phosphate (CSPD), and CDP and CDP- or other luminescent substrates well-known to those skilled in the art (such as chelates suitable for lanthanide elements (such as terbium (III) and europium (III))) and detected by measuring the presence or formation of chemiluminescence after conversion. The detection method is determined by the selected label. In the case where the label is a particle and accumulates to an appropriate degree, the appearance of the label or its reaction product can be seen with the naked eye or using an instrument (such as a spectrophotometer, luminometer, fluorometer, etc.), all of which are performed in accordance with standard specifications.

[0087] As used herein, the term "effector function" with respect to an antibody refers to those biological activities caused by the Fc region of the antibody (native sequence Fc region or amino acid sequence variant Fc region), which vary with the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis, downregulation of cell surface receptors (such as B cell receptors), and B cell activation. To exert effector functions, it can be said that the antibody recruits effector cells.

[0088] As used herein, the term "antigen-binding portion" refers to a fragment of an immunoglobulin (or intact antibody) and encompasses any polypeptide containing an antigen-binding fragment or antigen-binding domain. Preferably, the fragment is, for example, Fab, F(ab'), F(ab')2, Fv, scFv, Fd, disulfide-linked Fvs (sdFv), and other antibody fragments that retain antigen-binding function as described herein. Generally, such fragments will contain an antigen-binding domain and have the same properties as the antibodies described herein. Thus, the fragment preferably can also bind to the extracellular domain of NaPi2b.

[0089] As used herein, the term "specifically binds" refers to an antibody or its fragment or derivative that specifically binds to the NaPi2b protein but does not specifically bind to another protein. The antibody or its fragment or derivative according to the present invention binds to the NAPI2B protein through the variable domain of the antibody.

[0090] The pairing of VH and VL together forms a single antigen-binding site. The CH domain closest to VH is designated CH1. Each L chain is linked to the H chain by a covalent disulfide bond, and depending on the H chain isotype, the two H chains are linked to each other by one or more disulfide bonds. The VH and VL domains consist of four relatively conserved sequence regions (FR1, FR2, FR3, and FR4) called framework regions, which form the backbone for three hypervariable sequence regions (complementary determining regions, CDRs). The CDRs contain most of the residues responsible for the specific interaction of the antibody with the antigen. The CDRs are designated CDR1, CDR2, and CDR3. Thus, the CDR components on the heavy chain are called H1 or H-CDR1 (or CDR-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 CDR-L1), L2 or L-CDR2 (or CDR-L2), and L3 or L-CDR3 (or CDR-L3).

[0091] The term "variable" refers to the part of the immunoglobulin domain that shows sequence variability and is related to determining the specificity and binding affinity of a particular antibody (i.e., the "variable domain"). The variability is not evenly distributed throughout the variable domain of the antibody; it is concentrated in subdomains within the variable regions of each heavy and light chain. These subdomains are called "complementary determining regions" (CDRs).

[0092] The terms "CDR" and its plural "CDRs" refer to the complementary determining regions (CDRs), three of which make up the binding properties of the variable region of the light chain (L1-CDR, L2-CDR, and L3-CDR) and three of which make up the binding properties of the variable region of the heavy chain (H1-CDR, H2-CDR, and H3-CDR). The CDRs determine the functional activity of the antibody molecule and are separated by amino acid sequences containing a framework region or framework regions. The precisely defined CDR boundaries and lengths depend on different classification and numbering systems. Thus, the CDRs can be referred to by Kabat, Chothia, contact type, or any other boundary definition (including the numbering systems described herein). Although the boundaries are different, each of these systems has a degree of overlap in the parts that make up the so-called "hypervariable regions" within the variable sequences. Thus, the CDR definitions according to these systems can differ in length and boundary regions relative to the adjacent framework regions. However, numbering according to the so-called Kabat system is preferred.

[0093] The more conserved (i.e., less variable) portions of the variable domains are called "framework" regions (FRMs). The variable domains of naturally occurring heavy and light chains each contain four FRM regions, which mainly adopt a β-sheet conformation connected by three hypervariable regions that form loops connecting the β-sheet structure and in some cases form part of the β-sheet structure. The hypervariable regions in each chain are tightly bound together by the FRMs and together with the hypervariable regions from other chains contribute to the formation of the antigen-binding site (see Kabat et al., supra). 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.

[0094] The term "binding domain" as used in the present invention characterizes the domain of a polypeptide that specifically binds / interacts with a given target epitope. An "epitope" is antigenic, and thus the term epitope is sometimes also referred to herein as an "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 motif of a polypeptide that is capable of specifically interacting with a specific antigen or a specific set of antigens (such as the same antigen in different species). The said binding / interaction can also be understood as defining "specific recognition".

[0095] When used herein, the terms "antigen-binding domain", "antigen-binding portion", "antigen-binding fragment" and "antibody-binding region" refer to a part of an antibody molecule that contains the amino acids responsible for the specific binding between the antibody and the antigen. The portion of the antigen specifically recognized and bound by the antibody is called an "epitope" as described above. As mentioned above, the antigen-binding domain typically can contain the variable region of the antibody light chain (VL) and the variable region of the antibody heavy chain (VH).

[0096] The term "epitope" also refers to the site on an antigen (in the context of the present invention, the antigen is the NaPi2b protein) to which an antibody molecule binds. Preferably, the epitope is a site on a molecule (in the context of the present invention, the antigen is the NaPi2b protein) for which an antibody or its antigen-binding portion, preferably an antibody, will be produced and / or to which an antibody will bind. For example, an epitope can be recognized by an antibody or its antigen-binding portion. A "linear epitope" is an epitope in which the primary amino acid sequence contains the recognized epitope. A linear epitope typically includes at least 3, more typically at least 5, for example about 8 to about 10 amino acids in a unique sequence.

[0097] The term "cross-reactivity" can refer to the ability of an antibody to react with similar antigenic sites on different proteins.

[0098] The term "specifically" in this context means that the antibody or its antigen-binding portion binds to the target NaPi2b, but does not bind to another protein. The term "another protein" includes any protein, including a protein that is closely related or homologous to the NAPI2B protein targeted by the antibody or its antigen-binding portion. However, the term "another protein" does not include cross-reactivity of the antibody or its antigen-binding portion with a NaPi2b protein from another species that is different from the species from which the antibody or its antigen-binding portion is generated.

[0099] Accordingly, the present invention preferably contemplates cross-species specific antibodies or their antigen-binding portions directed against the NaPi2b protein.

[0100] The term "K D " can refer to the equilibrium dissociation constant (k off / k on ratio) between the antibody and its antigen or between the variable regions of one heavy chain and one light chain of the antibody or their fragments or derivatives and their antigen (such as NaPi2b, e.g., full-length NaPi2b and / or one or more of its fragments, preferably the one or more fragments comprise at least one extracellular domain (ECD) of the Napi2b (e.g., the ECD comprises amino acids 122-135 and / or amino acids 235-361 and / or 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., about 15 to about 30 amino acids in length), e.g., the full-length Napi2b and / or one or more of its fragments may or may not be fused with one or more protein tags (e.g., 6×His tag, FLAG, HA, V5, Fc fusion, MBP, SUMO, TEV, GFP, TST)), and is measured in vitro. K D is inversely correlated with affinity.

[0101] As used herein, the term "affinity" can refer to the binding strength between the variable regions of a heavy chain and a light chain of an antibody or fragments or derivatives thereof and their antigen (such as NaPi2b, for example full-length NaPi2b and / or one or more of its fragments, preferably one or more of said fragments comprising at least one extracellular domain (ECD) of said Napi2b (for example, said ECD comprises amino acids 122-135 and / or amino acids 235-361 and / or 429-485 and / or amino acids 547-552 of human Napi2b having SEQ ID NO:1) and / or one or more fragments of said ECD (for example, having a length of about 15 to about 30 amino acids), for example, said full-length NaPi2b and / or one or more of its fragments may or may not be fused with one or more protein tags (such as, 6×His tag, FLAG, HA, V5, Fc fusion, MBP, SUMO, TEV, GFP, TST)) and is measured in vitro. Affinity determines the strength of interaction between the epitope and the antigen-binding site of the antibody. Affinity can be calculated using the following formula:

[0102] KA = [AB-AG] / [AB]*[AG] = k on / k off

[0103] Where:

[0104] KA = association constant

[0105] [AB] = molar concentration of unoccupied binding sites on the antibody

[0106] [AG] = molar concentration of unoccupied binding sites on the antigen

[0107] [AB-AG] = molar concentration of the antibody-antigen complex.

[0108] The term "amino acid" or "amino acid residue" generally refers to an amino acid having its well-known definition in the art, such as an amino acid selected from the following: 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 (Ile 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, synthetic, or rare amino acids may be used as needed. Generally, amino acids can be grouped according to the non-polar side chains they possess (such as Ala, Cys, Ile, Leu, Met, Phe, Pro, Val); negatively charged side chains (such as Asp, Glu); positively charged side chains (such as Arg, His, Lys); or uncharged polar side chains (such as Asn, Cys, Gln, Gly, His, Met, Phe, Ser, Thr, Trp, and Tyr).

[0109] In this article, the term "polypeptide" is used interchangeably with the term "protein". A protein (including its fragments (preferably bioactive fragments) and peptides, which usually have fewer than 30 amino acids) contains one or more amino acids linked to each other by covalent peptide bonds (thus producing an amino acid chain). The term "polypeptide" as used herein describes a group of molecules composed of, for example, more than 30 amino acids. Polypeptides can further form multimers such as dimers, trimers, and higher oligomers, that is, composed of more than one polypeptide molecule. The polypeptide molecules forming such dimers or trimers, etc. can be the same or different. The corresponding higher-order structure of such a multimer is thus called a homodimer or heterodimer, homotrimer or heterotrimer, etc. An example of a heteromultimer is an antibody molecule, which in its naturally occurring form consists of two identical light polypeptide chains and two identical heavy polypeptide chains. The terms "polypeptide" and "protein" also refer to polypeptides / proteins that have been naturally modified, where such modifications are affected by, for example, post-translational modifications (such as glycosylation, acetylation, phosphorylation, etc.). These modifications are well known in the art.

[0110] The term "immune cell" refers to a cell capable of producing antibodies. Immune cells of particular interest herein are, for example, lymphoid cells derived from the spleen, peripheral blood lymphocytes (PBL), lymph nodes, inguinal nodes, Peyers patches, tonsils, bone marrow, cord blood, pleural effusion, and tumor infiltrating lymphocytes (TIL).

[0111] One type of antibody variant covered by the present invention is an amino acid substitution variant. These variants have at least one, two, three, four, five, six, seven, eight, nine or ten amino acid residues in the Napi2b antibody molecule replaced by a different residue. The sites of substitution mutagenesis of most interest include the CDRs of the heavy and / or light chains, particularly the hypervariable regions, but FR alterations in the heavy and / or light chains are also contemplated.

[0112] For example, if a CDR sequence consists of 6 amino acids, it is conceivable that one, two or three of these amino acids are substituted. Similarly, if a CDR sequence contains 15 amino acids, it is contemplated that one, two, three, four, five or six of these amino acids are substituted.

[0113] Generally, if amino acid substitutions are made in one or more or all of the CDRs of the heavy and / or light chains, preferably, the "substituted" sequence thus obtained 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%), more preferably 65%, even more preferably 70%, particularly preferably 75%, more particularly preferably 80% identical to the "original" CDR sequence. This means that the degree of identity with the "substituted" sequence depends on the length of the CDR. For example, a CDR with 5 amino acids is preferably 80% identical to its substituted sequence in order to have at least one amino acid substitution. Thus, the CDRs of the Napi2b antibody can have different degrees of identity with their substituted sequences, e.g., CDRL1 can have 80% while CDRL3 can have 90%.

[0114] Preferred substitutions (or replacements) are conservative substitutions. However, any substitution (including non-conservative substitutions or one or more of the "exemplary substitutions" listed in Table I herein) is contemplated, provided that the antibody retains its ability to specifically bind the Napi2b protein and / or the CDRs of the antibody have identity with 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%, more particularly preferably 80% identical to the "original" CDR sequence).

[0115] Conservative substitutions are shown under the heading "Preferred Substitutions" in Table I. If such substitutions result in an alteration of biological activity, larger-scale alterations (referred to as "exemplary substitutions" in Table I, or further described below with reference to amino acid types) can be introduced and the product screened for the desired characteristics.

[0116] Chemical modification

[0117] The antibodies or antigen-binding variants or fragments thereof used in the present invention can be modified. Typical modifications conceivable in the context of the present invention include, for example, chemical modifications as described below.

[0118] Possible chemical modifications of the antibody or its antigen-binding variant or fragment include acylation or acetylation at the amino terminus or amidation or esterification at the carboxyl terminus, or optionally, at both ends. The modification also affects the amino group on the lysine side chain or the hydroxyl group of threonine. Other suitable modifications include, for example, extending the amino group with a polypeptide chain of variable length (such as XTEN technology or ), N-glycosylation, O-glycosylation, and chemical conjugation of carbohydrates, such as hydroxyethyl starch (such as ) or polysialic acid (such as technology). Chemical modifications such as alkylation (such as methylation, propylation, butylation), arylation, and etherification are possible and are also contemplated.

[0119] Antibody-drug conjugate (ADC)

[0120] As used herein, the term antibody-drug conjugate (or ADC) can refer to any antibody according to the present invention conjugated to one or more drug moieties (e.g., cytotoxic payload). 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: (a) wherein the cytotoxic payload is selected from camptothecin, maytansine alkaloids, calicheamicin, duocarmycin, tubulysins, amanitin, dolastatin, and auristatins (such as monomethyl auristatin E (MMAE)), pyrrolobenzodiazepine dimers, indolobenzodiazepine Dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors and their analogs or prodrugs; and / or (b) wherein said cytotoxic payload is a camptothecin moiety C, selected from exatecan (such as CAS Nr: 171335-80-1), DXD, SN38, camptothecin, topotecan, irinotecan, belotecan, letotecan, rubitecan, silatecan, cositecan and gemitecan; and / or (c) wherein said cytotoxic payload is coupled via a cleavable linker (L), preferably wherein said linker L is cleavable by a protease, glucuronidase, sulfatase, phosphatase, esterase or by reduction of a disulfide, more preferably wherein said linker is cleavable by a protease, preferably by a cathepsin such as cathepsin B; and / or (d) wherein said linker (L) comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; and / or (e) wherein the cytotoxic payload is exatecan, which is coupled via a chemical valine-citrulline-PAB or valine-alanine-PAB release unit, wherein said release unit is cleavable by a protease. As used herein, "linker" (L) can refer to any chemical moiety capable of linking an antibody of the invention to one or more drug moieties (e.g., a cytotoxic moiety (e.g., a cytotoxic payload, e.g., a microtubule-disrupting agent, e.g., a topoisomerase-I inhibitor, e.g., an auristatin or a camptothecin, e.g., MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), e.g., exatecan); preferably, L is a phosphoamide ester linker; further preferably, the linker L comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; most preferably, the linker L is cleavable (e.g., enzymatically cleavable).

[0121] Sequence identity

[0122] As used herein, the term “% identity” or “% sequence identity” refers to the percentage of paired identical residues relative to the number of residues in the longer of the two sequences after (homologously) aligning the sequences of a polypeptide of the invention with the sequence in question. The percentage of identity is determined by dividing the number of identical residues by the total number of residues and multiplying the result by 100.

[0123] Sequence homology or percent sequence identity can be determined, for example, using BLASTP, version 2.2.5 (November 16, 2002; see Altschul, S.F. et al. (1997) Nucl. Acids Res. 25, 3389-3402). In this embodiment, the percent homology is based on an alignment of the complete polypeptide sequence including the propeptide sequence (matrix: BLOSUM 62; gap penalty: 11.1), and preferably the wild-type protein backbone is used as a reference in pairwise comparisons. The results output by the BLASTP program are expressed as a percentage calculated by dividing the number of "positives" (homologous amino acids) by the total number of amino acids selected by the program for the alignment.

[0124] The term "NaPi2b" refers to sodium-dependent phosphate transporter 2B, and generally includes all known isoforms. Preferably, the sodium-dependent phosphate transporter 2B has SEQ ID NO:1 or UniProtKB accession number: O95436.

[0125] vector

[0126] The nucleic acids of the present invention can also be in the form of a vector, can be present in a vector, and / or can be part of a vector.

[0127] The term "vector" refers to a nucleic acid molecule used as a vehicle for transporting (exogenous) genetic material into a host cell, and includes, but is not limited to, plasmids, viruses, cosmids, and artificial chromosomes (such as bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs)). Generally, engineered vectors contain an origin of replication, a multiple cloning site, and selectable markers. A vector itself is usually a nucleotide sequence, often a DNA sequence, which contains an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. In addition to the transgene insert and the backbone, a vector also includes additional elements: gene regulatory elements, genetic markers, antibiotic resistance, reporter genes, targeting sequences, or protein purification tags. In the context of the present invention, expression vectors (expression constructs) for expressing a transgene in a host cell are particularly contemplated, and such expression vectors generally contain gene regulatory sequences in addition to the transgene.

[0128] Generally, an expression vector is a vector that can provide expression of the antibodies 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 choice of the specific vector included depends on factors such as the host cell, the expected copy number of the vector, whether transient or stable expression of the antibodies of the present invention is contemplated, and the like.

[0129] "Transient expression" is produced by introducing nucleic acids that cannot replicate autonomously (e.g., linear or non-linear DNA or RNA molecules) or vectors into recipient host cells. Expression of the transgene occurs through transient expression of the introduced sequence.

[0130] However, "stable expression" of nucleic acid sequences as described herein is generally preferred and can be accomplished by stably integrating the nucleic acid sequence into the genome of the host cell or by introducing a vector containing the nucleic acid sequence of the invention and capable of autonomous replication into the host cell.

[0131] Specifically contemplated herein is that the vectors provided herein contain gene regulatory elements operably linked to the DNA sequences encoding the antibodies of the invention.

[0132] The term "gene regulatory element" refers to the DNA sequences required for expression of an operably linked coding sequence in a particular host organism. The term "gene regulatory element" includes controllable transcriptional promoters, operons, enhancers, silencers, transcriptional terminators, 5' and 3' untranslated regions that interact with host cell proteins for transcription and translation, and other elements that can control gene expression, including start and stop codons. The exact nature of the regulatory regions required for gene expression may vary between organisms. For example, prokaryotic gene regulatory elements include promoters, optionally operon sequences, and ribosome binding sites (RBS); while eukaryotic cell gene regulatory elements include promoters, polyadenylation (poly-A) signals, and enhancers.

[0133] It is contemplated that the gene regulatory element is "operably linked" to the gene to be expressed, i.e., that the gene regulatory element is in a functional relationship with the gene to be expressed. For example, if a promoter or enhancer affects the transcription of a sequence, it is "operably linked" to the coding nucleic acid sequence. The DNA sequences that are "operably linked" may or may not be contiguous. Ligation is typically accomplished at convenient restriction sites or at synthetic oligonucleotide adaptors or linkers.

[0134] Host cell

[0135] Also provided herein are host cells (e.g., recombinant and / or isolated host cells) containing the vectors described herein.

[0136] Many host cells can be used for expressing the 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 a recipient host cell is also referred to herein as "transformation" or "transfection". These terms are used interchangeably herein.

[0137] Host cell transformation generally involves opening transient pores or "holes" in the cell wall and / or cell membrane to allow uptake of materials. Exemplary examples of transformation protocols involve the use of calcium phosphate, electroporation, cell squeezing, dendrimers, liposomes, cationic polymers (such as DEAE-dextran or polyethylenimine), sonoporation, optical transfection, impalefection, nanoparticles (gene gun), magnetofection, particle bombardment, alkali metal cations (cesium, lithium), enzymatic digestion, glass bead agitation, viral vectors or others. The choice of method typically depends on the cell type being transformed, the vector to be introduced into the cell, and the conditions under which the transformation occurs.

[0138] As used herein, the term "host cell" refers to any cell or cell culture that serves as a recipient for a vector or isolated nucleic acid sequence encoding an Abs as described herein. Suitable host cells include prokaryotic or eukaryotic cells and also include, but are not limited to, bacterial, yeast, fungal, plant, and animal cells such as insect and mammalian cells (e.g., murine, rat, rhesus, or human).

[0139] For example, antibodies can be produced in bacteria. In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeasts are suitable cloning or expression hosts for the NAPI2B-antibodies of the present invention. Exemplary examples include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces hosts such as Kluyveromyces lactis, Kluyveromyces fragilis (ATCC 12424), Kluyveromyces bulgaricus (ATCC 16045), Kluyveromyces wickeramii (ATCC 24178), Kluyveromyces waltii (ATCC 56500), Kluyveromyces drosophilarum (ATCC 36906), Kluyveromyces thermotolerans, and Kluyveromyces marxianus; Yarrowia (EP 402226), Pichia pastoris (EP 183 070); Candida; Trichoderma reesei (EP 244 234); Neurospora crassa; Schwanniomyces (such as Schwanniomyces occidentalis); and filamentous fungi (such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as Aspergillus nidulans and Aspergillus niger).

[0140] Host cells suitable for expressing the glycosylated antibody constructs of the invention may also be derived from multicellular organisms. Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains and variants and corresponding permissive insect host cells have been identified, which are derived from hosts such as Spodoptera frugiperda (larvae), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. A variety of virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV.

[0141] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, Arabidopsis, and tobacco can also be used as hosts. Cloning and expression vectors for producing proteins in plant cell cultures are known to those skilled in the art.

[0142] Examples of useful mammalian host cell lines are the monkey kidney CV1 line transformed with SV40 (COS-7, ATCC CRL1651); human embryonic kidney 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 (CVl, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL1587); human cervical cancer cells (HELA, ATCC CCL 2); dog kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, 1413 8065); mouse mammary tumor (MMT 060562, ATCC CCL5 1); TRI cells; MRC 5 cells; FS4 cells; and human hepatoma cells (Hep G2).

[0143] patient

[0144] As used herein, the term "patient" or "subject" refers to a human or non-human animal, typically a mammal. Mammals are specifically contemplated, such as 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.

[0145] Treatment

[0146] All grammatical forms of the term "treatment" include therapeutic or prophylactic treatment. "Therapeutic or prophylactic treatment" includes prophylactic treatment aimed at completely preventing clinical and / or pathological manifestations, or therapeutic treatment aimed at improving or alleviating the clinical and / or pathological manifestations of a disease. Thus, the term "treatment" also includes the improvement or prevention of cancer.

[0147] In the context of the present invention, the term "treatment effect" generally refers to a satisfactory or beneficial effect of treatment, such as the improvement or alleviation of disease manifestations. The term "manifestations" of a disease is used herein to describe its perceivable manifestations, including clinical manifestations and pathological manifestations, where the clinical manifestations are defined hereinafter as disease indications (i.e., symptoms) detectable upon physical examination and / or perceivable by the patient, and the pathological manifestations refer to the manifestations of the disease at the cellular and molecular levels. The treatment effect of treatment with the NaPi2b-ADC of the present invention can be evaluated using conventional methods in the art, such as measuring the leukemia burden by blood / bone marrow analysis (cytomorphology, flow cytometry, genetics), clinical chemistry or radiological procedures (such as CT). Additionally or alternatively, the general manifestations of each patient (such as health status, well-being) can also be evaluated, which will also help the skilled medical staff to evaluate whether a treatment effect has been achieved. Those skilled in the art know many other methods suitable for observing the treatment effect of the compounds of the present invention.

[0148] Dose

[0149] Preferably, a therapeutically effective amount of the compounds described herein is administered. A "therapeutically effective amount" means the amount of the compounds described herein that produces a treatment effect. The exact dose of the Ab-NaPi2b-ADC of the present invention depends on the purpose of treatment (such as remission, induction, maintenance) and will be determined by those skilled in the art using known techniques. Adjustments may be necessary for the route of administration, age, weight, general health status, gender, diet, time of administration, drug interactions and severity of the condition, and such adjustments can be determined by those skilled in the art using routine experimentation.

[0150] Administration

[0151] The compounds of the present invention can be administered by a variety of routes, including but not limited to oral, topical, transdermal, subcutaneous, intravenous, intraperitoneal, intramuscular or intraocular, preferably subcutaneous and / or intravenous. However, if desired, those skilled in the art can easily select any other route.

[0152] Composition

[0153] It is contemplated to administer the NAPI2B antibody and / or ADC of the present invention in the form of a pharmaceutical composition.

[0154] The term "pharmaceutical composition" particularly refers to a composition suitable for administration to humans, i.e., preferably a sterile and / or pharmaceutically acceptable composition. However, compositions suitable for administration to non-human animals are also contemplated herein. Preferably, the pharmaceutical composition comprises the Ab-NaPi2b-ADC of the present invention and one or more pharmaceutical excipients. The term "excipient" includes fillers, binders, disintegrants, coatings, adsorbents, anti-adhesives, glidants, preservatives, antioxidants, flavoring agents, coloring agents, sweetening agents, solvents, co-solvents, buffers, chelating agents, viscosity imparting agents, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers or tonicity regulators. The pharmaceutical compositions of the present invention can be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, particularly 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 a form particularly suitable for the desired method of administration.

[0155] The pharmaceutical compositions of the present invention may further comprise one or more additional agents. Preferably, the agent is therapeutically effective for the treatment of the diseases described herein and is present in the composition in a therapeutically effective amount.

[0156] In view of the above, the present invention thus also provides a pharmaceutical composition comprising one or more NaPi2b antibodies and / or ADCs of the present invention. The pharmaceutical composition is particularly intended for use in methods of therapeutic and / or prophylactic treatment of cancer.

[0157] Kit

[0158] A kit is also provided herein. The kit can be a kit having two or more parts, which comprises preferably a therapeutically effective amount and a pharmaceutically acceptable form of the NaPi2b antibody and / or ADC of the present invention. The components of the kit can be contained in containers or vials. It is contemplated that the kit contains additional agents useful in the treatment of cancer.

[0159] ***

[0160] In some aspects / embodiments, the present invention relates to 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)), wherein the NaPi2b antibody is capable of: (a) 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 approximately the same K D , and further preferably the antibody is selected from: AV-25, AV-15, AV-18, AV-21 and AV-29 antibodies), and further most preferably, the approximately 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) cross-reacting with rat NaPi2b (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO:2), (c) cross-reacting with cynomolgus monkey (e.g., Macaca fascicularis) NaPi2b (e.g., having UniProtKB accession number: A0A2K5UHY1 or SEQ ID NO:3), (d) internalizing, preferably internalizing by antigen-mediated antibody internalization; (e) optionally, not having a dipeptide deamidation site in CDR2 of the heavy chain variable region (V H ), preferably the missing dipeptide deamidation site is NG (Asn-Gly) in V H CDR2.

[0161] In some aspects / embodiments, the present invention relates to monoclonal human or humanized IgG1 anti-NaPi2b antibodies, and the antibody preferably comprises a κ light chain.

[0162] In some aspects / embodiments, the present invention relates to hybridomas, wherein the hybridomas produce the antibodies of the present invention.

[0163] In some aspects / embodiments, the present invention relates to nucleic acids encoding the antibodies of the present invention.

[0164] In some aspects / embodiments, the present invention relates to expression vectors comprising at least one of the nucleic acid molecules of the present invention.

[0165] In some aspects / embodiments, the present invention also relates to isolated host cells (e.g., isolated recombinant host cells) comprising the vectors and / or the nucleic acids of the present invention.

[0166] In some aspects / embodiments, the present invention relates to an antibody-drug conjugate (ADC) comprising the anti-NaPi2b antibody of the present invention.

[0167] In some aspects / embodiments, the present invention relates to the antibody-drug conjugate (ADC) of the present invention, which comprises the anti-NaPi2b antibody of the present invention (e.g., a humanized monoclonal NaPi2b-specific IgG1 antibody) conjugated to a cytotoxic payload: (a) wherein the cytotoxic payload is selected from camptothecins, maytansine alkaloids, calicheamicins, duocarmycins, tubulysins, amanitins, dolostatins, and auristatins (such as monomethyl auristatin E (MMAE)), pyrrolobenzodiazepine dimers, indolinobenzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors, and their analogs or prodrugs; and / or (b) wherein the cytotoxic payload is a camptothecin moiety C selected from exatecan, DXD, SN38, camptothecin, topotecan, irinotecan, belotecan, letotecan, rubitecan, silatecan, cositecan, and gemitecan; and / or (c) wherein the cytotoxic payload is conjugated through a cleavable linker (L), preferably wherein the linker L is cleavable by protease, glucuronidase, sulfatase, phosphatase, esterase, or by reduction of a disulfide bond, more preferably wherein the linker is cleavable by protease, preferably by a cathepsin such as cathepsin B; and / or (d) wherein the linker (L) comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; and / or (e) wherein the cytotoxic payload is exatecan, which is conjugated through a chemical valine-citrulline-PAB or valine-alanine-PAB release unit, wherein the release unit is cleavable by protease.

[0168] In some aspects / embodiments, the present invention relates to cancer. The cancer can be any cancer. Preferably, the cancer is solid cancer and / or metastatic cancer, and further preferably, the cancer is selected from: lung cancer, ovarian cancer, thyroid cancer, non-squamous non-small cell lung cancer, non-mucinous ovarian cancer, papillary thyroid cancer, renal cancer, endometrial cancer, uterine cancer, ureteral cancer, bladder cancer, and fallopian tube cancer.

[0169] In some aspects / embodiments, the present invention relates to a composition or kit comprising the anti-NaPi2b, antibody-drug conjugate (ADC), hybridoma, nucleic acid, expression vector, and / or host cell of the present invention.

[0170] In some aspects / embodiments, the present invention relates to a method for synthesizing the antibody-drug conjugate (ADC) of the present invention.

[0171] In some aspects / embodiments, the present invention relates to methods of treatment (e.g., methods of treating a patient) and uses of the antibodies, antibody-drug conjugates (ADCs), nucleic acids, expression vectors, host cells, compositions, and / or kits of the present invention.

[0172] In some aspects of the present invention, the antibody of the present invention is expressed as Fc-silent (LALA mutant) IgG1 in CHO cells and purified by protein A chromatography.

[0173] The present invention also relates to the following items:

[0174] 1. An anti-NaPi2b antibody (e.g., an antibody against NPT2B_human sodium-dependent phosphate transporter 2B (e.g., having UniProt accession number: O95436 or SEQ ID NO:1), and / or an antibody against NPT2B_rat sodium-dependent phosphate transporter 2B (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO:2)), wherein the NaPi2b antibody is capable of:

[0175] a) 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 and rat NaPi2b has approximately the same K D , more preferably the antibody is selected from: AV-25, AV-15, AV-18, AV-21, and AV-29 antibodies), even more preferably, the approximately 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%);

[0176] b) cross-reacting with rat NaPi2b (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO:2), preferably, with the parental antibody (e.g., comprising SEQ ID NO:54 and 55, e.g., as Figure 1The cross-reactivity is increased (e.g., by at least 10%) compared to the corresponding cross-reactivity with rat Napi2b (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO:2). Further preferably, the cross-reactivity with rat Napi2b is measured using endogenous rat Napi2b. Most preferably, the endogenous Napi2b is located on the cell surface;

[0177] c) Cross-reacts with cynomolgus monkey (e.g., Macaca fascicularis) NaPi2b (e.g., having UniProtKB accession number: A0A2K5UHY1 or SEQ ID NO:3);

[0178] d) Internalizes, preferably by antigen-mediated antibody internalization. Further preferably, the internalization is increased (e.g., by at least 10%, e.g., 15%) compared to the corresponding internalization of the parental antibody (e.g., comprising SEQ ID NO:54 and 55, e.g., as Figure 1 shown);

[0179] e) Optionally, does not have a deamidated dipeptide site in CDR2 of the heavy chain variable region (V H ). Preferably, the missing deamidated dipeptide site is NG (Asn - Gly) in V H CDR2.

[0180] 2. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody does not have a deamidated dipeptide site in CDR2 of the heavy chain variable region (V H ). Preferably, the missing deamidated dipeptide site is NG (Asn - Gly) in V H CDR2. Further preferably, the deletion of the deamidated site reduces the post-translational modification of the corresponding V H CDR2 (e.g., improves the homogeneity of the antibody and / or simplifies its production process).

[0181] 3. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody is cross-reactive with rat NaPi2b (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO:2). Preferably, compared to the parental antibody (e.g., comprising SEQ ID NO:54 and 55, e.g., as Figure 1The cross-reactivity is increased (e.g., by at least 10%, such as 15%) compared to the corresponding cross-reactivity with rat Napi2b (e.g., having UniProt accession number: Q9JJ09 or SEQ ID NO: 2). Further preferably, the cross-reactivity with rat Napi2b is measured using endogenous rat Napi2b. Most preferably, the endogenous Napi2b is located on the cell surface.

[0182] 4. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody is capable of internalization, preferably by antigen-mediated antibody internalization; further preferably, the internalization is increased (e.g., by at least 10%, such as 15%) compared to the corresponding internalization of the parental antibody (e.g., comprising SEQ ID NOs: 54 and 55, such as as Figure 1 shown).

[0183] 5. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody binds to the human NaPi2b (e.g., SEQ ID NO: 1) and / or rat NaPi2b (e.g., SEQ ID NO: 2) with an increased (e.g., by at least 10%, such as 15%) K Figure 1 compared to the corresponding K D of the parental antibody (e.g., comprising SEQ ID NOs: 54 and 55, such as as D shown).

[0184] 6. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody can bind (e.g., specifically bind) to the extracellular domain of NaPi2b (the extracellular domain of NaPi2b, for example, comprises amino acids 122 - 135 and / or amino acids 235 - 361 and / or 429 - 485 and / or amino acids 547 - 552 of human Napi2b having SEQ ID NO: 1).

[0185] 7. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody has one or more of the following characteristics:

[0186] a) (e.g., in OVCAR-3 cells (e.g., HTB-161#, ATCC) that endogenously express Napi2b) the K D for endogenously expressed human NaPi2b is about 0.01 to about 10 nmol / L, preferably about 1 to about 10 nmol / L, more preferably about 1 to about 7 nmol / L, further preferably about 1 to about 4 nmol / L. More preferably, the K DMeasured by the method of FACS determination, and further most preferably, the K D is from about 2.661 to about 6.644 nmol / L; and / or

[0187] b) Optionally, the K D for the immobilized exogenous full-length NaPi2b and / or one or more of its fragments is from about 0.01 to about 10 nmol / L. Preferably, the one or more fragments comprise at least one extracellular domain (ECD) of the NaPi2b (e.g., the ECD comprises amino acids 122 - 135 and / or amino acids 235 - 361 and / or 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 (such as having a length of about 15 to about 30 amino acids), wherein the full-length Napi2b and / or one or more of its fragments are fused or not fused with one or more protein tags (e.g., 6×His tag, FLAG, HA, V5, Fc-fusion, MBP, SUMO, TEV, GFP, TST). Preferably, the K D is measured by the method of ELISA assay, preferably from about 0.05 to about 0.2 nmol / L, and further preferably, the K D is from about 0.071 to about 0.147 nmol / L.

[0188] 8. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody is an antibody comprising:

[0189] a) A heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence having 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%) identity with SEQ ID NO:4, and the light chain variable region having an amino acid sequence having 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%) identity with SEQ ID NO:5; preferably, the anti-NaPi2b antibody is the AV-25 antibody, which comprises: a light chain comprising SEQ ID NO:6 and a heavy chain comprising SEQ ID NO:7;

[0190] b) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having 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%) identity to SEQ ID NO:8, and the light chain variable region has an amino acid sequence having 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%) identity to SEQ ID NO:9; Preferably, the anti-NaPi2b antibody is the AV-15 antibody, which comprises: a light chain comprising SEQ ID NO:10 and a heavy chain comprising SEQ ID NO:11;

[0191] c) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having 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%) identity to SEQ ID NO:12, and the light chain variable region has an amino acid sequence having 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%) identity to SEQ ID NO:13; Preferably, the anti-NaPi2b antibody is the AV-18 antibody, which comprises: a light chain comprising SEQ ID NO:14 and a heavy chain comprising SEQ ID NO:15;

[0192] d) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having 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%) identity to SEQ ID NO: 16, and the light chain variable region has an amino acid sequence having 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%) identity to SEQ ID NO: 17; preferably, the anti-NaPi2b antibody is the AV-21 antibody, which comprises: a light chain comprising SEQ ID NO: 18 and a heavy chain comprising SEQ ID NO: 19;

[0193] e) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having 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%) identity to SEQ ID NO: 20, and the light chain variable region has an amino acid sequence having 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%) identity to SEQ ID NO: 21; preferably, the anti-NaPi2b antibody is the AV-29 antibody, which comprises: a light chain comprising SEQ ID NO: 22 and a heavy chain comprising SEQ ID NO: 23.

[0194] 9. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody is:

[0195] a) An antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 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 the light chain variable region comprises 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;

[0196] b) An antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 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 the light chain variable region comprises 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;

[0197] c) An antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 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 the light chain variable region comprises 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;

[0198] d) An antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 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 the light chain variable region comprises 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;

[0199] e) An antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 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 the light chain variable region comprises 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.

[0200] 10. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody has one or more of the following characteristics:

[0201] a) Monoclonal antibody;

[0202] b) Chimeric antibody and / or humanized antibody;

[0203] c) Specifically recognizes NaPi2b overexpressed on cancer cells;

[0204] d) Human IgG antibody, preferably human IgG1 antibody;

[0205] e) Comprises a kappa (κ) light chain;

[0206] f) Comprises a lambda (λ) light chain;

[0207] g) Comprises an Fc silent mutation, such as the substitution of leucine (L) with alanine (A) at positions 234 and 235 (LALA mutation), wherein the LALA mutation can reduce immune cell effector function;

[0208] h) Can be internalized by target cells (such as cancer cells) expressing NaPi2b; preferably, the internalized antibody is directed to lysosomes;

[0209] i) Tumor-selective antibody, preferably the tumor is a liquid and / or solid tumor;

[0210] j) Malignant cell-selective antibody;

[0211] k) Binds to the human and / or rat NaPi2b in a glycosylation-dependent manner, wherein the antibody binds to the glycosylated form of the NaPi2b protein;

[0212] l) (e.g., in OVCAR-3 cells (e.g., HTB-161#, ATCC) endogenously expressing Napi2b) the K of endogenously expressed human NaPi2bD is from about 0.01 to about 10 nmol / L, preferably from about 1 to about 10 nmol / L, more preferably from about 1 to about 7 nmol / L, further preferably from about 1 to about 4 nmol / L, and more preferably, the K D is measured by the method of FACS assay. Even more preferably, the K D is from about 2.661 to about 6.644 nmol / L;

[0213] m) Optionally, the K D for the immobilized exogenous full-length NaPi2b and / or one or more of its fragments is from about 0.01 to about 10 nmol / L. Preferably, one or more of the fragments comprise at least one extracellular domain (ECD) of the NaPi2b (e.g., the ECD comprises amino acids 122 - 135 and / or amino acids 235 - 361 and / or 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 (such as having a length of about 15 to about 30 amino acids), wherein the full-length Napi2b and / or one or more of its fragments are fused or not fused with one or more protein tags (e.g., 6×His tag, FLAG, HA, V5, Fc-fusion, MBP, SUMO, TEV, GFP, TST). Preferably, the K D is measured by the method of ELISA assay, preferably from about 0.05 to about 0.2 nmol / L. Even more preferably, the K D is from about 0.071 to about 0.147 nmol / L.

[0214] 11. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody is a monoclonal antibody.

[0215] 12. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody is a chimeric antibody and / or a humanized antibody.

[0216] 13. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody is capable of specifically recognizing NaPi2b overexpressed on cancer cells.

[0217] 14. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody is a human IgG antibody, preferably a human IgG1 antibody.

[0218] 15. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody comprises a kappa (κ) light chain.

[0219] 16. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody comprises a lambda (λ) light chain.

[0220] 17. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody comprises an Fc silent mutation, such as the substitution of leucine (L) with alanine (A) at positions 234 and 235 ((LALA mutation (as in the Fc region of the antibody)), wherein the LALA mutation is capable of reducing immune cell effector function (e.g., wherein the antibody comprises a mutated (e.g., according to any one of the preceding items) Fc region (or fragment crystallizable region), e.g., the tail region of the antibody that interacts with cell surface Fc receptors when unmutated (e.g., wherein the Fc region of an immunoglobulin molecule consists of the constant region of the heavy chain, e.g., and is capable of binding to antibody receptors (Fc receptors) on cells and the Clq component of complement when unmutated)).

[0221] 18. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody can be internalized by target cells (e.g., cancer cells) expressing NaPi2b; preferably, the internalized antibody is directed to lysosomes.

[0222] 19. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody is a tumor-selective antibody, preferably the tumor is a liquid and / or solid tumor.

[0223] 20. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody is a malignant cell-selective antibody.

[0224] 21. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody can bind to the human and / or rat NaPi2b in a glycosylation-dependent manner, wherein the antibody binds to the glycosylated form of the NaPi2b protein.

[0225] 22. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody (e.g., in OVCAR-3 cells (e.g., HTB-161#, ATCC) endogenously expressing Napi2b) to the K of endogenously expressed human NaPi2b Dis from about 0.01 to about 10 nmol / L, preferably from about 1 to about 10 nmol / L, more preferably from about 1 to about 7 nmol / L, still more preferably from about 1 to about 4 nmol / L, and even more preferably, the K D is measured by the method of FACS assay. Even more preferably, the K D is from about 2.661 to about 6.644 nmol / L.

[0226] 23. The anti-NaPi2b antibody according to any one of the preceding items, wherein the K D of the anti-NaPi2b antibody against immobilized exogenous full-length NaPi2b and / or one or more of its fragments is from about 0.01 to about 10 nmol / L. Preferably, the one or more fragments comprise at least one extracellular domain (ECD) of the NaPi2b (e.g., the ECD comprises amino acids 122-135 and / or amino acids 235-361 and / or 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 (such as having a length of about 15 to about 30 amino acids), wherein the full-length Napi2b and / or one or more of its fragments are fused or not fused with one or more protein tags (e.g., 6×His tag, FLAG, HA, V5, Fc-fusion, MBP, SUMO, TEV, GFP, TST). Preferably, the K D is measured by the method of ELISA assay, preferably from about 0.05 to about 0.2 nmol / L. Even more preferably, the K D is from about 0.071 to about 0.147 nmol / L.

[0227] 24. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody is conjugated with a labeling group.

[0228] 25. The anti-NaPi2b antibody according to any one of the preceding items, wherein the anti-NaPi2b antibody is obtainable by a hybridoma (e.g., the antibody is a recombinant antibody).

[0229] 26. The anti-NaPi2b antibody according to any one of the preceding items, which is obtained according to Example 1 or 2 herein and / or has the characteristics as described in Examples 1, 2 or 3 herein (e.g., Figures 1 - 33 , in particular Figure 3 , 4 , 5, 6, 7, 8, 9, 19 and / or 20).

[0230] 27. The anti-NaPi2b antibody according to any one of the preceding items, wherein the antibody comprises one or more (e.g., 2) CDRs, heavy chain variable regions, light chain variable regions, heavy chains, light chains, and / or signal sequences according to any one of the preceding items, preferably comprising one or more selected from: SEQ ID NOs: 4-53.

[0231] 28. The anti-NaPi2b antibody according to any one of the preceding items, wherein the antibody preferably comprises at least one (e.g., 2) heavy chain and light chain according to any one of the preceding items.

[0232] 29. The anti-NaPi2b antibody according to any one of the preceding items, which comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions (or mutations), and the amino acid substitutions are preferably located in one or more regions selected from the following: CDRs (e.g., CDR1, CDR2, CDR3, e.g., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and / or CDR-L3, e.g., those according to any one of the preceding items, e.g., as in the sequence listing disclosed herein), V H (heavy chain variable region), V L (light chain variable region), C H (heavy chain constant region) or C L (light chain constant region), F(ab) and / or Fc region (e.g., as defined herein Figure 1 ).

[0233] 30. The anti-NaPi2b antibody according to any one of the preceding items, which comprises one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) highly conservative, conservative, or equivalent amino acid substitutions (or mutations), for example, "conservative or equivalent substitution" refers to the substitutions listed as "exemplary substitutions" in Table I below, and the "highly conservative" substitutions used herein refer to the substitutions shown under the title "Preferred substitutions" in Table I below:

[0234] Table I: Amino Acid Substitutions

[0235] Original Exemplary Substitutions Preferred Substitutions Ala(A) val; leu; ile Val Arg(R) lys; gin; asn lys Asn(N) gln; his; asp, lys; arg gln Asp(D) glu; asn glu Cys(C) ser; ala ser Gln(Q) asn; glu asn Glu(E) asp; gln asp Gly(G) ala ala His(H) asn; gln; lys; arg arg Ile(I) leu; val; met; ala; phe; leu Leu(L) Norleucine; ile; val; met; ala; ile Lys(K) arg; gin; asn arg Met(M) leu; phe; ile leu Phe(F) leu; val; ile; ala; tyr tyr Pro(P) ala ala Ser(S) thr thr Thr(T) ser ser Trp(W) tyr; phe tyr Tyr(Y) trp; phe; thr; ser Phe Val(V) ile; leu; met; phe; ala; leu

[0236] Preferably, the one or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10) highly conservative, conservative, or equivalent amino acid substitutions (or mutations) are located in one or more regions selected from: CDRs (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 according to any of the foregoing items, e.g., as in the sequence listing disclosed herein), V H (variable region heavy chain), V L (variable region light chain), C H (constant region heavy chain) or C L (constant region light chain), F(ab) and / or Fc region (e.g., as defined herein Figure 1 ).

[0237] 31. The antibody according to any of the foregoing items, wherein the % HMWS (high molecular weight substance) of the antibody is less than 5% (e.g., as Figure 20 shown).

[0238] 32. A hybridoma, wherein the hybridoma produces a monoclonal antibody according to any of the foregoing items.

[0239] 33. A nucleic acid encoding an antibody according to any of the foregoing items.

[0240] 34. An expression vector comprising at least one nucleic acid molecule according to any of the foregoing items.

[0241] 35. An isolated host cell (e.g., an isolated recombinant host cell) comprising a vector and / or nucleic acid according to any of the foregoing items.

[0242] 36. An antibody-drug conjugate (ADC) comprising an anti-NaPi2b antibody according to any of the foregoing items.

[0243] 37. An antibody-drug conjugate (ADC) according to any one of the preceding items, wherein the 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, further most preferably 6 to 10, further most preferably 7 to 10, further most preferably 4 or 8, most preferably 8) cytotoxic moieties (e.g., cytotoxic payloads, e.g., tubulin disruptors, e.g., topoisomerase-I inhibitors (class), e.g., auristatins or camptothecin, e.g., MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), e.g., irinotecan), preferably via one or more linkers, more preferably via one or more phosphoroamidate linkers.

[0244] 38. An antibody-drug conjugate (ADC) according to any one of the preceding items, wherein the 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, further most preferably 6 to 10, further most preferably 7 to 10, further most preferably 4 or 8, most preferably 8) camptothecin (such as irinotecan) cytotoxic moieties, preferably via one or more linkers, more preferably via one or more phosphoroamidate linkers.

[0245] 39. An antibody-drug conjugate (ADC) according to any one of the preceding items, wherein the 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, further most preferably 6 to 10, further most preferably 7 to 10, further most preferably 4 or 8, most preferably 8) irinotecan cytotoxic moieties via one or more linkers, preferably via one or more phosphoroamidate linkers.

[0246] 40. An antibody-drug conjugate (ADC) according to any one of the preceding items, wherein the ADC comprises a humanized monoclonal NaPi2b-specific IgG1 antibody conjugated to a cytotoxic payload:

[0247] a) wherein the cytotoxic payload is selected from camptothecin, maytansine, calicheamicin, duocarmycin, laulimalide, amanitin, dolastatin and auristatin (such as monomethyl auristatin E (MMAE)), pyrrolobenzodiazepine dimer, indolobenzodiazepine Dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors and their analogs or prodrugs; and / or

[0248] b) wherein the cytotoxic payload is camptothecin moiety C, selected from exatecan, DXD, SN38, camptothecin, topotecan, irinotecan, belotecan, letotecan, rubitecan, seliciclib, cositecan and gemtuzumab; and / or

[0249] c) wherein the cytotoxic payload is conjugated via a cleavable linker (L), preferably wherein the linker L is cleavable by protease, glucuronidase, sulfatase, phosphatase, esterase or by reduction of disulfide, more preferably wherein the linker is cleavable by protease, preferably by a cathepsin such as cathepsin B; and / or

[0250] d) wherein the linker (L) comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; and / or

[0251] e) wherein the cytotoxic payload is exatecan, which is conjugated via a chemical valine-citrulline-PAB or valine-alanine-PAB release unit, wherein the release unit is cleavable by protease.

[0252] 41. The antibody-drug conjugate (ADC) according to any one of the preceding items, wherein the 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, further most preferably 6 to 10, further most preferably 7 to 10, further most preferably 4 or 8, most preferably 8) camptothecin (such as exatecan) cytotoxic moieties via an ethynylphosphonamide ester linker / conjugation (e.g., to all 8 interchain cysteine residues), preferably each phosphonamide ester linker carries at least one PEG24 moiety (e.g., to prevent ADC aggregation), and further preferably, the ADC carries up to the 8 linker payload moieties and 8 PEG24 moieties.

[0253] 42. The antibody-drug conjugate (ADC) according to any one of the preceding items, wherein the ADC has a formula selected from the following:

[0254] a) Formula I:

[0255]

[0256] b) Formula II:

[0257]

[0258] where n ranges from 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); c) Formula III:

[0259]

[0260] where n ranges from 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); d) Formula IV:

[0261]

[0262] where n ranges from 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); e) Formula V:

[0263]

[0264] where n ranges from 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); f) Formula VI:

[0265]

[0266] where n ranges from 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); g) Formula VII:

[0267]

[0268] where n ranges from 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8); h) Formula VIII:

[0269] where n ranges from 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8);

[0270] i) Formula IX:

[0271] where n ranges from 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8);

[0272] j) Formula X:

[0273] where n ranges from 0 to 20 (for example, 1, 2, 3, 4, 5, 6, 7, or 8);

[0274] k) Formula XI:

[0275]

[0276] where n ranges from 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8);

[0277] l) Formula XII:

[0278]

[0279] where n ranges from 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8);

[0280] m) Formula XIII:

[0281]

[0282] where n ranges from 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8);

[0283] n) Formula XIV:

[0284]

[0285] where n ranges from 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8);

[0286] o) Formula XV:

[0287]

[0288] where n ranges from 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8);

[0289] p) Formula XVI:

[0290]

[0291] where n ranges from 0 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, or 8).

[0292] 43. An antibody-drug conjugate (ADC) according to any one of the preceding items, wherein:

[0293] a) the anti-NaPi2b monoclonal antibody is capable of specifically recognizing human NaPi2b (e.g., SEQ ID NO:1) and / or rat NaPi2b (e.g., SEQ ID NO:2) overexpressed on cancer cells; and

[0294] b) when the ADC binds to the NaPi2b overexpressed on cancer cells, the ADC can be internalized by the cell and transported to the lysosomal compartment, where preferably lysosomal proteases (e.g., cathepsin B) can release the cytotoxic payload from the ADC.

[0295] 44. An antibody-drug conjugate (ADC) according to any one of the preceding items, wherein the % HMWS (high molecular weight species) of the antibody is less than 5% (e.g., as shown in Figure 20 .

[0296] 45. A method for producing an antibody-drug conjugate (ADC), the method comprising:

[0297] (a) conjugating an antibody according to any one of the preceding items with 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, further most preferably 6 to 10, further most preferably 7 to 10, further most preferably 4 or 8, most preferably 8) cytotoxic moieties (e.g., cytotoxic payloads, e.g., microtubule disrupting agents, e.g., topoisomerase-I inhibitors, e.g., auristatins or camptothecins, e.g., MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), e.g., irinotecan (e.g., CAS Nr: 171335-80-1)), preferably via one or more linkers, more preferably via one or more phosphonamidate linkers.

[0298] 46. A method for producing an antibody-drug conjugate (ADC) according to any one of the preceding items, wherein the anti-NaPi2b antibody is conjugated with 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, further most preferably 6 to 10, further most preferably 7 to 10, further most preferably 4 or 8, most preferably 8) camptothecin (e.g., irinotecan (e.g., CAS Nr: 171335-80-1)) cytotoxic moieties, preferably via one or more linkers, more preferably via one or more phosphonamidate linkers.

[0299] 47. A method for producing an antibody-drug conjugate (ADC) according to any one of the preceding items, wherein the anti-NaPi2b antibody is conjugated with 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, further most preferably 6 to 10, further most preferably 7 to 10, further most preferably 4 or 8, most preferably 8) irinotecan cytotoxic moieties via one or more linkers, preferably via one or more phosphonamidate linkers.

[0300] 48. A method for producing an antibody-drug conjugate (ADC) according to any one of the preceding items, wherein the 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, further most preferably 6 to 10, further most preferably 7 to 10, further most preferably 4 or 8, most preferably 8) camptothecin-based (such as irinotecan) cytotoxic moieties via an ethynylphosphonamidate linker / conjugation (e.g., to all 8 interchain cysteine residues), preferably each phosphonamidate linker carries at least one PEG moiety up to 24 PEG units (e.g., to prevent ADC aggregation), and further preferably, the ADC carries up to the 8 linker payload moieties and 8 PEG24 moieties.

[0301] 49. A method for producing an antibody-drug conjugate (ADC) according to any one of the preceding items, wherein the ADC comprises a humanized monoclonal NaPi2b-specific IgG1 antibody conjugated to a cytotoxic payload:

[0302] a) wherein the cytotoxic payload is selected from camptothecin, maytansine alkaloids, calicheamicin, duocarmycin, tubulysin, amanitin, dolastatin (such as monomethyl auristatin E (MMAE)), pyrrolobenzodiazepine dimers, indolobenzodiazepine dimers, radioisotopes, therapeutic proteins and peptides (or fragments thereof), nucleic acids, PROTACs, kinase inhibitors, MEK inhibitors, KSP inhibitors and their analogs or prodrugs; and / or

[0303] b) wherein the cytotoxic payload is a camptothecin moiety C selected from irinotecan, DXD, SN38, camptothecin, topotecan, irinotecan, belotecan, letotecan, rubitecan, selicetecan, cositecan and gemtitecan; and / or

[0304] c) wherein the cytotoxic payload is conjugated via a cleavable linker (L), preferably wherein the linker L is cleavable by protease, glucuronidase, sulfatase, phosphatase, esterase or by reduction of a disulfide bond, more preferably wherein the linker is cleavable by protease, preferably by a cathepsin such as cathepsin B; and / or

[0305] d) wherein the linker (L) comprises a valine-citrulline-PAB moiety or a valine-alanine-PAB moiety; and / or

[0306] e) wherein the cytotoxic payload is irinotecan (e.g., CAS Nr: 171335-80-1), which is conjugated via a chemical valine-citrulline-PAB or valine-alanine-PAB linker unit, wherein said linker unit is cleavable by a protease.

[0307] 50. An antibody-drug conjugate (ADC) produced by the method according to any one of the preceding items.

[0308] 51. The antibody-drug conjugate (ADC) according to any one of the preceding items, wherein the % HMWS (high molecular weight species) of the antibody is 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) tissues.

[0309] 52. The antibody-drug conjugate (ADC) according to any one of the preceding items, wherein the drug-to-antibody ratio (DAR) 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 even most preferably in the range of 4 to 8.

[0310] 53. The antibody-drug conjugate (ADC) according to any one of the preceding items, wherein the DAR of the ADC is 4 or 8, preferably 8.

[0311] 54. A composition or kit comprising an anti-NaPi2b antibody-drug conjugate (ADC), hybridoma, nucleic acid, expression vector, and / or host cell according to any one of the preceding items.

[0312] 55. The composition according to any one of the preceding items, wherein the composition is a pharmaceutical composition and / or a diagnostic composition.

[0313] 56. The composition or kit according to any one of the preceding items, wherein the ratio of the drug (e.g., cytotoxic moiety (e.g., cytotoxic payload, e.g., microtubule-disrupting agent, e.g., topoisomerase-I inhibitor, e.g., auristatin or camptothecin, e.g., MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), e.g., irinotecan)) to the antibody (DAR) 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 even most preferably in the range of 4 to 8.

[0314] 57. A composition or kit comprising an anti-NaPi2b antibody, antibody-drug conjugate (ADC), hybridoma, nucleic acid, expression vector, and / or host cell according to any one of the preceding items.

[0315] 58. A method for treating, ameliorating, preventing, and / or diagnosing cancer, preferably wherein the cancer is solid cancer and / or metastatic cancer, and further preferably wherein the cancer is selected from: 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 comprising: administering a therapeutically or prophylactically effective amount of an antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition, or kit according to any one of the preceding items.

[0316] 59. An antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition, or kit according to any one of the preceding items, for use as a medicament and / or for treatment.

[0317] 60. An antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition, or kit according to any one of the preceding items, for use in one or more of the following methods:

[0318] (a) A method for treating, ameliorating, preventing, and / or diagnosing cancer, preferably wherein the cancer is solid cancer and / or metastatic cancer, and further preferably wherein the cancer is selected from: 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;

[0319] (b) A method for monitoring cancer progression and / or for evaluating the efficacy of cancer therapy;

[0320] (c) A method for screening candidate compounds having anti-cancer activity;

[0321] (d) A method for altering the chemoresistance of cancer cells;

[0322] (e) A method for sensitizing cancer cells to chemotherapy;

[0323] (f) A method for inhibiting the growth of cancer cells expressing NaPi2b;

[0324] (g) A method for producing or preparing an antibody;

[0325] (h) A method for immunizing non-human animals;

[0326] (i) A method for preparing hybridomas;

[0327] (j) A method according to any one of the preceding items;

[0328] (k) The method according to any one of (a)-(j), wherein the method is an in vivo, in vitro or ex vivo method.

[0329] 61. Use of the antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition or kit according to any one of the foregoing items for one or more of the following:

[0330] (a) Treating, ameliorating, preventing and / or diagnosing cancer, preferably the cancer is solid cancer and / or metastatic cancer, more preferably the cancer is selected from: 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;

[0331] (b) Monitoring cancer progression and / or evaluating the efficacy of cancer therapy;

[0332] (c) Screening for candidate compounds with anti-cancer activity;

[0333] (d) Altering the resistance of cancer cells to chemotherapy;

[0334] (e) Sensitizing cancer cells to chemotherapy;

[0335] (f) Inhibiting the growth of cancer cells expressing NaPi2b;

[0336] (g) Production or preparation of antibodies;

[0337] (h) Immunizing non-human animals;

[0338] (i) Preparation of hybridomas;

[0339] (j) In the method according to any one of the foregoing items;

[0340] (k) The use according to any one of (a)-(k), wherein the use is an in vivo, in vitro or ex vivo use.

[0341] ***

[0342] It should be noted that, as used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a reagent" includes one or more such different reagents, and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art, which may be modified or substituted for the methods described herein.

[0343] Unless otherwise specified, the term "at least" before a series of elements shall be understood to refer to each element in the series. Those skilled in the art will recognize or be able to use, without more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are also intended to be encompassed by the present invention.

[0344] Wherever used herein, the term "and / or" includes the meanings of "and", "or", and "any other combination, whole or part, of the elements connected by said term".

[0345] As used herein, the terms "about" or "approximately" mean within 20% of a given value or range, preferably within 10%, and more preferably within 5%.

[0346] The terms "less than" or "greater than" do not include the specific number.

[0347] For example, less than 20 means less than the indicated number. Similarly, greater than or higher than means greater than or higher than the indicated number. For example, greater than 80% means greater than or higher than the indicated number 80%.

[0348] Throughout the specification and the following claims, unless the context requires otherwise, the word "comprise" and its variations such as "comprises" and "comprising" shall be understood to imply the inclusion of the stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. When used herein, the term "comprising" may be replaced by the term "containing" or "including", or sometimes when used herein by the term "having". When used herein, "consisting of" excludes any element, step or ingredient not specified in the claim elements.

[0349] The term "including" means "including but not limited to". "Including" and "including but not limited to" are used interchangeably.

[0350] It should be understood that the present invention is not limited to the specific methods, protocols, materials, reagents, substances, etc. described herein and may vary. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present invention, which is defined only by the claims.

[0351] All publications (including all patents, patent applications, scientific publications, manufacturer's specifications, etc.) cited throughout this specification, whether above or below, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosures by virtue of prior invention. To the extent that the material incorporated by reference conflicts or is inconsistent with this specification, this specification will supersede any such material.

[0352] The contents of all documents and patent documents cited herein are hereby incorporated by reference in their entirety into this text.

[0353] Embodiments of the Invention

[0354] The present invention and its advantages can be better understood 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.

[0355] Example 1:

[0356] General Information

[0357] Chemicals, Solvents and Antibodies

[0358] 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. Anhydrous solvents were purchased from Merck (Merck Group, Germany). PEG24 was purchased from BiochemPEG (Pure Chemistry Scientific, USA).

[0359] Preparative HPLC

[0360] In Pure C-850 Flash-Prep system ( Preparative HPLC was carried out on a Labortechnik AG (Switzerland), using a VP 250 / 10 Macherey-Nagel Nucleodur C18 HTec Spum column (Macherey-Nagel GmbH & Co. Kg, Germany) for small-scale preparation. 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 and the following gradient was applied: 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.

[0361] LC-MS

[0362] The Waters H-Class instrument equipped with a quaternary solvent manager, Waters Sample Manager-FTN, Waters PDA detector and a Waters column manager equipped with an Acquity UPLC Protein BEH C4 column 1.7 μm, 2.1 mm x 50 mm) was used to analyze small molecules, linker-payloads, antibodies and ADCs. Here, the samples were eluted at a column temperature of 80 °C. The following gradient was used: A: H2O solution of 0.1% formic acid; B: MeCN solution of 0.1% formic acid. 25% B 0 - 1 min, 0.4 mL / min, 25 - 95% B 1 - 3.5 min 0.2 mL / min, 95% B 3.5 - 4.5 min 0.2 mL / min, 95 - 25% B 4.5 - 5 min 0.4 mL / min, 25 - 95% B 5 - 5.5 min 0.4 mL / min, 95 - 25% B 5.5 - 7.5 min 0.4 mL / min. Mass analysis was performed using a Waters XEVO G2-XS QTof analyzer. Proteins were ionized in positive ion mode with a cone voltage of 40 kV. The raw data were analyzed using MaxEnt 1. Using an Acquity UPLC-BEH C18 column ( 1.7μm, 2.1mm x50mm) for analysis of small molecules and linker-payloads. Here, the sample was eluted at a flow rate of 0.4mL / min at a column temperature of 45°C. The following gradient was used: A: 0.1% formic acid in H2O; B: 0.1% formic acid in MeCN. 2% B 0-1min, 2-98% B 1-5min, 98% B 5-5.5min, 98-2% B 5.5-6min, 2% B 6-7min.

[0363] CDR mutagenesis and antibody expression

[0364] For the CDR mutagenesis activity of the parent antibody, 22 × 10 8 Phage display library of 10 sequences (66% functional): homology modeling of antibody Fv regions, screening of surface CDR residues that may be involved in antigen binding (identified VH: 18 positions, VK: 16 positions) and analysis of NGS data (approximately 2×10 6 The amino acid usage frequency at each position of the IgG sequence) Figure 1 ). To select antibodies with improved affinity, the panning of the CDR mutant phage display library was carried out on cells expressing antigen / NaPi2b with 4 rounds of continuous antigen restriction, which was achieved by increasing cell dilution. 384 clones (a total of 1536 clones) were selected for each round of panning, produced as soluble scFv antibodies in Escherichia coli, and the combination with HEK293 cells expressing human NaPi2b and parental HEK293 cells was screened using flow cytometry analysis. The signal-to-noise ratio (N / S) between the positive cell line MFI and the negative cell line MFI was selected to be greater than 12 as the hit selection criteria. According to the selection criteria, 375 clones were identified and DNA sequencing was performed on them. From these results, 255 unique antibody sequences were identified, and the dissociation rates of these clones were further analyzed using BLI technology. In detail, the biotinylated NaPi2b antigen was fixed to a streptavidin sensor, and the binding and dissociation of scFv were measured. Dissociation rate kinetics were modeled and clones that met the following criteria were ranked: significant binding response and high modeling accuracy were achieved. Five antibody clones / sequences were selected for further in-depth analysis: AV-15, AV-18, AV-21, AV-25, and AV-29 ( Figure 1 ).

[0365] Then, the Expi-CHO transfection system (Thermo Fisher) was used to transiently express the antibody in Expi-CHO-S cells (Thermo Fisher) by co-transfecting the cells with pcDNA3.4 expression plasmids (Thermo Fisher) encoding the heavy and light chains of the corresponding sequences at a ratio of 1:1. The cells were harvested by centrifugation at 300 g for 5 minutes at 4°C. To remove particulates from the supernatant, the supernatant was centrifuged at 4000–5000 g for 30 minutes at 4°C. For further clarification, the supernatant was passed through a 0.22 μm filter. The antibody was purified from the clarified and filtered supernatant by protein A chromatography and analyzed by HPLC-SEC, HPLC-HIC, LC-MS, and SDS-PAGE.

[0366] Preparative size exclusion chromatography

[0367] Using an Pure FPLC system (GE Healthcare, USA) equipped with an F9-C fraction collector, proteins were purified by size exclusion chromatography.

[0368] ADC concentration determination

[0369] In a 96-well plate, the ADC concentration was determined using the Pierce TM Rapid Gold BCA Protein Assay Kit (Thermo Fisher Scientific, USA) and Bradford reagent B6916 (Merck, Germany) with a pre-diluted protein assay standard with bovine γ-globulin (Thermo Fisher Scientific, USA). The results of the two assays were averaged arithmetically.

[0370] Sample preparation of ADC and antibody for MS

[0371] For antibody and ADC deglycosylation, 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 0.2 mg / mL antibody or ADC in PBS solution, and the solution was incubated at 37°C for at least 2 hours. Glycosylated mAb and ADC were incubated with 10 mM DTT at a concentration of 0.2 mg / ml for 1 hour. LC / MS assays were performed on the samples, with 2 μl of each sample injected.

[0372] Analytical size exclusion chromatography

[0373] The analytical size-exclusion chromatography (A-SEC) of ADC was performed on a Vanquish Flex UHPLC system equipped with a DAD detector, a split sampler FT (4 °C), a column compartment H (25 °C), and a binary pump F (Thermo Fisher Scientific, USA), using a MAbPac SEC-1 4 × 300 mm column (Thermo Fisher Scientific, USA) with a flow rate of 0.15 mL / min. A phosphate buffer at pH 7 (20 mM Na2HPO4 / NaH2PO4), 300 mM NaCl, and 5% v / v isopropanol were used as the mobile phase, and the separation of different ADC / mAb populations was achieved during a 30-minute isocratic gradient. 8 μg of ADC / mAb was loaded onto the column for A-SEC analysis. UV chromatograms were recorded at 220 and 280 nm.

[0374] Analytical hydrophobic interaction chromatography

[0375] Determination was performed on a Vanquish Flex UHPLC system (2.9) using a MabPac HIC butyl 4.6 × 100 mm column (Thermo Fischer Scientific, USA). The separation of different ADC / antibodies was achieved with the following gradient: A: 1 M (NH4)2SO4, 500 mM NaCl, 100 mM NaH2PO4 pH 7.4; B: 20 mM NaH2PO4, 20% (v / v) isopropanol, 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, with a flow rate of 700 μL / min. 15 μg of the sample was loaded onto the column for each analysis. UV chromatograms were recorded at 220 and 280 nm.

[0376] Charge variant analysis

[0377] Assays were performed on a Vanquish Flex UHPLC system (2.9) using a ProPac Elite WCX 5 μm 4 × 150 mm column (Thermo Fisher Scientific). Separation of antibody charge variants was carried out with the following gradient: 1× CX-1 buffer pH 5.6 B:1× CX-1 buffer pH 10.2 (Thermo Fisher Scientific). 0-100% B: 0-60 min, at a flow rate of 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. Shown are the changes relative to day 0 after incubation at 40 °C for 7 days (decrease % of the main peak, increase % of acidic and basic species)( Figure 2 ).

[0378] SDS-PAGE

[0379] Samples for SDS-PAGE were prepared by incubation in SDS sample buffer (BioRad) supplemented with 25 mM DTT (95 °C, 5 min) and separated on a 4-20% polyacrylamide gel (BioRad 4-20% Mini Protean TGX), followed by Coomassie staining (Thermo Fisher Scientific Imperial Protein Stain).

[0380] ADC synthesis: A general method for conjugating a linker-payload construct based on P5 to an antibody to obtain a DAR of 8.

[0381] To 50 μl of a 10.0 mg / ml solution of each anti-NaPi2b antibody (parent, AV15, AV18, AV21, AV25, AV29) in P5-conjugation buffer (50 mM Tris, 1 mM EDTA, 100 mM NaCl, pH 8.3 at room temperature) was added 3.33 μl of a 10 mM TCEP solution in P5-conjugation buffer. Immediately thereafter, 1.67 μl of a DMSO solution of 40 mM P5-irinotecan construct was added. The mixture was shaken at 350 rpm and 25 °C for 16 h. The reaction mixture was purified by preparative size exclusion chromatography on a 25 ml Superdex TM 200 Increase 10 / 300 GL (Cytiva, Sweden) at a flow rate of 0.8 ml / min and eluted with sterile PBS (Merck, Germany). Fractions containing the antibody were pooled and concentrated by spin filtration( Ultra-2mL MWCO: 30 kDa, Merck, Germany).

[0382] Evaluation of binding to human and rat NaPi2b by flow cytometry

[0383] HEK293 cells stably overexpressing human and rat NaPi2b were generated by stably integrating a human or rat full-length NaPi2b-mCherry-(GGGS)3x-mCherry expression construct controlled by the human EF1 promoter and a puromycin selection cassette controlled by the CMV promoter into the parental HEK293 cell line. Briefly, cells were transfected with linearized plasmid using Lipofectamine 2000 (ThermoFisher) and selected with puromycin antibiotic. Single cell clones were then plated into 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. Target- and mCherry-positive clones were expanded and used for further experiments. To determine the equilibrium binding constant (K D ), HEK293 cells stably expressing human (A and C), cynomolgus monkey (B), or rat (D) full-length NaPi2b-mCherry were incubated with antibodies at concentrations ranging from 0.002 - 200 nM, stained with an Alexa-dye-labeled anti-human IgG H+L secondary antibody (Thermo Fisher Scientific), and analyzed by flow cytometry. Antibody binding was evaluated on mCherry-positive cells. Mean fluorescence intensity (MFI) ratios were normalized against a secondary antibody control. The assay was performed in duplicate, and data points were analyzed by non-linear regression using a single-site specific binding model with Prism 9 software to obtain the K D value. Mean ± SEM of n = 2 is shown in the figure ( Figure 3 ).

[0384] Evaluation of binding to immobilized human NaPi2b by ELISA

[0385] In an ELISA setup, the binding of increasing concentrations of parental mAb and AV mAb clones to purified recombinant immobilized human NaPi2b antigen was tested.

[0386] To determine the binding of parental and AV mAb clones to the NaPi2b antigen, 96-well plates (Nunclon, Thermo Fisher Scientific) with a flat bottom surface were coated with 1 μg / mL of purified recombinant human NaPi2b antigen, which consists of the NaPi2b extracellular loop 2 expressed as an Fc-fusion-6-His-tagged protein in Expi-HEK293 cells. After blocking with 2% bovine serum albumin (Carl Roth) in 1× PBS-Tween 20 (0.05%), increasing antibody concentrations (0.00015, 0.00046, 0.00137, 0.00412, 0.01235, 0.03704, 0.11111, 0.33333, 1.00000, 3.00000, 9.00000 μg / ml) were allowed to bind for 2 hours at room temperature. Bound antibodies were detected by incubation with a goat anti-human κ light chain secondary antibody conjugated to HRP (diluted 1:10,000 in the blocking solution) for 1 hour at RT. Ultra-TMB (Thermo Fisher, 34028) substrate was added and incubated for 15 to 30 minutes at room temperature, after which 100 μl / well of 1 M sulfuric acid was added. Absorbance at 450 nm was measured using a microplate reader Infinite M1000Pro (Tecan) within 10 minutes of adding the acid. The apparent dissociation constant (K D ) was obtained by non-linear regression using Prism 9 software with a single-site specific binding model. K D , in μg / ml, was 0.02209 for the parental mAb, 0.01581 for AV15, 0.01494 for AV18, 0.01248 for AV21, 0.01060 for AV25, 0.01238 for AV29. Mean ± SEM of n = 2 are shown in the figure ( Figure 4 ).

[0387] Internalization assessed by flow cytometry

[0388] To study internalization based on pHrodo, according to the manufacturer's instructions, pHrodo TMThe Deep Red antibody labeling kit (Thermo Fisher Scientific) was used to label the goat anti-human IgG Fcγ fragment-specific secondary antibody (Jackson ImmunoResearch). In the presence of equimolar amounts of pHrodo Deep Red-labeled secondary antibody, NaPi2b-positive OVCAR-3 cells were incubated with 5 μg / ml of the parental mAb and AV mAb clones at 37 °C for 1 hour, 5 hours, and 24 hours. An increase in MFI indicated the presence of the AV antibody in the late endosomal and lysosomal compartments. The MFI ratio was measured by dividing the MFI of the pHrodo-incubated cells by the MFI of the unstained cells ( Figure 5 ).

[0389] Melting curves evaluated by NanoDSF

[0390] The thermal stability of proteins was determined using nano differential scanning fluorimetry (nanoDSF), which measures the temperature-dependent changes in the intrinsic fluorescence of tryptophan and tyrosine residues (Tycho NT.6, NanoTemper Technologies). For this purpose, a PBS solution of 1 μM protein was aspirated into a capillary and then placed in the reader. The protein was then incubated at elevated temperatures while measuring the intrinsic protein fluorescence at 330 nM and 350 nM simultaneously. The change in the fluorescence signal indicated a transition in the folded state of the protein, and the temperature at which the transition occurred was called the inflection point temperature (Ti) or melting temperature (Tm) (Haffke, M et al., Label-free Thermal Unfolding Assay of G Protein-Coupled Receptors for Compound Screening and Buffer Composition Optimization. 2016) ( Figure 6 ).

[0391] In vitro cytotoxicity evaluated by resazurin assay

[0392] To study the direct cytotoxicity of the ADC, cells were seeded in 96-well plates (flat bottom, 5000 cells / well, suspended in 100 μl of medium) and incubated with increasing concentrations of the ADC (0 - 3 μg / ml) in the medium for 7 days to generate a dose-response curve. Before analyzing viability, the supernatant was removed from adherent cells and replaced with fresh medium. Subsequently, resazurin (Sigma-Aldrich) was used as a cell viability dye, and killing was analyzed at a final concentration of 55 μM. Fluorescence emission at 590 nM was measured on a microplate reader Infinite M1000 Pro (Tecan). Cell viability was measured by dividing the fluorescence of cells treated with the ADC by the fluorescence of control cells treated with only the medium in the same manner. The figure shows the mean ± SEM for n = 2 ( Figure 7 ).

[0393] Bystander killing

[0394] To analyze the bystander activity of the ADC against target-negative cells, 20,000 NaPi2B-positive cells (OVCAR-3) were incubated with increasing concentrations of the ADC (0 - 3 μg / ml). After 5 days, half of the volume of the cell culture supernatant was transferred to 5,000 NaPi2B-negative cells (SW-620) and incubated for another 5 days. Killing was analyzed by resazurin-based viability measurements as described above ( Figure 8 ).

[0395] In vitro inhibition of topoisomerase-I by SN-38 delivery via ADC

[0396] Inhibition of topoisomerase-I by SN-38 delivery via TUB-040 ADC induced DNA damage markers. OVCAR-3 cells were treated with 5 μg / mL TUB-040 or 5 nM free SN-38 for 72 hours. Cells were stained with a live / dead stain and analyzed by flow cytometry for the DNA damage markers active caspase-3, cleaved PARP, and phosphorylated H2A.X (Ser-139). The figure shows the mean ± SEM for n = 2 ( Figure 9 ).

[0397] In vivo PK assessment of an ADC

[0398] In vivo PK experiments were performed with AV25 and AV25-P5(PEG24)-VC-PAB-SN-38. Female Sprague Dawley rats were treated with 10 mg / kg of the uncoupled AV25 antibody or the ADC. Blood samples were taken at different time points, and the amount of the ADC was quantified in total antibody and intact ADC ELISA assays.

[0399] To evaluate the PK of the ADC in vivo, the total antibody concentration in the serum of SD rats treated with the ADC was measured at different time points. The total humanized anti-NaPi2B antibody in rat serum was analyzed in the range of 2000 - 15.6 ng / ml. Coat a Nunc 96-well plate with (100 μl / well) NaPi2B diluted in PBS (required concentration: 0.25 μg / ml), and seal it with a PCR foil. Incubate the plate overnight in the refrigerator at a temperature maintained between 2 - 8 °C. Wash the coated plate 3 times with 300 μl of PBST. Add 200 μl / well of the blocking solution (PBST solution with 2% albumin), seal the plate and incubate it at room temperature for 1 hour. Wash the coated plate 3 times with 300 μl of PBST. Add 100 μl / well of the prepared standards (corresponding ADC, QC at 2000 - 15.6 ng / ml) and test samples, seal the plate and incubate it at room temperature for 1 hour. Wash the plate 3 times with 300 μl of PBST. Add 100 μl / well of anti-human IgG (γ-chain specific)-peroxidase antibody (diluted 1:60000 in PBS) and incubate it at room temperature for 1 hour. Wash the plate 3 times with 300 μl of PBST. Add 50 μl / well of TMB, seal the plate and incubate it at room temperature for 15 minutes. Add 50 μl / well of 1M sulfuric acid. Measure the absorbance at a wavelength of 450 nm using a Tecan plate reader.

[0400] To evaluate the stability of the ADC in vivo, the intact ADC concentration in the serum of SD rats treated with the ADC was measured at different time points. The intact ADC in rat serum was analyzed in the range of 2000 - 15.6 ng / ml. Coat a Nunc 96-well plate with (100 μl / well) rabbit anti-irinotecan mAb diluted in PBS (required concentration: 1 μg / ml), and seal it with a PCR foil. Incubate the plate overnight in the refrigerator at a temperature maintained between 2 - 8 °C. Wash the coated plate 3 times with 300 μl of PBST. Add 200 μl / well of the blocking solution (PBST solution with 2% albumin), seal the plate and incubate it at room temperature for 1 hour. Wash the coated plate 3 times with 300 μl of PBST. Add 100 μL / well of the prepared standards (corresponding ADC, QC at 2000 - 15.6 ng / ml) and test samples, seal the plate and incubate it at room temperature for 1 hour. Wash the plate 3 times with 300 μL of PBST. Add 100 μL / well of pre-adsorbed goat anti-human IgG (H+L) (diluted 1:25000 in PBS) and incubate it at room temperature for 1 hour. Wash the plate 3 times with 300 μl of PBST. Add 100 μL / well of TMB, seal the plate and incubate it at room temperature for 10 minutes. Add 100 μl / well of 1M sulfuric acid. Measure the absorbance at a wavelength of 450 nm using a Tecan plate reader ( Figure 10 ).

[0401] Adapter-payload conjugation

[0402] General method 2 for the synthesis of PEGylated P5 building blocks via the Staudinger phosphite reaction

[0403]

[0404] Under an argon atmosphere, 267 mg of bis(diisopropylamino)chlorophosphine (1.00 mmol, 1.00 equiv) was added to a 25 mL Schlenk flask, cooled to 0 °C, and 2.20 mL of ethynylmagnesium bromide solution (0.5 M in THF, 1.10 mmol, 1.10 equiv) was added dropwise. The pale yellow solution was warmed to room temperature and stirred for an additional 30 minutes. 3.00 mmol (3.0 equiv) of the desired PEG-alcohol, dissolved in 5.56 mL of 1H-tetrazole solution (0.45 M MeCN solution, 2.50 mmol, 2.50 equiv), was added, and the white suspension was stirred overnight at room temperature. The formation of the desired phosphite was monitored by 31 P-NMR. 1.0 mmol (1.0 equiv) of the desired azide, dissolved in 2 mL of DMF, THF, or MeCN, was added, and the suspension was stirred at room temperature for a further 24 hours. The crude reaction mixture was purified by preparative HPLC.

[0405] P5(PEG12)-OSu

[0406]

[0407] The title compound was synthesized according to General Method 2 from 19.5 mg of bis(diisopropylamino)chlorophosphine (73 μmol, 1.00 equiv), 146 μL of ethynylmagnesium bromide solution (0.5 M in THF, 73 μmol, 1.00 equiv), 100 mg of dodecaglycol (183 μmol, 2.50 equiv), 400 μL of 1H-tetrazole solution (0.45 M MeCN solution, 183 μmol), and 19 mg of 4-azidobenzoic acid N-hydroxysuccinimide ester (73 μmol, 1.00 equiv). After preparative HPLC (Method D) and lyophilization, a colorless oil was obtained. (42.5 mg, 50 μmol, 68%). 11H NMR (300 MHz, acetonitrile-d3) δ 8.06 (d, J = 8.7 Hz, 2H), 7.32 (d, J = 8.8 Hz, 2H), 4.40–4.14 (m, 2H), 3.79–3.69 (m, 2H), 3.66–3.47 (m, 40H), 3.21 (d, J = 13.1 Hz, 1H), 2.86 (s, 4H), 1.30 (m, 2H), 1.13–0.79 (m, 2H). 13 13C NMR (151 MHz, CDCl3) δ 169.77, 169.46, 161.66, 161.47, 152.75, 146.09, 132.90, 132.24, 117.82, 113.97, 113.29, 89.25, 88.92, 77.27, 77.06, 76.85, 74.69, 72.57, 71.19, 70.62, 70.54, 70.51, 70.47, 70.44, 70.36, 70.27, 70.20, 69.74, 69.70, 68.14, 65.77, 65.73, 61.63, 61.60, 40.72, 30.34, 25.68. 31 31P NMR (122 MHz, acetonitrile-d3) δ -10.87. HRMS C 37 H 60 N2O 19 P + Calcd: 851.3573 [M+H] + , 851.3571.

[0408] P5(PEG12)-COOH

[0409]

[0410] The title compound was synthesized according to General Method 2 from 40 mg of bis(diisopropylamino)chlorophosphine (150 μmol, 1.00 equiv), 360 μL of ethynylmagnesium bromide solution (0.5 M in THF, 180 μmol, 1.2 equiv), 245 mg of PEG12 (450 μmol, 3.0 equiv), 0.83 mL of 1H-tetrazole solution (0.45 M in MeCN, 450 μmol, 2.5 equiv) and 39 mg of 4-azidobenzoic acid (150 μmol, 1.00 equiv). After preparative HPLC (Method D) and lyophilization, a colorless oil was obtained. (25 mg, 34 μmol, 23%). C 33 H 57 NO 16 P + [M+H] +HR-MS calculated value: 754.3410, measured value 754.3398.( Figure 11 UV trace in).

[0411] P5(PEG24)-OSu

[0412]

[0413] The title compound was synthesized according to General Method 2 from 41 mg of bis(diisopropylamino)chlorophosphine (159 μmol, 1.00 equiv), 370 μL of ethynylmagnesium bromide solution (0.5 M in THF, 185 μmol, 1.2 equiv), 450 mg of PEG24 (388 μmol, 2.50 equiv), 1.02 mL of 1H-tetrazole solution (0.45 M in MeCN, 466 μmol, 3.0 equiv) and 40 mg of 4-azidobenzoic acid N-hydroxysuccinimide ester (155 μmol, 1.00 equiv). After preparative HPLC (Method D) and lyophilization, a colorless oil product was obtained. (79 mg, 57 μmol, 37%). C 61 H 109 N2O 30 P 2+ [M+2H] 2+ MS calculated value of: 690.3396, measured value: 690.81.( Figure 12 UV trace in).

[0414] NH2-VC-PAB-Irinotecan TFA salt

[0415]

[0416] 34.3 mg of irinotecan mesylate (0.0645 mmol, 1.0 equiv) was added to a screw-cap vial and suspended in 645 μL of anhydrous DMSO. 241 μL of an anhydrous DMSO solution of 0.4 mol / L Fmoc-VC-PAB-PNP (0.0967 mmol, 1.5 equiv), 64.5 μL of an anhydrous DMSO solution of 1 mol / L HOBt hydrate (0.0645 mmol, 1.0 equiv) and 113 μL of DIPEA (0.645 mmol, 10.0 equiv) were added. The yellow solution was then stirred at 50 °C for 2 h. Then, 425 μL of an anhydrous DMSO solution of 50% diethanolamine (w / w) was added and the reaction mixture was stirred at room temperature for an additional 30 min. 1.5 mL of MeCN and 2.5 mL of H2O were added and the yellow solution was purified directly by preparative HPLC using Method D. After lyophilization, 47.3 mg (76.7%, 0.0495 mmol) of a light yellow solid was obtained as the TFA salt.

[0417] C 43 H 50 FN8O9 + [M+H] + HR-MS calculated value: 841.3680, measured value: 841.3696 ( Figure 13 UV trace in).

[0418] NH2-VA-PAB-Irinotecan TFA salt

[0419]

[0420] 1.23 mg of irinotecan mesylate (0.00232 mmol, 1.0 equiv) was added to a screw-cap vial and suspended in 23 μL of anhydrous DMSO. 8.7 μL of an anhydrous DMSO solution of 0.4 mol / L Fmoc-VA-PAB-PNP (0.00348 mmol, 1.5 equiv), 2.3 μL of an anhydrous DMSO solution of 1 mol / L HOBt hydrate (0.00232 mmol, 1.0 equiv) and 4 μL of DIPEA (0.0232 mmol, 10.0 equiv) were added. The yellow solution was stirred overnight at room temperature. Then, 15 μL of an anhydrous DMSO solution of 50% diethanolamine (w / w) was added, and the reaction mixture was stirred for an additional 30 minutes at room temperature. 1.5 mL of MeCN and 2.5 mL of H2O were added, and the yellow solution was purified directly by preparative HPLC using method D. After lyophilization, 1.01 mg (50.0%, 0.00116 mmol) of a light yellow solid was obtained as the TFA salt.

[0421] C 40 H 44 FN6O8 + [M+H] + HR-MS calculated value: 755.3200, measured value: 755.3201. ( Figure 14 UV trace in).

[0422] P5(PEG2)-VC-PAB-Irinotecan

[0423]

[0424] Add 23.4 μL of an anhydrous DMSO solution of 200 mM NH2-VC-PAB-irinotecan TFA salt (0.00468 mmol, 1.0 equiv), 46.8 μL of a solution of 200 mM 2-(2-hydroxyethoxy)ethyl-N-(4-benzoyl-N-hydroxysuccinimide ester)-P-ethynylphosphoramidate (P5(PEG2)-COOSu, 0.00936 mmol, 2.0 equiv) and 4.08 μL of DIPEA (0.0234 mmol, 5.0 equiv) to a screw-cap vial. Shake the solution at 50 °C for 5 h, cool to room temperature, add 1.5 mL of MeCN and 2.5 mL of H2O, and purify the solution directly by preparative HPLC using Method C. After lyophilization, 1.33 mg (25.0%, 0.00117 mmol) of a pale yellow solid was obtained.

[0425] C 56 H 64 FN9O 14 P + [M+H] + HR-MS calculated for: 1136.4289, found: 1136.4306. ( Figure 15 UV trace in

[0426] P5(PEG12)-VC-PAB-irinotecan

[0427]

[0428] Add 51 μL of an anhydrous DMSO solution of 200 mM NH2-VC-PAB-irinotecan TFA salt (0.0102 mmol, 1.0 equiv), 102 μL of an anhydrous DMSO solution of 200 mM PEG12-N-(4-benzoyl)-P-ethynylphosphoramidate (P5(PEG12)-COOH, 0.0204 mmol, 2.0 equiv), 102 μL of an anhydrous DMSO solution of 250 mM Pybop (0.0255 mmol, 2.5 equiv) and 8.89 μL of DIPEA (0.051 mmol, 5.0 equiv) to a screw-cap vial. Shake the solution at room temperature for 2 h, add 1.5 mL of MeCN and 2.5 mL of H2O, and purify the solution directly by preparative HPLC using Method D. After lyophilization, 15.91 mg (99.0%, 0.0101 mmol) of a pale yellow solid was obtained.

[0429] C 76 H 105 FN9O 24 P 2+ [M+H] 2+HR-MS calculated value: 788.8492, measured value: 788.8485.( Figure 16 UV trace in).

[0430] P5(PEG24)-VC-PAB-Irinotecan

[0431]

[0432] Add 102 μL of an anhydrous DMSO solution of 200 mM NH2-VC-PAB-Irinotecan TFA salt (0.0204 mmol, 1.0 equivalent), 204 μL of an anhydrous DMSO solution of 200 mM (P5(PEG24)-OSu (0.0408 mmol, 2.0 equivalents), and 17.78 μL of DIPEA (0.102 mmol, 5.0 equivalents) to a screw-cap vial. Shake the solution overnight at room temperature, add 1.5 mL of MeCN and 2.5 mL of H2O, and purify the solution directly by preparative HPLC using Method D. After lyophilization, 25.76 mg (60.0%, 0.01224 mmol) of a light yellow solid was obtained.

[0433] C 100 H 153 FN9O 36 P 2+ [M+H] 2+ HR-MS calculated value: 1053.5081, measured value: 1053.50833.( Figure 17 UV trace in).

[0434] P5(PEG12)-VA-PAB-Irinotecan

[0435]

[0436] Add 11.6 μL of an anhydrous DMSO solution of 100 mM NH2-VA-PAB-irinotecan TFA salt (0.00116 mmol, 1.0 equiv), 8.7 μL of an anhydrous DMSO solution of 200 mM PEG12-N-(4-benzoic acid)-P-ethynylphosphonamidate (P5(PEG12)-COOH, 0.00174 mmol, 1.5 equiv), 11.6 μL of an anhydrous DMSO solution of 200 mM Pybop (0.00232 mmol, 2.0 equiv), and 2.02 μL of DIPEA (0.0116 mmol, 10.0 equiv) to a screw-cap vial. Shake the solution at room temperature for 2 h, add 1.5 mL of MeCN and 2.5 mL of H2O, and purify the solution directly by preparative HPLC using Method C. After lyophilization, 0.56 mg (32.2%, 0.000375 mmol) of a light yellow solid was obtained.

[0437] C 73 H 99 FN7O 23 P 2+ [M+H] 2+ HR-MS calculated for: 745.8252, found: 745.8255. ([ Figure 18 UV trace in).

[0438] P5(PEG12)-irinotecan

[0439]

[0440] Add 50 μL of an anhydrous DMSO suspension of 100 mM irinotecan mesylate (0.005 mmol, 1.0 equiv), 20 μL of an anhydrous DMSO solution of 250 mM PEG12-N-(4-benzoic acid)-P-ethynylphosphonamidate (P5(PEG12)-COOH, 0.005 mmol, 1.0 equiv), 20 μL of an anhydrous DMSO solution of 300 mM Pybop (0.006 mmol, 1.2 equiv), and 4.33 μL of DIPEA (0.025 mmol, 5.0 equiv) to a screw-cap vial. Shake the solution at room temperature for 2 h, add 1.5 mL of MeCN and 2.5 mL of H2O, and purify the solution directly by preparative HPLC using Method C. After lyophilization, 2.63 mg (45.0%, 0.0023 mmol) of a light yellow solid was obtained.

[0441] C 57 H 77 FN4O 19 P + [M+H] +HR-MS calculated value: 1171.4899, measured value: 1171.4852.( Figure 19 UV-traces in).

[0442] mAb and ADC characterization

[0443] Analytical overview of the synthesized mAb and the ADC synthesized from P5(PEG24)-VC-PAB-irinotecan( Figure 20 )

[0444] As described above, %HMWS was determined by analytical size exclusion chromatography. % represents the peak area with a lower retention time relative to the monomeric species. Charge variant analysis was performed as described above. Shown are the changes relative to day 0 after incubation at 40 °C for 7 days. As described above, the ADC retention time was measured by hydrophobic interaction chromatography. As described above, Ti was obtained from the melting curve measured by Nano DSF. Binding affinity was determined by flow cytometry (FACS of OVCAR-3 cells) or by ELISA of the above isolated proteins. As described above, cytotoxicity was evaluated by the resazurin assay. As described above, internalization was expressed as the %MFI ratio of pH rodo after 5 - 6 hours relative to time point 0.

[0445] Analytical raw data of the exemplary monoclonal antibody( Figure 21 )

[0446] (A - D) Analytical characterization of one of the mAbs. As described above, the antibody was expressed in Expi-CHO cells and purified by protein A chromatography. The mAb was analyzed by HLPC-SEC (A), LC-MS (B), HLPC-HIC (C), and reducing SDS-PAGE (D).

[0447] LC / MS analysis of the antibody clones

[0448] Parent( Figure 22 )

[0449] LC calculated value: 23473, measured value: 23472; HC calculated value: 48705, measured value: 48706.

[0450] AV15( Figure 23 )

[0451] LC calculated value: 23459, measured value: 23458; HC calculated value: 48705, measured value: 48706.

[0452] AV18( Figure 24 )

[0453] LC calculated value: 23473, measured value: 23472; HC calculated value: 48632, measured value: 48634.

[0454] AV21( Figure 25 )

[0455] LC calculated value: 23473, measured value: 23472; HC calculated value: 48564, measured value: 48566.

[0456] AV25( Figure 26 )

[0457] LC calculated value: 23443, measured value: 23442; HC calculated value: 48632, measured value: 48634.

[0458] AV29( Figure 27 )

[0459] LC calculated value: 23443, measured value: 23442; HC calculated value: 48634, measured value: 48636.

[0460] Analysis raw data of the exemplary ADC( Figure 28 )

[0461] Figure 28 . (A-D) Analytical characterization of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR8) synthesized and purified as described above. A) Analytical size exclusion chromatography, B) LC-MS of the ADC product, C) Analytical hydrophobic interaction chromatography after conjugation. The data indicate that the ADC is fully conjugated to DAR 8 and only a small amount of aggregates are present after purification.

[0462] LC / MS analysis of the ADC

[0463] Parent-P5(PEG24)-VC-PAB-irinotecan DAR 8( Figure 29 )

[0464] LC calculated value: 25579, measured value: 25577; HC calculated value: 55023, measured value: 55021.

[0465] AV15-P5(PEG24)-VC-PAB-irinotecan DAR 8( Figure 30 )

[0466] LC calculated value: 25565, measured value: 25563; HC calculated value: 55023, measured value: 55022.

[0467] AV18-P5(PEG24)-VC-PAB-irinotecan DAR 8( Figure 31 )

[0468] LC calculated value: 25579, measured value: 25577; HC calculated value: 54950, measured value: 54947.

[0469] AV21-P5(PEG24)-VC-PAB-Irinotecan DAR 8( Figure 32 )

[0470] LC calculated value: 25579, measured value: 25577; HC calculated value: 54882, measured value: 54881.

[0471] AV25-P5(PEG24)-VC-PAB-Irinotecan DAR 8( Figure 33 )

[0472] LC calculated value: 25549, measured value: 25547; HC calculated value: 54950, measured value: 54950.

[0473] AV29-P5(PEG24)-VC-PAB-Irinotecan DAR 8( Figure 34 )

[0474] LC calculated value: 25549, measured value: 25547; HC calculated value: 54952, measured value: 54950.

[0475] Example 2: Ex vivo serum stability, in vivo efficacy in a cell line-derived xenograft model (CDX), and in vivo efficacy in a patient-derived xenograft model (PDX) of the exemplary ADCs of the present invention:

[0476] Ex vivo serum stability:

[0477] Serum samples of each species were mixed with AV25-P5(PEG24)-VC-PAB-Irinotecan DAR 8 at a concentration of 0.2 mg / ml, which accounted for at least 80% of the serum. The samples were sterile filtered using a UFC30GV0S centrifugal filter device (Merck, Germany) and incubated at 37 °C for 1, 2, 3, 5, and 7 days. The samples on day 0 were directly subjected to subsequent processing.

[0478] 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 the NaPi2B-conjugated bead suspension. The beads were incubated with 100 μl of the serum-ADC mixture (premixed with 200 μl of PBS) at room temperature for 2 hours. Then, the supernatant was removed, and the resin was washed 2 times with 1 mL of PBS-T. Then, it was incubated with 10 μl of 100 mM glycine buffer (pH 2.5) at room temperature for 15 minutes. By using 75 μL of ZebaTM A rotating desalting column (7K MWCO) (Thermo Fisher Scientific, USA) was used to buffer the solution back into PBS. As described above, the samples were further processed for MS-analysis. The drug-to-antibody ratio (DAR) was calculated from the MS intensities of the light chain adducts conjugated to 0 or 1 molecule of P5(PEG24)-VC-PAB-irinotecan and the heavy chain adducts conjugated to 0 - 3 molecules of P5(PEG24)-VC-PAB-irinotecan.

[0479] The results clearly showed that the linker between the AV25 antibody and the irinotecan drug molecule was highly stable in sera of different species, with no significant loss of payload (unchanged drug - antibody ratio (DAR)) after incubating the ADC for several days. Figure 35 )

[0480] In vivo efficacy of the ADC in a cell line-derived xenograft model (CDX):

[0481] All animal experiments were conducted in accordance with the German Animal Welfare Act and approved by the local authorities. Briefly, 1×10 7 OVCAR-3 cells (100 μL + 100 μL Matrigel) were subcutaneously injected into CB17-Scid mice. Treatment was initiated when the tumors reached an average tumor volume of 0.1 - 0.15 cm 3 15 days after implantation. After randomization into treatment and control groups, 5 animals per group were injected intravenously once on day 0 with 1 mg / kg, 3 mg / kg, or 5 mg / kg of AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8, isotype-P5(PEG24)-VC-PAB-irinotecan DAR 8, or vehicle. Tumor volume, body weight, and general health status were recorded throughout the study.

[0482] Complete tumor regression was observed for the targeted AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 at all dose levels (1, 3, and 5 mg / kg) after a single injection. In contrast, only a minor effect was observed for the non-targeted isotype control isotype-P5(PEG24)-VC-PAB-irinotecan DAR 8 (left) at the highest dose of 5 mg / kg. Figure 36)。This clearly demonstrates the significant effect of targeting NaPi2b with AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8. Comparison with ADCs targeting NaPi2B known in the literature (such as lifastuzumab vedotin) in in vivo models derived from the same OVCAR3 cell line shows that AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 exhibits clear advantages at all tested dose levels. AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 at a single dose as low as 1 mg / kg did not show any tumor recurrence until the end of the observation period, which is very rare compared to other ADCs targeting NaPi2b (even at higher doses and more frequent administrations) in the same OVCAR3 model.

[0483] In vivo efficacy of ADCs in patient-derived xenograft models (PDX):

[0484] All animal experiments were conducted in accordance with the German Animal Welfare Act and approved by the local authorities. Briefly, 3×3 mm samples of patient-derived tumor samples were subcutaneously implanted into the flanks of female immunodeficient NMRI nu / nu mice. Treatment was initiated when the tumors reached an average tumor volume of 0.1 - 0.15 cm 3 . Each group of animals was administered a single intravenous injection of 10 mg / kg AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 or vehicle on day 0. Tumor volume, body weight, and general health status were recorded throughout the study.

[0485] Data clearly demonstrate very high and durable efficacy in patient-derived in vivo tumor models of different tumor types such as lung cancer, endometrial cancer, and ovarian cancer compared to vehicle control ( Figure 37 ).

[0486] In vivo toxicity in cynomolgus monkeys:

[0487] Toxicology studies were conducted in cynomolgus monkeys. Two animals per group were intravenously administered 10 and 20 mg / kg AV25-P5(PEG24)-VC-PAB-irinotecan on days 1 and 22, and a terminal necropsy was performed on day 43. Three analytes in animal plasma samples, including total ADC, intact ADC, and free irinotecan, were analyzed by ELISA and LC / MS-MS. The exposure and pharmacokinetic (PK) parameters of each analyte were evaluated. No significant changes in body weight, clinical chemistry, hematology, and histopathology were observed at these dose levels. The highest non-severe toxic dose (HNSTD) was defined as at least 20 mg / kg in this study.

[0488] For PK analysis, total ADC and intact ADC were measured by ELISA. The first ELISA setup was used to quantify the human antibody (total antibody / total mAb).

[0489] The first ELISA setup was used to quantify the human antibody (total antibody / total mAb). For this purpose, the recombinant purified NaPi2B antigen was immobilized on a protein-binding plate. Samples and standard dilutions were added to the wells, and AV25-P5(PEG24)-VC-PAB-irinotecan would be captured regardless of its DAR. A specific human detection antibody recognizing κ-LC was added to the wells to enable quantification of the total captured antibody.

[0490] The second ELISA setup was designed to specifically measure intact ADC (conjugated with linker-payload). For this purpose, an anti-payload specific antibody was immobilized on the binding plate. Only AV25-P5(PEG24)-VC-PAB-irinotecan with (at least one) conjugated payload molecule could be detected. Samples and standard dilutions were added to the wells, and only payload-conjugated AV25-P5(PEG24)-VC-PAB-irinotecan could be captured. A specific human detection antibody recognizing κ-LC was added to the wells to enable quantification of the total captured antibody.

[0491] The amount of free irinotecan in plasma samples of cynomolgus monkeys treated with AV25-P5(PEG24)-VC-PAB-irinotecan was determined by liquid chromatography mass spectrometry (LCMS). A quantitative method was established on an Xevo G2-XS qTOF instrument using the MRM (multiple reaction monitoring) acquisition mode and specific mass transitions of irinotecan. Deuterated irinotecan-d5 was used as an internal standard in all measurements. To quantify the amount of free irinotecan in serum samples from different treatment groups and time points, a liquid extraction operation was performed to separate the protein components from the lipophilic small molecules. To prepare the calibration curve and control samples, pre-dilutions of irinotecan and irinotecan-d5 were prepared at concentrations of 100 nM and 10 nM respectively in ACN:H2O 1:1. This analysis was performed according to the D×D quantitative analysis previously described by Nagai et al. 2018.

[0492] PK analysis showed a good exposure curve, with no enhanced in vivo clearance in cross-reactive species ( Figure 38 ).

[0493] In this study, the HNSTD (highest non-severe toxic dose) was defined as at least 20 mg / kg. This dose was at least 6-fold higher than the doses explored in toxicology studies of other ADCs targeting NaPi2b ( Figure 38 ).

[0494] This significant improvement in ADC tolerability and excellent PK properties can be attributed to the improved properties of the antibody clones described herein, such as the reduced aggregation tendency of the AV25 clone described above. Antibody and ADC aggregation have been shown to be one of the major off-target toxicity drivers of ADCs.

[0495] Example 3: Further Characterization of AV25 mAb and ADCs Based Thereon

[0496] For all data shown, if not explicitly labeled as AV25-wt, AV25-P5(PEG24)-VC-PAB-irinotecan and AV25 refer to the LALA mutant form of the AV25 mab.

[0497] Synthesis of Anti-NaPi2b Comparative Antibody

[0498] Synthesize the DNA encoding the light chain (SEQ ID NO:56

[0499]

[0500] shown in italics)

[0501] and the heavy chain (SEQ ID NO:57

[0502]

[0503] shown in italics) (Geneart, Thermo Fisher). Add the signal sequence of the heavy chain (SEQ ID NO:58, MDWTWRILFLVAAATGAHS) and the signal sequence of the light chain (SEQ ID NO:59, MLPSQLIGFLLLWVPASRG). Clone the light chain and heavy chain sequences into the pcDNA3.4-TOPO (Thermo Fisher) expression plasmid. Then, using the Expi-CHO transfection system (Thermo Fisher), co-transfect the pcDNA3.4 expression plasmids (Thermo Fisher) encoding the heavy chain and light chain of the corresponding sequences into cells at a ratio of 1:1 for transient expression of the antibody in Expi-CHO-S cells (ThermoFisher). Harvest the cells by centrifugation at 300g for 5 minutes at 4°C. To remove particulates from the supernatant, centrifuge the supernatant at 4000–5000g for 30 minutes at 4°C. For further clarification, pass the supernatant through a 0.22 μm filter. Purify the antibody from the clarified and filtered supernatant by protein A chromatography.

[0504] Synthesis of Anti-NaPi2b Comparative ADC

[0505] Mix 50 μl of a solution of Dulbecco's-PBS (Merck KGaA) containing 10 mg / mL of anti-NaPi2b comparator antibody (66.67 μM) with 1.33 μl of TCEP solution (0.5 mM, in buffer solution, Merck KGaA diluted to 10 mM with PBS, 4 equivalents of TCEP relative to the antibody). After incubating at room temperature for 30 minutes, add 1.66 μl of a solution of 20 mM maleimidopropionyl valine citrulline p-aminobenzyl carbamate-monomethyl auristatin E (MC-VC-PAB-MMAE) in DMSO (10.0 equivalents relative to the antibody). Shake the mixture at room temperature (25 °C) at 350 RPM for 1 hour. Purify the reaction mixture by preparative size exclusion chromatography using 25 ml of Superdex TM 200 Increase 10 / 300GL (Cytiva, Sweden) and elute with sterile PBS (Merck, Germany) at a flow rate of 0.8 ml / min. Combine the fractions containing the antibody and concentrate by spin filtration ( Ultra-2mL MWCO: 30 kDa, Merck, Germany) and analyze by MS as described above. MS analysis was performed as described in Example 1.

[0506] Figure 39 MS analysis of the anti-NaPi2b comparator ADC synthesized by the above method is shown. Signals are labeled with mass in daltons and absolute intensity. An exemplary spectrum from the conjugation reaction is shown. The ratio of the finally conjugated drug to the antibody was estimated from the MS signals to be 3.5 to 4.0. LC: light chain of the anti-NaPi2b comparator antibody, HC: heavy chain of the anti-NaPi2b comparator antibody.

[0507] Binding to human NaPi2a, NaPi2b, and NaPi2c was evaluated by flow cytometry

[0508] To determine concentration-dependent binding, HEK293 cells transiently or stably expressing human Napi2a-, Napi2b-, and Napi2c-mCherry were incubated with the antibody or ADC at concentrations ranging from 0.002 to 200 nM, stained with an Alexa-dye-labeled anti-human IgG H+L secondary antibody (Thermo Fisher Scientific), and analyzed by flow cytometry. The mean fluorescence intensity (MFI) ratio was normalized against a non-specific binding control. The assay was performed in duplicate, and data points were analyzed by non-linear regression using Prism9 software and a single-point specific binding model. The mean ± SD for n = 2 is shown in the figure.

[0509] In this experiment, the specificity of AV25 and AV25-P5(PEG24)-VC-PAB-irinotecan for NaPi2b was determined. Notably, AV25 only showed binding to HEK293 cells overexpressing NaPi2b (SLC34A2), but not to HEK293 cells overexpressing Napi2a (SLC34A1) or Napi2c (SLC34A3). Both Napi2a and Napi2c are members of the SLC34 family and have 45.5 - 50.9% sequence homology with NaPi2b.

[0510] This experiment clearly demonstrated the highest selectivity of AV25 for the target NaPi2b relative to other NaPi2 proteins. This highlights an important feature of the antibodies disclosed herein for selective tumor targeting.

[0511] Figure 40 Binding of unmodified AV25 mAb and AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 ADC, expressed as MFI ratio, to HEK293 cells transfected with Napi2a (left), Napi2b (middle), and Napi2c (right) is shown. AV25 mAb and AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 ADC specifically bind to NaPi2b. n = 2 ± SD is shown in the figure (note that the error bars are too small to be visible).

[0512] In vitro cytotoxicity of synthetic anti-NaPi2b comparator ADC and AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 evaluated by resazurin assay

[0513] To study the direct cytotoxicity of the 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 the ADC (0 - 12 μg / ml) in the medium for 7 days to generate a dose-response curve. Before the viability assay, the supernatant was removed from the adherent cells and replaced with fresh medium. Thereafter, resazurin (Sigma-Aldrich) was used as a cell viability dye and the killing was analyzed at a final concentration of 55 μM. Fluorescence emission at 590 nM was measured on a microplate reader Infinite M200 Pro (Tecan). Cell viability was measured by dividing the fluorescence of the cells treated with the ADC by the fluorescence of the control cells treated only with the medium in the same manner. The mean ± SEM of n = 2 is shown in the figure.

[0514] In this experiment, the cytotoxicity of AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 targeting NaPi2b was studied compared to an anti-NaPi2b comparator ADC on NaPi2b 高 -expressing cell lines. Against HCC-78, AV25-P5(PEG24)-VC-PAB-irinotecan showed a significant increase in selective potency, with an IC50 concentration 15.5-fold lower than that of the anti-NaPi2b comparator ADC. No non-specific toxicity was observed on target-negative cells.

[0515] Figure 41 Cytotoxicity-dose responses of AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 and an anti-NaPi2b comparator ADC on 3 different cell lines are shown. Mean and SD of two measurements are shown, as well as dose-response fits. The % fluorescence of the media control equals the % of live cells.

[0516] In vitro inhibition of topoisomerase-I by irinotecan delivered by ADC

[0517] As detected by the accumulation of cleaved PARP, active caspase 3, and phosphorylated histone 2AX (pH2AX), topoisomerase-I inhibition resulting from the delivery of irinotecan by the AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 ADC induced DNA damage. OVCAR-3 and HCC-78 cells (both NaPi2b 高 ) were treated with increasing concentrations (0.05–12 μg / ml) of AV25-P5(PEG24)-VC-PAB-irinotecan or isotype control (isotype-P5(PEG24)-VC-PAB-irinotecan) for 72 h, cells were stained with a live / dead stain (Thermo Fisher Scientific), and after fixation and permeabilization using a fixation / permeabilization kit (BD Biosciences), analysis was performed by flow cytometry for DNA damage markers - active caspase-3, cleaved PARP, and pH2A.X (Ser-139) (all from BD Biosciences). Mean ± SEM of n = 2 is shown in the figure.

[0518] As shown by the increase in cleaved PARP-, active caspase 3-, and pH2AX-positive HCC-78 (A) and OVCAR-3 (B) cells, AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 induced a concentration-dependent accumulation of DNA damage, while the isotype control remained inactive.

[0519] This experiment clearly demonstrated that the ADC selectively delivered irinotecan to target cells and induced cell killing through topoisomerase-I inhibition.

[0520] Figure 42 A dose-dependent induction of DNA-damage and apoptosis markers in response to increasing concentrations of AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 treatment was shown. The corresponding non-targeting isotype control conjugate was included in the experiment as a negative control. HCC-78 cells (A, NaPi2b 高 ) and OVCAR-3 cells (B, NaPi2b 高 ) were treated with increasing concentrations (0.05–12 μg / ml) of AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 or isotype ADC (isotype-P5(PEG24)-VC-PAB-irinotecan DAR 8) for 72 h, cells were stained for cleaved PARP (left), Caspase 3 (middle) and pH2AX (right), and analyzed by flow cytometry. Mean ± SEM of n = 2 is shown in the figure.

[0521] Interaction with complement factor C1q

[0522] To reduce or even prevent the interaction of the IgG1 backbone AV25 antibody with complement factors or Fc receptors (FcR) that may trigger unwanted immune activation and / or FcR-mediated cellular uptake, the Fc portion of the AV25 antibody and the derived ADC was silenced by two-point mutations L234A and L235A (LALA). AV25 - P5(PEG24) - VC - PAB - Irinotecan DAR8 of the Fc portion.

[0523] In this experiment, LALA-silenced AV25

[0524] (HC-LALA: SEQ ID NO:60

[0525]

[0526] HC shown in italics)

[0527] was compared with Fc wild-type AV25 (HC-wt: SEQ ID NO:61:

[0528]

[0529]

[0530] The interaction of HC (shown in italics) with complement factor C1q was analyzed. The C1q interaction study was performed using the HTRF Human C1q Binding Kit (Cisbio) according to the manufacturer's instructions. Briefly, AV25 HC-wt and HC-LALA, standards, and an anti-MHC-I positive control (Invivogen) known to interact with C1q were captured and aggregated by streptavidin-conjugated anti-human IgG Fab-biotin, which binds to streptavidin-labeled d2 (fluorescent acceptor). If the antibody binds human C1q, an anti-C1q antibody labeled with europium cryptate (fluorescent donor) can come into close proximity to the fluorescent acceptor and trigger fluorescence resonance energy transfer (FRET). The fluorescence emission intensity at 665 nm was measured on a microplate reader Infinite M200 Pro (Tecan). The mean ± SD for n = 2 is shown in the figure.

[0531] Although the binding ability of AV25 HC-wt to C1q was comparable to that of the standards and the anti-MHC-I positive control, for AV25 HC-LALA, the interaction with C1q completely disappeared. This reduction in the interaction with C1q is expected to reduce the unwanted activation of the innate immune system.

[0532] Figure 43 The binding of the Fc region of the AV25 HC-LALA antibody to the recombinant hexameric C1q complement protein was shown relative to the AV25 HC-wt antibody, which was measured according to the manufacturer's instructions in a HTRF (homogeneous time-resolved fluorescence)-based human C1q binding assay (HTRF Human C1q Binding Kit, Cisbio). Briefly, serial dilutions of all test antibodies (AV25 HC-wt, AV25 HC-LALA, α-MHC-I positive, IgG1 kit standard) from 280 nM to 11.6 nM were measured. The HTRF ratio was calculated by dividing the receptor emission signal at 665 nm by the donor emission signal at 620 nm and multiplying by 10,000. The HTRF ratio at a concentration of 70 nM is shown in the figure, where the background (diluent only) HTRF ratio was subtracted. The mean ± SD for n = 2 is shown in the figure.

[0533] Interaction with FcR

[0534] To reduce or even prevent the interaction of the IgG1 backbone AV25 antibody with complement factors or Fc receptors (FcR) that may trigger unwanted immune activation and / or FcR-mediated cellular uptake, the Fc portion of the AV25 antibody and the derived ADC AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 was silenced by two-point mutations L234A and L235A (LALA).

[0535] In this experiment, the interaction of LALA-silenced AV25 (HC-LALA: SEQ ID NO.62:

[0536]

[0537]

[0538] HC shown in italics)

[0539] and AV25-P5(PEG24)-VC-PAB-irinotecan DAR8 with respect to Fc-wild-type AV25 (HC-wt: SEQ ID NO:63,

[0540] HC shown in italics) with Fc gamma receptors (FcγR) was analyzed. The Lumit TM 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, was used to conduct the FcγR interaction study according to the manufacturer's instructions. Briefly, AV25 HC-wt, HC-LALA and AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8, standards and trastuzumab as a positive control were incubated with Tracer-LgBiT and FcγR-SmBiT. In the absence of the antibody analyte, or if the tested antibody does not interact with FcγR, Tracer-LgBiT binds to the FcγR-SmBiT target, generating the maximum luminescence signal. In the case of successful interaction with FcγR, the tested antibody / ADC will compete with Tracer-LgBiT for binding to the FcγR target, resulting in a concentration-dependent decrease in the luminescence signal. The luminescence signal was measured on a microplate reader Infinite M200Pro (Tecan). n = 1 in the figure.

[0541] Although the binding of AV25 HC-wt to all FcγRs is comparable to that of the IgG1 positive control, for AV25 HC-LALA and AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8, their interaction with FcγRs is greatly reduced or completely eliminated in most cases. Interestingly, compared with the positive control, the positive control trastuzumab even shows a reduced interaction with all FcγRs. In the FcγRn interaction experiment, murine AV25 was used as another negative control.

[0542] This experiment clearly shows that the introduction of the LALA mutation into the AV25 antibody mediates a reduction in the unwanted interaction with FcγRI (CD64), CD16, and CD32. The reduction in interaction with these receptors is expected to reduce the unwanted uptake of AV25-related ADC conjugates into non-target cells, thereby reducing unwanted toxicity. The interaction with FcRn is only slightly reduced.

[0543] Figure 44 The dose-dependent binding of the Fc regions of the AV25-HC-LALA antibody and the AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 ADC to recombinant human FcRn and FcγRs relative to the AV25-HC-wt antibody was shown, using Lumit TM FcγR binding immunoassay (FcγRn, FcγRI, FcγRIIa / CD32 R131 / H131 polymorphism, FcγRIIIa / CD16 V158 / F158 polymorphism; Promega), measured according to the manufacturer's instructions. Serial dilutions of AV25 HC-wt, AV25 HC-LALA, and AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8, standards, and trastuzumab as a positive control were incubated with Tracer-LgBiT and FcγR-SmBiT. In the absence of the antibody analyte, or if the tested antibody does not interact with FcγR, Tracer-LgBiT binds to the FcγR-SmBiT target, generating a maximum luminescence signal. In the case of successful interaction with FcγR, the tested antibody / ADC will compete with Tracer-LgBiT for binding to the FcγR target, resulting in a concentration-dependent decrease in the luminescence signal. Luminescence was measured on a microplate reader Infinite M200Pro (Tecan). n = 1 in the figure.

[0544] ADCC

[0545] To reduce or even prevent the interaction of the IgG1 backbone AV25 antibody with complement factors or Fcγ receptors (FcRγ) that may trigger unwanted immune activation and / or FcR-mediated cellular uptake, the Fc portion of the AV25 antibody and the derived ADC AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 was silenced by two-point mutations L234A and L235A (LALA).

[0546] In this experiment, the Fc-wild-type AV25 mAb (HC-wt: SEQ ID NO:64,

[0547]

[0548]

[0549] HC shown in italics)

[0550] and the LALA-silenced AV25 mAb (HC-LALA: SEQ ID NO:65,

[0551]

[0552] HC shown in italics)

[0553] and the antibody-dependent cellular cytotoxicity (ADCC) of AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 relative to isotype and anti-MHC-I positive controls (Invivogen), which is mediated after interaction with FcγRIIIa (CD16). For the calcein release-based antibody-dependent cellular cytotoxicity (ADCC) assay, LeucosSep tubes (Greiner Bio-One) and -1077 (density 1.077 g / ml, Merck) density gradient method was used to isolate peripheral blood mononuclear cells (PBMCs) from the buffy coat of healthy donors (purchased from DONAS GmbH) according to the standard protocol. Then, according to the manufacturer's instructions, natural killer (NK) cells were MACS (magnetic cell separation) sorted by negative selection using a human NK cell isolation kit (Miltenyi Biotec) to obtain non-contact (untouched) human primary NK cells. NaPi2b-positive target cells OVCAR-3 and HCC-78 (both NaPi2b 高)。In the presence of 15 μg / ml antibody or ADC, 40,000 NK cells and 10,000 calcein-stained target cells were incubated at a ratio of 4:1. Cells permeabilized with 2.5% Triton X 100 (Merck) were used as a positive control for maximum calcein release. After 4 hours, the supernatant was transferred to a flat-bottom black non-binding 96-well plate (Greiner Bio-One), and the fluorescence intensity was measured at 485 / 535 nM by an Infinite M200 Pro reader (Tecan). The percentage of specific lysis was calculated by subtracting the background calcein release (NK + target) from the calcein released by antibody-mediated killing and dividing by the calcein released by Triton X-permeabilized cells (maximum lysis) minus the background calcein release (target only). The mean ± SEM for n = 2 is shown in the figure.

[0554] Anti-MHC-I positive control and AV25 HC-wt triggered high ADCC activity in human NK cells, while all LALA-silenced antibodies and ADCs (AV25 HC-LALA, AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 and isotype control) did not induce ADCC or induced only very minimal ADCC. The expected reduction in ADCC may reduce the unwanted toxicity of AV25-related antibodies.

[0555] Figure 45 An antibody-dependent cell cytotoxicity (ADCC) assay based on calcein release is shown. Co-cultures of NK cells derived from healthy donors (HD) and calcein-stained target positive tumor cells (OVCAR-3 and HCC-78) at a ratio of 4:1 were incubated with 15 μg / ml of the indicated antibody or ADC (anti-MHC-I antibody was used as a positive control). The percentage of specific lysis was calculated by dividing the calcein released by antibody-mediated cell killing by the calcein released by Triton X-permeabilized cells (maximum lysis). The mean ± SEM for n = 2 is shown in the figure.

[0556] AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 dose response and exposure PDX study (Lu7700)

[0557] All animal experiments were conducted in accordance with the German Animal Welfare Act and approved by the local authorities. Briefly, 3×3 mm samples of patient-derived tumor samples (Lu7700 non-small cell lung cancer (NSCLC) model, EPO Experimentelle Pharmakologie&Onkologie Berlin-Buch GmbH) were subcutaneously implanted into the flanks of female immunodeficient NMRI nu / nu mice. Treatment was initiated when the tumors reached an average tumor volume of 0.1 - 0.15 cm 3 Each group of animals was administered a single intravenous injection of 5, 3, or 1 mg / kg AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8, 5 mg / kg isotype-P5(PEG24)-VC-PAB-irinotecan DAR8, or vehicle on day 0. Tumor volume, body weight, and general health status were recorded throughout the study.

[0558] Data demonstrated that all tested ADC dose levels showed the highest and most durable efficacy in an in vivo NSCLC PDX model compared to the vehicle control. Taking the non-targeted isotype control ADC group at the highest dose as an example, the above effect was highly specific for the targeting anti-TPBG antibody.

[0559] Figure 46 Results of in vivo efficacy analysis of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8), a representative ADC of the present invention, in a patient-derived NSCLC xenograft model (PDX, Lu7700) are shown. Shown on the left is the change in tumor volume over time relative to the untreated (vehicle) group after a single administration on day 0 with different dose levels of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8) (1, 3, and 5 mg ADC / kg body weight), isotype control (5 mg / kg) carrying the same amount of linker-payload. Shown on the right is the body weight of animals treated with different dose levels of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8), isotype control carrying the same amount of linker-payload relative to the untreated (vehicle) animals. All results are presented as the mean and SEM of 4 animals per group.

[0560] AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 Dose Response and Exposure PDX Study (Ov6668)

[0561] All animal experiments were conducted in accordance with the German Animal Welfare Act and approved by the local authorities. Briefly, 3×3 mm samples of patient-derived tumor samples (Ov6668 ovarian cancer model, EPO Experimentelle Pharmakologie&Onkologie Berlin-Buch GmbH) were subcutaneously implanted into the flanks of female immunodeficient NMRI nu / nu mice. Treatment was initiated when the tumors reached an average tumor volume of 0.1 - 0.15 cm 3 . Animals in each group were administered a single intravenous injection of 5, 3, or 1 mg / kg AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 or vehicle on day 0. Tumor volume, body weight, and general health status were recorded throughout the study.

[0562] Data demonstrated that all tested ADC dose levels showed the highest and most durable efficacy in the PDX in vivo model compared to the vehicle control. Dose proportionality of exposure had been demonstrated in the PK data. PK analysis had been completed as described in Example 1.

[0563] Complete tumor regression was observed at 5 and 3 mg / kg dose levels of the targeted AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 in ovarian cancer PDX after a single injection. The 1 mg / kg dose also showed strong tumor regression in the ovarian cancer model, followed by partial tumor regrowth after day 40. PK analysis demonstrated dose-proportional exposure profiles and high stability of the AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 ADC at the tested dose levels of 5, 3, and 1 mg / kg.

[0564] Data demonstrated that AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 at all tested ADC dose levels had high and durable efficacy in the PDX in vivo model compared to the vehicle group. The lack of body weight loss indicated the highest tolerance. PK analysis demonstrated dose-proportional exposure profiles and high stability of the AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 ADC at the tested dose levels of 5, 3, and 1 mg / kg.

[0565] Figure 47The results of the in vivo efficacy analysis of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8), which is a representative ADC of the present invention, in a patient-derived ovarian cancer xenograft model (PDX, Ov6668) are shown in Figure A. Shown on the left is the change in tumor volume over time relative to the untreated (vehicle) group after a single administration of different dose levels of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8) (1, 3, and 5 mg ADC / kg body weight) on day 0. Shown on the right is the body weight of animals treated with different dose levels of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8) relative to the untreated (vehicle) animals. All results are presented as the mean and SEM of 9 animals per group. Figure B shows the in vivo PK evaluation of total antibody and intact ADC at three dose levels in a dose-response efficacy study, and the results are given as the mean and SD of 3 measurements at each time point for mice that were administered different dose levels of AV25-P5(PEG24)-VC-PAB-irinotecan (1, 3, and 5 mg ADC / kg body weight) on day 0. The PK analysis was completed as described in Example 1.

[0566] AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 PDX study (10 mg / kg)

[0567] This figure shows the results of the in vivo efficacy analysis of a single dose of 10 mg / kg of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8), which is a representative ADC of the present invention, or isotype-AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8) in three patient-derived ovarian cancer xenograft models (PDX) with different NaPi2b expression levels and BRCA mutation statuses. The PDX experiments were conducted as described in Example 2.

[0568] The data clearly demonstrate that, compared to the vehicle control, there is very high and durable efficacy in patient-derived in vivo models of ovarian cancer tumors, even in very refractory BRCA-mutated PDX models and models showing low NaPi2b target expression.

[0569] Figure 48Shows the in vivo efficacy analysis results of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8), which is a representative ADC of the present invention, in a patient-derived ovarian cancer xenograft model. Shown at the top is the change in tumor volume over time relative to the untreated (vehicle) group after a single administration of 10 mg ADC / kg body weight of AV25-P5(PEG24)-VC-PAB-irinotecan (DAR 8) or isotype-P5(PEG24)-VC-PAB-irinotecan (DAR 8) on day 0. Shown at the bottom are BRCA-mutant models with low NaPi2B expression (lower left) and high NaPi2B expression (lower right). All results are presented as the mean and SEM of 3 animals per group.

[0570] Binding to NaPi2b-expressing cell lines OVCAR-3 and HCC-78 evaluated by flow cytometry - Upifitamab vs AV25

[0571] Upifitamab is an antibody having the same sequence as the parental mAb of the present disclosure. Upifitamab was purchased from MedChemExpressed and used for the following experiments.

[0572] To determine concentration-dependent binding, NaPi2b-positive OVCAR-3 and HCC-78 were incubated with antibodies at concentrations ranging from 0.00075 to 30 μg / ml, stained with an Alexa-dye-labeled anti-human IgG H+L secondary antibody (Thermo Fisher Scientific), and analyzed by flow cytometry. The mean fluorescence intensity (MFI) ratio was normalized against a non-specific binding control. The assay was performed in duplicate, and data points were analyzed by non-linear regression using Prism 9 software and a single-point specific binding model. Shown in the figure are the mean ± SD for n = 2.

[0573] In this experiment, the binding of AV25 and upifitamab on NaPi2b-positive cell lines was compared, and the K D values were determined. Notably, compared to upifitamab (K D of 1.11 μg / ml on OVCAR-3 and K D of 1.27 μg / ml on HCC-78), AV25 (K D = 0.44 μg / ml on OVCAR-3 and K D of 0.42 μg / ml on HCC-78) showed improved binding and lower K D values.

[0574] This experiment clearly demonstrated the binding superiority of AV25 over Upifitamab.

[0575] Figure 49 Dose-dependent binding of increasing concentrations of Upifitamab or AV25, normalized to non-specific binding control, is shown on two different NaPi2b-positive cell lines (OVCAR-3, left, HCC78, right), expressed as MFI ratio. Mean and SD of two measurements are shown, as well as dose-response fitting.

[0576] In vitro cytotoxicity evaluated by resazurin assay: Upifitamab-P5(PEG24)-VC-PAB-irinotecan vs AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8

[0577] Upifitamab is an antibody having the same sequence as the parental mAb of the present disclosure. Upifitamab was purchased from MedChemExpressed and used for the following experiments. As described in Example 1, Upifitamab was further conjugated with P5(PEG24)-VC-PAB-irinotecan and purified to a DAR 8 conjugate.

[0578] To study the direct cytotoxicity of the ADCs, 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 ADCs (0 - 12 μg / ml) in medium for 7 days to generate a dose-response curve. Before viability analysis, the supernatant was removed from adherent cells and replaced with fresh medium. Thereafter, resazurin (Sigma-Aldrich) was used as a cell viability dye and cytotoxicity was analyzed at a final concentration of 55 μM. Fluorescence emission at 590 nM was measured on a microplate reader Infinite M200 Pro (Tecan). Cell viability was measured by dividing the fluorescence of cells treated with ADC by the fluorescence of control cells treated with medium only in the same manner. Mean ± SEM of n = 2 is shown in the figure.

[0579] In this experiment, AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 against NaPi2b was studied 高The cytotoxicity of the expressed cell lines (OVCAR-3 and HCC-78) was compared head-to-head with Upifitamab-P5(PEG24)-VC-PAB-irinotecan DAR 8. In both cell lines, AV25-P5(PEG24)-VC-PAB-irinotecan had an IC50 concentration 3.5 to 3.8 times lower than that of upifitamab-P5(PEG24)-VC-PAB-irinotecan, indicating an increase in selective potency. No non-specific toxicity was observed for the non-targeting isotype control.

[0580] This experiment clearly demonstrated the killing superiority of AV25-P5(PEG24)-VC-PAB-irinotecan compared to upifitamab-P5(PEG24)-VC-PAB-irinotecan, indicating an increased response to targeted therapy with the AV25 antibody-based agent in cancer patients compared to the parental mAb or Upifitamab.

[0581] Figure 50 The cytotoxicity dose-response of AV25-P5(PEG24)-VC-PAB-irinotecan DAR 8 compared to Upifitamab-P5(PEG24)-VC-PAB-irinotecan DAR 8 and the isotype control Isotype-P5(PEG24)-VC-PAB-irinotecan DAR 8 was shown on two different cell lines. The mean and SD of two measurements were shown, as well as the dose-response fit. The % fluorescence of the media control was equal to the % of live cells.

[0582] ***

[0583] Those skilled in the art will readily appreciate that the present invention is well-suited to achieving the stated objectives and obtaining the mentioned results and advantages, as well as those inherent therein. Additionally, various substitutions and modifications can be made to the invention disclosed herein without departing from the scope and spirit of the invention, which will be apparent to those skilled in the art. The compositions, methods, processes, treatments, molecules, and specific compounds described herein are representative of some of the presently exemplary embodiments and are not intended to limit the scope of the invention. Variations and other uses will occur to those skilled in the art, which are encompassed within the spirit of the invention as defined by the scope of the claims. The listing or discussion of previously published documents in this specification should not necessarily be construed as an admission that such documents are part of the prior art or common general knowledge.

[0584] The invention described illustratively herein may be practiced in the absence of any element or elements, or one or more limitations not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. should be construed in a broad and non-limiting sense. Additionally, the terminology and expressions employed herein are descriptive rather than limiting, and there is no intention of using such terminology and expressions to exclude equivalents of any shown and described features or portions thereof, but rather it is recognized that various modifications may be subsumed within the scope of the invention as claimed. Accordingly, it should be understood that although the invention has been specifically disclosed by way of exemplary embodiments and optional features, those skilled in the art may seek modifications and variations of the invention as embodied herein, and such modifications and variations are considered to be within the scope of the invention.

[0585] The invention has been described herein in general terms and generically. Each narrower genus and subgeneric group falling within the scope of the general disclosure forms part of the invention. This includes the general description of the invention with additional conditions or negative limitations removing any subject matter from the genus, whether or not the removed material is part of that specifically described in the invention. All documents cited herein, including patent applications and scientific publications, are incorporated herein by reference for all purposes.

[0586] Other embodiments are within the following claims. Additionally, when features or aspects of the invention are described in terms of a Markush group, those skilled in the art will recognize that the invention can also be described in terms of any single member or subgroup of members of the Markush group.

Claims

1. An anti-NaPi2b antibody (such as an antibody against NPT2B_human sodium-dependent phosphate transporter 2B (such as having UniProt accession number: O95436 or SEQ ID NO: 1), and / or an antibody against NPT2B_rat sodium-dependent phosphate transporter 2B (such as having UniProt accession number: Q9JJ09 or SEQ ID NO: 2)), wherein the NaPi2b antibody is capable of: a) bind to human NaPi2b (such as SEQ ID NO: 1) and / or rat NaPi2b (such as SEQ ID NO: 2), preferably the binding to the human and rat NaPi2b has approximately the same K D , further preferably the binding to the human and rat NaPi2b is the binding to endogenous NaPi2b (such as located on the cell surface), most preferably, the antibody is selected from: AV-25, AV-15, AV-18, AV-21 and AV-29 antibodies), further most preferably, the approximately the same K D has a difference of up to 50% (such as, 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) cross-react with rat NaPi2b (such as having UniProt accession number: Q9JJ09 or SEQ ID NO: 2), preferably, it is carried out by antigen-mediated antibody internalization; further preferably, compared with the corresponding internalization of the parental antibody (such as, comprising SEQ ID NO: 54 and 55, such as, as shown in Figure 1), the internalization is increased (such as, at least 10%, such as 15%); c) cross-react with cynomolgus monkey (such as Macaca fascicularis) NaPi2b (such as having UniProtKB accession number: A0A2K5UHY1 or SEQ ID NO: 3); d) internalize, preferably internalize by antigen-mediated antibody internalization; and e) does not have a dipeptide deamidation site in CDR2 of the heavy chain variable region (V H ), preferably the missing dipeptide deamidation site is NG (Asn-Gly) in V H CDR2.

2. The anti-NaPi2b antibody according to any one of the preceding claims, wherein the anti-NaPi2b antibody does not have a dipeptide deamidation site in CDR2 of the heavy chain variable region (V H ), preferably the missing dipeptide deamidation site is V HNG (Asn-Gly) in CDR2.

3. The anti-NaPi2b antibody according to any one of the preceding claims, wherein the anti-NaPi2b antibody is an antibody comprising: a) a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence having 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%) identity to SEQ ID NO:4, and the light chain variable region having an amino acid sequence having 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%) identity to SEQ ID NO:5; preferably, the anti-NaPi2b antibody is the AV-25 antibody, which comprises: a light chain comprising SEQ ID NO:6 and a heavy chain comprising SEQ ID NO:7; b) a heavy chain variable region and a light chain variable region, the heavy chain variable region having an amino acid sequence having 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%) identity to SEQ ID NO:8, and the light chain variable region having an amino acid sequence having 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%) identity to SEQ ID NO:9; preferably, the anti-NaPi2b antibody is the AV-15 antibody, which comprises: a light chain comprising SEQ ID NO:10 and a heavy chain comprising SEQ ID NO:11; c) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having 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%) identity to SEQ ID NO: 12, and the light chain variable region has an amino acid sequence having 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%) identity to SEQ ID NO: 13; preferably, the anti-NaPi2b antibody is the AV-18 antibody, which comprises: a light chain comprising SEQ ID NO: 14 and a heavy chain comprising SEQ ID NO: 15; d) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having 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%) identity to SEQ ID NO: 16, and the light chain variable region has an amino acid sequence having 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%) identity to SEQ ID NO: 17; preferably, the anti-NaPi2b antibody is the AV-21 antibody, which comprises: a light chain comprising SEQ ID NO: 18 and a heavy chain comprising SEQ ID NO: 19; e) A heavy chain variable region and a light chain variable region, wherein the heavy chain variable region has an amino acid sequence having 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%) identity to SEQ ID NO:20, and the light chain variable region has an amino acid sequence having 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%) identity to SEQ ID NO:21; preferably, the anti-NaPi2b antibody is the AV-29 antibody, which comprises: a light chain comprising SEQ ID NO:22 and a heavy chain comprising SEQ ID NO:

23.

4. The anti-NaPi2b antibody according to any one of the preceding claims, wherein the anti-NaPi2b antibody is: a) An antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO:24, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:25, and a heavy chain CDR3 having an amino acid sequence as shown in SEQ ID NO:26, and the light chain variable region comprises a light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:27, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:28, and a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO:29; b) An antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 having an amino acid sequence as shown in SEQ ID NO:30, a heavy chain CDR2 having an amino acid sequence as shown in SEQ ID NO:31, and a heavy chain CDR3 having an amino acid sequence as shown in SEQ ID NO:32, and the light chain variable region comprises a light chain CDR1 having an amino acid sequence as shown in SEQ ID NO:33, a light chain CDR2 having an amino acid sequence as shown in SEQ ID NO:34, and a light chain CDR3 having an amino acid sequence as shown in SEQ ID NO:35; c) An antibody comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises 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 the light chain variable region comprises 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 and a light chain variable region, wherein the heavy chain variable region comprises 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 the light chain variable region comprises 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 and a light chain variable region, wherein the heavy chain variable region comprises 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 the light chain variable region comprises 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.

5. The anti-NaPi2b antibody according to any one of the preceding claims, wherein the anti-NaPi2b antibody has one or more of the following characteristics: a) A monoclonal antibody; b) A chimeric antibody and / or a humanized antibody; c) Specifically recognizes NaPi2b overexpressed on cancer cells; d) A human IgG antibody, preferably a human IgG1 antibody; e) Comprises a kappa (κ) light chain; f) Comprises a lambda (λ) light chain; g) Comprising Fc silent mutations, such as the substitution of leucine (L) with alanine (A) at positions 234 and 235 (LALA mutation), wherein said LALA mutation is capable of reducing immune cell effector functions; h) Capable of being internalized by target cells (such as cancer cells) expressing NaPi2b; preferably, said internalized antibody is directed to lysosomes; i) Tumor-selective antibody, preferably said tumor is a liquid and / or solid tumor; j) Malignant cell-selective antibody; k) Binding to said human and / or rat NaPi2b in a glycosylation-dependent manner, wherein said antibody binds to the glycosylated form of the NaPi2b protein; l) (e.g., in OVCAR-3 cells (such as HTB-161#, ATCC) endogenously expressing Napi2b) the K of endogenously expressed human NaPi2b D is about 0.01 to about 10 nmol / L, preferably about 1 to about 10 nmol / L, more preferably about 1 to about 7 nmol / L, further preferably about 1 to about 4 nmol / L, more preferably, said K D is measured by means of FACS assay, and even more preferably, said K D is about 2.661 to about 6.644 nmol / L; m) Optionally, the K for immobilized exogenous full-length NaPi2b and / or one or more of its fragments D is about 0.01 to about 10 nmol / L, preferably, said one or more fragments comprise at least one extracellular domain (ECD) of said NaPi2b (e.g., said ECD comprises amino acids 122 - 135 and / or amino acids 235 - 361 and / or 429 - 485 and / or amino acids 547 - 552 of human Napi2b having SEQ ID NO:1) and / or one or more fragments of said ECD (such as having a length of about 15 to about 30 amino acids), wherein said full-length Napi2b and / or one or more of its fragments are fused or not fused with one or more protein tags (such as 6×His tag, FLAG, HA, V5, Fc-fusion, MBP, SUMO, TEV, GFP, TST), preferably, said K D is measured by means of ELISA assay, preferably about 0.05 to about 0.2 nmol / L, and even more preferably, said K D is about 0.071 to about 0.147 nmol / L; n) Cross-reacting with rat NaPi2b (such as having UniProt accession number: Q9JJ09 or SEQ ID NO:2); and / or o) In the complementarity-determining region 2 (CDR2) of the heavy-chain variable region (V H ), there is no dipeptide deamidation site, and preferably, the missing dipeptide deamidation site is NG (Asn-Gly) in V H CDR2.

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

7. A nucleic acid that encodes an antibody according to any one of the preceding claims.

8. An expression vector that contains at least one nucleic acid molecule according to any one of the preceding claims.

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

10. An antibody-drug conjugate (ADC) that contains an anti-NaPi2b antibody according to any one of the preceding claims.

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

12. The composition according to any one of the preceding claims, wherein the composition is a pharmaceutical composition and / or a diagnostic composition.

13. A method for treating, ameliorating, preventing, and / or diagnosing cancer, preferably, the cancer is solid cancer and / or metastatic cancer, and further preferably, the cancer is selected from: lung cancer, ovarian cancer, thyroid cancer, non-squamous non-small cell lung cancer, non-mucinous ovarian cancer, papillary thyroid cancer, renal cancer, endometrial cancer, uterine cancer, ureteral cancer, bladder cancer, and fallopian tube cancer. The method includes: Administer a therapeutically or prophylactically effective amount of an antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition or kit according to any one of the preceding claims.

14. A method for producing an antibody-drug conjugate (ADC), the method includes: Conjugate an antibody according to any one of the preceding claims with one or more (such as 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, further most preferably 6 to 10, further most preferably 7 to 10, further most preferably 4 or 8, most preferably 8) cytotoxic moieties (such as a cytotoxic payload, such as a microtubule-disrupting agent, such as a topoisomerase-I inhibitor, such as auristatins or camptothecins, such as MMAE (monomethyl auristatin E) or MMAF (monomethyl auristatin F), such as irinotecan), preferably via one or more linkers, more preferably via one or more phosphonamidate linkers.

15. The antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition, or kit according to any one of the preceding claims, which is used as a drug and / or for treatment.

16. The antibody-drug conjugate (ADC), nucleic acid, host cell, composition, or kit according to any one of the preceding claims, which is used in one or more of the following methods: (a) A method for treating, ameliorating, preventing, and / or diagnosing cancer, preferably wherein the cancer is solid cancer and / or metastatic cancer, and further preferably wherein the cancer is selected from: lung cancer, ovarian cancer, thyroid cancer, non-squamous non-small cell lung cancer, non-mucinous ovarian cancer, papillary thyroid cancer, renal cancer, endometrial cancer, uterine cancer, ureteral cancer, bladder cancer, and fallopian tube cancer; (b) A method for monitoring cancer progression and / or for evaluating the efficacy of cancer therapy; (c) A method for screening candidate compounds having anti-cancer activity; (d) A method for altering the chemoresistance of cancer cells; (e) A method for sensitizing cancer cells to chemotherapy; (f) A method for inhibiting the growth of cancer cells expressing NaPi2b; (g) A method for producing or preparing an antibody; (h) A method for immunizing a non-human animal; (i) A method for preparing a hybridoma; (j) A method according to any one of the preceding claims; (k) A method according to any one of (a)-(j), wherein the method is an in vivo, in vitro, or ex vivo method.

17. Use of an antibody, antibody-drug conjugate (ADC), nucleic acid, expression vector, host cell, composition, or kit according to any one of the preceding claims for one or more of the following: (a) Treating, ameliorating, preventing, and / or diagnosing cancer, preferably wherein the cancer is solid cancer and / or metastatic cancer, and further preferably wherein the cancer is selected from: lung cancer, ovarian cancer, thyroid cancer, non-squamous non-small cell lung cancer, non-mucinous ovarian cancer, papillary thyroid cancer, renal cancer, endometrial cancer, uterine cancer, ureteral cancer, bladder cancer, and fallopian tube cancer; (b) Monitoring cancer progression and / or evaluating the efficacy of cancer therapy; (c) Screening candidate compounds having anti-cancer activity; (d) Altering the chemoresistance of cancer cells; (e) Sensitizing cancer cells to chemotherapy; (f) Inhibiting the growth of cancer cells expressing NaPi2b; (g) Production or preparation of an antibody; (h) Immunizing a non-human animal; (i) Preparation of a hybridoma; (j) In a method according to any one of the preceding claims; (k) Use according to any one of (a)-(j), wherein the use is an in vivo, in vitro, or ex vivo use.

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