Anti-NaPi2b antibodies and methods of use

Abstract

Described herein are antibody constructs that bind to human NaPi2b (or SLC34A2), and antibody-drug conjugates (ADCs) comprising anti-NaPi2b antibody constructs conjugated to drugs such as cytotoxins or immunomodulators, and their use as therapeutic or diagnostic agents, such as in the treatment or diagnosis of cancer.

Description

[Technical Field]

[0001] Field The present disclosure relates to the field of antibody therapeutics, and in particular to antibodies that target human sodium-dependent phosphate transporter 2B (hNaPi2b). [Background technology]

[0002] background Sodium-dependent phosphate transporter 2B (NaPi2b) is a transmembrane protein encoded by the SLC34A2 gene. The NaPi2b polypeptide is 690 amino acids long, with a limited extracellular domain (amino acids 188–361) exposed on the cell surface. It is widely expressed in normal tissues and overexpressed in various cancers, including ovarian, endometrial, and lung cancers.

[0003] Given the overexpression of NaPi2b in certain types of cancer, NaPi2b-targeting agents have been tested in clinical trials for the treatment of cancer, but mixed results have been reported. Mersana Therapeutics conducted a Phase I / II clinical trial testing upifytamavrilsodutin, an antibody-drug conjugate (ADC) consisting of the NaPi2b-targeting antibody MX-35 and an auristatin-F payload (Draflexin platform) in patients with platinum-resistant ovarian cancer or non-small cell lung cancer (NSCLC). The NSCLC arm of the trial was discontinued due to lack of efficacy, but upifytamavrilsodutin received fast track designation for the treatment of platinum-resistant ovarian cancer patients who had received three to four prior lines of therapy. Mersana also completed a Phase I / II clinical trial of XMT-1592 in ovarian cancer. XMT-1592 is a site-specific ADC consisting of the antibody MX-35 conjugated to an auristatin-F payload using its Draflexin platform. However, development of this ADC has been discontinued. Rifastuzumab vedotin, an ADC of rifastuzumab with an MMAE payload, was tested in a clinical trial sponsored by Genentech in patients with ovarian cancer or NSCLC, but the trial was subsequently discontinued.

[0004] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily intended, nor should it be construed, that any of the preceding information constitutes prior art against the claimed invention. Summary of the Invention

[0005] overview

[0003] One aspect of the present disclosure relates to an antibody construct comprising an antigen-binding domain that binds to human NaPi2b (sodium-dependent phosphate transport protein 2B), wherein the antigen-binding domain comprises a heavy chain CDR1 (HCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO:7, a heavy chain CDR2 (HCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO:8, and a heavy chain CDR3 (HCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO:9, as well as a light chain CDR1 (LCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO:19, a light chain CDR2 (LCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO:20, and a light chain CDR3 (LCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO:18.

[0006] Another aspect of the present disclosure pertains to a polynucleotide or set of polynucleotides that encodes the anti-NaPi2b constructs described herein.

[0007] Another aspect of the present disclosure relates to an expression vector or a set of expression vectors comprising a polynucleotide or a set of polynucleotides encoding an anti-NaPi2b antibody construct described herein. Another aspect of the present disclosure relates to a host cell comprising the expression vector or a set of expression vectors.

[0008] Another aspect of the present disclosure relates to antibody-drug conjugates comprising an anti-NaPi2b construct described herein conjugated to one or more drug moieties.

[0009] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an anti-NaPi2b antibody construct described herein or an antibody-drug conjugate described herein, and a pharmaceutically acceptable carrier or diluent.

[0010] Another aspect of the present disclosure relates to an anti-NaPi2b antibody construct described herein or an antibody-drug conjugate described herein for use in therapy, such as the treatment of cancer.

[0011] Another aspect of the present disclosure relates to the use of an anti-NaPi2b antibody construct described herein or an antibody-drug conjugate described herein in the manufacture of a medicament for the treatment of cancer.

[0012] Another aspect of the present disclosure relates to a method of inhibiting the growth of NaPi2b-positive tumor cells, comprising contacting the cells with an anti-NaPi2b antibody construct described herein or an antibody-drug conjugate described herein.

[0013] Another aspect of the present disclosure relates to a method of treating a subject having cancer, comprising administering to the subject an effective amount of an anti-NaPi2b antibody construct described herein or an antibody-drug conjugate described herein. [Brief explanation of the drawings]

[0014] [Figure 1] Figure 1A shows the mouse heavy chain variable domain CDR sequences of the chimeric anti-NaPi2b antibody v23855 grafted onto the human VH germline (IGHV1-46*03), and Figure 1B shows the mouse light chain variable domain CDR sequences of the chimeric antibody v23855 grafted onto the human VL framework (IGKVID-39*01). CDRs are assigned using the AbM definition and are marked in bold and underlined. [Figure 2A] Non-reducing (NR) SDS-PAGE profiles of all humanized variants and the parent chimeric variant 23855 are shown. [Figure 2B] Reducing (R) SDS-PAGE profiles of all humanized variants and the parental chimeric variant 23855 are shown. [Figure 2C] 1 shows the UPLC-SEC profile of the parental mouse-human chimeric antibody v23855. [Figure 2D] 1 shows the UPLC-SEC profile of a representative humanized antibody v29456. [Figure 3A] 1 illustrates the binding of humanized antibody variants v29456, MX-35 (v18992), and rifastuzumab (v18993) to human NaPi2b. [Figure 3B] 1 illustrates the binding of humanized antibody variants v29456, MX-35 (v18992), and rifastuzumab (v18993) to cynomolgus monkey NaPi2b. [Figure 3C] 1 illustrates the binding of humanized antibody variants v29456, MX-35 (v18992), and rifastuzumab (v18993) to mouse NaPi2b. [Figure 4A] N-curve analysis of binding of v29814 to NaPi2b expressed in IGROV-1 cells is shown. For each panel, the right curve shows data for a constant binding partner of 500 pM, and the left curve shows data for a constant binding partner of 50 pM. [Figure 4B] N-curve analysis of binding of v36123 to NaPi2b expressed in IGROV-1 cells is shown. For each panel, the right curve shows data for a constant binding partner of 500 pM, and the left curve shows data for a constant binding partner of 50 pM. [Figure 4C] N-curve analysis of binding of v36124 to NaPi2b expressed in IGROV-1 cells is shown. For each panel, the right curve shows data for a constant binding partner of 500 pM, and the left curve shows data for a constant binding partner of 50 pM. [Figure 5A] A comparison of the ability of v23855 (parent chimera), v29456 (H1L2), v18992 (MX35), and v18993 (rifastuzumab) to internalize into HCC-78 cells is shown. [Figure 5B] A comparison of the ability of v23855 (parent chimera), v29456 (H1L2), v18992 (MX35), and v18993 (rifastuzumab) to internalize into NCI-H441 cells is shown. [Figure 6] Binding of MX35 and rifastuzumab ADCs to IGROV-1 cells, as well as binding of the parent chimeric antibody (v23855), humanized antibody variants v29452 and v29456, are depicted. [Figure 7A]Figure 1 illustrates the ability of humanized antibody variants v29456 and v29456 conjugated to DL1 or DL2 to bind to IGROV-1 cells. [Figure 7B] Illustrates the ability of v29456 and v29456 conjugated to DL1 or DL2 to bind to HCC-78 cells. [Figure 8A] 1 shows the cytotoxicity of ADCs of humanized antibody variant v29456, as well as ADCs of reference antibodies MX35 and rifastuzumab in OVCAR-3 cells. [Figure 8B] Figure 1 shows the cytotoxicity of ADCs of v29456, as well as ADCs of reference antibodies MX35 and rifastuzumab in IGROV-1 cells. [Figure 8C] 1 shows the cytotoxicity of ADCs of v29456, as well as ADCs of reference antibodies MX35 and rifastuzumab in HCC-78 cells. [Figure 9A] 1 illustrates the in vivo efficacy of the ADC of the parental chimeric v23855 compared to the ADC of the reference antibody v18992 (MX35) in the OVCAR-3 xenograft model of ovarian cancer. [Figure 9B] 1 illustrates the in vivo efficacy of the ADC of the parental chimeric v23855 compared to the ADC of the reference antibody v18993 (rifastuzumab) in the OVCAR-3 xenograft model of ovarian cancer. [Figure 10] 1 illustrates the in vivo efficacy of ADC of v29456 at 1, 3, and 10 mg / kg in the OVCAR-3 xenograft model of ovarian cancer. [Figure 11A] 1 shows the in vivo efficacy of ADC of v29456 at 1, 3, and 10 mg / kg in the NCI-H441 xenograft model of lung cancer. [Figure 11B] 1 shows the in vivo efficacy of the ADC of v29456 at 0.3 and 1 mg / kg in the NCI-H441 xenograft model of lung cancer. [Figure 12]Figure 12A illustrates the results of a Membrane Proteome Array™ assay using v38591. Figure 12B illustrates validation data for CLDN3, showing weak binding to CLDN3. [Figure 13] 1 illustrates the binding of humanized antibody variants v38591 and v29456 to IGROV-1 and TOV-21G cells compared to the binding of rifastuzumab. [Figure 14] Figure 1 shows the PK profiles of v29456 and v18993 (rifastuzumab)-MC-VC-PABC-MMAE(DL3) in Tg32 mice. DETAILED DESCRIPTION OF THE INVENTION

[0015] Detailed Description The present disclosure relates to antibody constructs that bind to human sodium-dependent phosphate transporter 2B (NaPi2b). In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure can also bind to cynomolgus monkey NaPi2b.

[0016] The present disclosure also relates to antibody-drug conjugates (ADCs) comprising the anti-NaPi2b antibody constructs described herein conjugated to a drug, such as a cytotoxin or an immunomodulator. The anti-NaPi2b antibody constructs and ADCs of the present disclosure may find use, for example, as therapeutic or diagnostic agents. Certain aspects of the present disclosure relate to therapeutic methods and uses of the anti-NaPi2b antibody constructs and ADCs, such as in the treatment of cancer. Some aspects relate to diagnostic methods and uses of the anti-NaPi2b antibody constructs and ADCs, such as in the diagnosis or analysis of cancer.

[0017] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0018] As used herein, the term "about" refers to approximately a + / - 10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0019] The use of the words "a" or "an," when used herein in conjunction with the term "comprising," can mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more."

[0020] As used herein, the terms "comprising," "having," "including," and "containing," and grammatical variations thereof, are inclusive, i.e., open-ended, and do not exclude additional, unrecited elements and / or method steps. When used herein in connection with a composition, use, or method, the term "consisting essentially of" means that additional elements and / or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method, or use functions. When used herein in connection with a composition, use, or method, the term "consisting of" excludes the presence of additional elements and / or method steps. A composition, use, or method described herein as including particular elements and / or steps may also, in certain embodiments, consist essentially of those elements and / or steps, and in other embodiments, consist of those elements and / or steps, whether or not those embodiments are specifically mentioned.

[0021] "Complementarity-determining regions" or "CDRs" are amino acid sequences that contribute to antigen-binding specificity and affinity. "Framework" regions (FRs) may help maintain the proper conformation of the CDRs to promote binding between the antigen-binding region and the antigen. From the N-terminus to the C-terminus, both the light chain variable region (VL) and heavy chain variable region (VH) of an antibody typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three heavy chain CDRs are referred to herein as HCDR1, HCDR2, and HCDR3, and the three light chain CDRs are referred to as LCDR1, LCDR2, and LCDR3. CDRs provide the majority of contact residues for antibody binding to an antigen or epitope. In many cases, three heavy chain CDRs and three light chain CDRs are required for antigen binding. However, in some cases, even a single variable domain can confer antigen-binding specificity. Furthermore, as is known in the art, in some cases, antigen binding may also occur through a combination of at least one or more CDRs, e.g., HCDR3s, selected from the VH and / or VL domains.

[0022] Several different definitions of CDR sequences are commonly used, including those described by Kabat et al. (1983, Sequences of Proteins of Immunological Interest, NIH Publication No. 369-847, Bethesda, MD), Chothia et al. (1987, J Mol Biol, 196:901-917), and IMGT, AbM (University of Bath), and Contact (MacCallum, et al., 1996, J Mol Biol, 262(5):732-745). By way of example, the definitions of CDRs according to Kabat, Chothia, IMGT, AbM, and Contact are provided in Table 1 below. Thus, as will be readily apparent to one skilled in the art, the exact numbering and arrangement of CDRs may vary based on the numbering system used. However, it should be understood that the disclosure of a VH herein includes disclosure of the associated (unique) heavy chain CDRs (HCDRs), as defined by any of the known numbering systems. Similarly, the disclosure of a VL herein includes disclosure of the associated (unique) light chain CDRs (LCDRs), as defined by any of the known numbering systems.

[0023] (Table 1) Common CDR definitions 1 TIFF2025535238000002.tif168165

[0024] The term "identical" in the context of two or more polynucleotide or polypeptide sequences refers to two or more sequences or subsequences that are the same. Sequences are "substantially identical" if they have the same percentage of amino acid residues or nucleotides (e.g., about 80%, about 85%, about 90%, about 95%, or about 98% identity over a designated region) when compared and aligned for maximum correspondence over a comparison window or over a designated region, as measured using one of the commonly used sequence comparison algorithms known to those skilled in the art or by manual alignment and visual inspection. For sequence comparison, a test sequence is typically compared to a designated reference sequence. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0025] A "comparison window" refers to a segment of a sequence that includes contiguous amino acid or nucleotide positions, for example, about 10 to about 600 contiguous amino acid or nucleotide positions, or about 10 to about 200, or about 10 to about 150 contiguous amino acid or nucleotide positions, and a test sequence can be compared to a reference sequence over the same number of contiguous positions after the two sequences are optimally aligned. Methods for aligning sequences for comparison purposes are known to those of skill in the art. Optimal sequence alignment for comparison can be performed, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. Appl. Math., 2:482c, by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443, by the search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444, or by computer implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, or TFASTA (Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI)), or by manual alignment and visual inspection (see, for example, Ausubel et al., Current Protocols in Molecular Biology, (1995 supplement), Cold Spring Harbor Laboratory Press). Examples of available algorithms suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., 1997, Nuc. Acids Res., 25:3389-3402, and Altschul et al., 1990, J. Mol. Biol., 215:403-410, respectively. Software for performing BLAST analyses is publicly available from the website of the National Center for Biotechnology Information (NCBI).

[0026] As used herein, the term "subject" refers to an animal, in some embodiments a mammal, that is the object of treatment, observation, or experiment. The animal may be a human, a non-human primate, a companion animal (e.g., dog, cat, etc.), a livestock animal (e.g., cow, sheep, pig, horse, etc.), or a laboratory animal (e.g., rat, mouse, guinea pig, non-human primate, etc.). In certain embodiments, the subject is a human.

[0027] It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method, use, or composition disclosed herein, and vice versa.

[0028] Particular features, structures, and / or characteristics described in connection with an embodiment disclosed herein may be combined in any suitable manner with features, structures, and / or characteristics described in connection with other embodiments disclosed herein to provide one or more additional embodiments.

[0029] It should also be understood that the affirmative recitation of a feature in one embodiment serves as a basis for the exclusion of that feature in an alternative embodiment. For example, where a list of alternatives is presented for a given embodiment or claim, it should be understood that one or more alternatives may be deleted from the list, and that the shortened list may form an alternative embodiment, whether or not such alternative embodiment is specifically mentioned.

[0030] Anti-NaPi2b antibody construct The present disclosure relates to an antibody construct that specifically binds to human NaPi2b (hNaPi2b). In this context, the term "antibody construct" refers to a polypeptide or set of polypeptides that includes one or more antigen-binding domains, each of which specifically binds to an epitope or antigen. When an antibody construct includes two or more antigen-binding domains, each of the antigen-binding domains may bind to the same epitope or antigen (i.e., the antibody construct is monospecific), or they may bind to different epitopes or antigens (i.e., the antibody construct is bispecific or multispecific). The antibody construct may further include a scaffold, and one or more antigen-binding domains may be fused or covalently linked to the scaffold, optionally via a linker.

[0031] According to the present disclosure, an anti-NaPi2b antibody construct comprises at least one antigen-binding domain that specifically binds to hNaPi2b. "Specifically binds" to hNaPi2b means that the antibody construct binds to hNaPi2b but does not exhibit significant binding to either NaPi2a or NaPi2c. In certain embodiments, the anti-NaPi2b antibody construct of the present disclosure can bind to NaPi2b from one or more non-human species. In certain embodiments, the anti-NaPi2b antibody construct of the present disclosure can bind to cynomolgus monkey NaPi2b.

[0032] Human NaPi2b is also known as human "solute carrier family 34 member 2" or "SLC34A2." Protein sequences for hNaPi2b from various sources are known in the art and readily available from publicly accessible databases such as GenBank or UniProtKB. Exemplary hNaPi2b sequences include those provided under NCBI reference numbers NP_006415.3, NP_001171470.2, and NP_001171469.2. An exemplary hNaPi2b protein sequence is provided in Table 2 as SEQ ID NO: 1 (UniProt ID: 095436). An exemplary cynomolgus monkey NaPi2b protein sequence is also provided in Table 2 (SEQ ID NO: 2, UniProt ID: A0A2K5UHY1), as is an exemplary mouse NaPi2b protein sequence (SEQ ID NO: 3, UniProt ID: Q9DBP0).

[0033] Table 2. Human, cynomolgus, and mouse NaPi2b protein sequences TIFF2025535238000003.tif147165TIFF2025535238000004.tif145165TIFF2025535238000005.tif151165

[0034] Specific binding of an antigen-binding domain to a target antigen or epitope can be measured, for example, by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR) technology (e.g., using a BIAcore instrument) (Liljeblad et al., 2000, Glyco J, 17:323-329), flow cytometry, or conventional binding assays (Heeley, 2002, Endocr Res, 28:217-229). In certain embodiments, specific binding can be defined as binding to a non-target protein (e.g., hNaPi2a or hNaPi2c) that is less than about 5% to 10% of the binding to hNaPi2b, as measured, for example, by ELISA or flow cytometry.

[0035] Dissociation constant (K D or Kd The term "K" as used herein is intended to refer to the equilibrium dissociation constant of a particular ligand-protein interaction. As used herein, a ligand-protein interaction refers to, but is not limited to, a protein-protein interaction or an antibody-antigen interaction. D measures the tendency of two proteins (e.g., AB) complexed with each other to reversibly dissociate into their components (A+B), which is called the “off rate (k off ) and the dissociation rate constant or "on rate (k on )" Therefore, K D is k off / k on and is expressed as molar concentration (M). D The smaller the K, the stronger the binding affinity. D A decrease in K indicates an increase in affinity. D has a K of 1 nM D The affinity is expressed as K A or K a It is sometimes measured as K D or K d The K between an antibody and its antigen D can be determined using methods well established in the art. D One method for determining K is by using surface plasmon resonance (SPR), typically using a biosensor system such as a Biacore® system. Isothermal titration calorimetry (ITC) can be used to determine K D The Octet™ system may also be used to measure the affinity of an antibody for a target antigen.

[0036] In certain embodiments, the specific binding of the antibody construct to NaPi2b has a dissociation constant (Kd or K) of <1 μM, e.g., <500 nM, <250 nM, <100 nM, <50 nM, or <10 nM. DIn certain embodiments, specific binding of an antibody construct to a particular antigen or epitope may be defined by a -6 M or less, e.g., 10 -7 M or less, or 10 -8 The dissociation constant (K D In some embodiments, the specific binding of an antibody construct to a particular antigen or epitope may be defined by a -6 M~10 -9 M, e.g., 10 -7 M~10 -9 Dissociation constant of M (K D ) As is known in the art, the numerical value of the dissociation constant obtained may vary depending on the test method. For example, the expression level of NaPi2b in the cell line, the format of the antibody construct (i.e., monovalent or bivalent), and the type of assay (i.e., ELISA or flow cytometry) may affect the numerical value of the dissociation constant when measured in a cell-based assay. The data provided in the Examples illustrate this general point.

[0037] In some embodiments, the anti-NaPi2b antibody constructs of the present disclosure have a Kd lower than that of the reference antibody rifastuzumab and equivalent to that of the reference antibody MX35, as measured by flow cytometry in cells expressing high levels of NaPi2b. Thus, in these embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain with an affinity for human NaPi2b higher than that of the reference antibody rifastuzumab and equivalent to that of the reference antibody MX35.

[0038] In certain embodiments, the anti-NaPi2b antibody constructs exhibit comparable levels of internalization to the reference antibody MX35 and higher levels of internalization compared to the reference antibody rifastuzumab in high and medium NaPi2b-expressing cells. In some embodiments, internalization is measured 4 hours, 5 hours, or 24 hours after treatment.

[0039] Antibody internalization can be measured using methods known in the art, for example, the direct internalization method according to the protocol detailed in Schmidt, M. et al., 2008, Cancer Immunol. Immunother., 57:1879-1890, or using commercially available fluorescent dyes such as pHAb dyes (Promega Corporation, Madison, WI), pHrodo iFL and Deep Red Dye (ThermoFisher Scientific Corporation, Waltham, MA), and Incucyte® Fabfluor-pH antibody labeling reagent (Sartorius AG, Goettingen, Germany), and analytical techniques such as microscopy, FACS, high-content imaging, or other plate-based assays.

[0040] Expression of NaPi2b varies depending on cell type, as shown throughout this disclosure, and NaPi2b expression levels are referred to herein as "high," "medium," "low," or "negative." These terms are used by reference to generally describe expression levels, as designated in Table 10.1 of Example 10, and are not intended to be limited to any particular number of average NaPi2b proteins per cell contained therein.

[0041] antigen-binding domain The anti-NaPi2b antibody constructs of the present disclosure comprise at least one antigen-binding domain capable of binding to hNaPi2b. The at least one antigen-binding domain capable of binding to hNaPi2b is typically an immunoglobulin-based binding domain such as an antigen-binding antibody fragment. Examples of antigen-binding antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, single-chain Fabs (scFabs), single-chain Fvs (scFvs), and single-domain antibodies (sdAbs).

[0042] A "Fab fragment" contains the light and heavy chain variable domains (VL and VH, respectively), as well as the light chain constant domain (CL) and the first heavy chain constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of a few amino acid residues at the C-terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab fragments may also be single-chain Fab molecules, i.e., Fab molecules in which the Fab light chain and Fab heavy chain are connected by a peptide linker to form a single peptide chain. For example, in a single-chain Fab molecule, the C-terminus of the Fab light chain may be connected to the N-terminus of the Fab heavy chain.

[0043] An "scFv" comprises an antibody heavy chain variable domain (VH) and light chain variable domain (VL) in a single polypeptide chain. An scFv may optionally further comprise a polypeptide linker between the VH and VL domains, which enables the scFv to form the desired structure for antigen binding. For example, an scFv may comprise a VL connected from the C-terminus of the VL to the N-terminus of the VH by a polypeptide linker. Alternatively, an scFv may comprise a VH connected via the C-terminus of the VH to the N-terminus of the VL by a polypeptide linker (see review in Plückthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)).

[0044] The "sdAb" format refers to a single immunoglobulin domain. sdAbs may, for example, be of camelid origin. Camelid antibodies lack light chains and their antigen-binding site consists of a single domain called "VHH". sdAbs form three CDR / hypervariable loops that form the antigen-binding site: CDR1, CDR2 and CDR3. sdAbs are fairly stable and are easily expressed, for example, as fusions with the Fc chain of an antibody (e.g., Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., 77(1):13-22).

[0045] In those embodiments in which the anti-NaPi2b antibody construct comprises two or more antigen-binding domains, each additional antigen-binding domain can independently be an immunoglobulin-based domain, e.g., an antigen-binding antibody fragment, or a non-immunoglobulin-based domain, e.g., a non-immunoglobulin-based antibody mimetic, or other polypeptide or small molecule, e.g., a natural or engineered ligand, capable of specifically binding to its target. Non-immunoglobulin-based antibody mimetic formats include, for example, anticalins, finomers, affimers, alphabodies, DARPins, and avimers.

[0046] The present disclosure describes herein the identification of a murine antibody that specifically binds to hNaPi2b. A murine-human chimeric variant of this antibody is identified as variant 23855. The anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain derived from this murine antibody or its humanized antibody variants. Representative humanized antibody variants of the murine antibody (v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460) are also described. In certain embodiments, the anti-NaPi2b antibody constructs described herein specifically bind to human NaPi2b having the sequence set forth in SEQ ID NO: 1.

[0047] In certain embodiments, the anti-NaPi2b antibody construct competes for binding to human NaPi2b with any one of humanized antibody variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460, or with the parent chimeric antibody v23855. When assessing competition as described below, each of variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, v29460, and v23855 is referred to as a competing reference antibody.

[0048] Using competitive assays known in the art, it can be determined whether an antibody construct competes with variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460, or the parent chimeric antibody v23855, for binding to hNaPi2b. For example, a competitive reference antibody is first bound to hNaPi2b under saturating conditions, and then the ability of a test antibody construct to bind to hNaPi2b is measured. If the test antibody construct can bind to hNaPi2b simultaneously with the competitive reference antibody, the test antibody construct is considered to bind to a different epitope than the competitive reference antibody. Conversely, if the test antibody construct is unable to bind to hNaPi2b simultaneously with the competing reference antibody, the test antibody construct is considered to bind to the same epitope, an overlapping epitope, or an epitope adjacent to the epitope bound by the competing reference antibody. Such competition assays can be performed using techniques such as ELISA, radioimmunoassay, surface plasmon resonance (SPR), biolayer interferometry, flow cytometry, and the like. An "antibody that competes with" a reference antibody refers to an antibody that blocks binding of the competing reference antibody to its epitope by 50% or more in a competition assay.

[0049] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise at least one antigen-binding domain that specifically binds to hNaPi2b, and the antigen-binding domain comprises a set of CDRs based on the CDRs of the parent chimeric antibody v23855 described herein. The CDR sequences of the parent chimeric antibody v23855 and representative humanized antibody variants are shown in Table 3.

[0050] Table 3. CDR sequences of anti-NaPi2b antibody constructs TIFF2025535238000006.tif165165

[0051] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain having heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) comprising the sequences set forth in SEQ ID NOs: 7, 8, and 9, and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) comprising the sequences set forth in SEQ ID NOs: 19, 20, and 18.

[0052] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain having: (i) an HCDR1 amino acid sequence selected from the HCDR1 amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460; an HCDR2 amino acid sequence selected from the HCDR2 amino acid sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, and an HCDR3 amino acid sequence selected from the HCDR3 amino acid sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460; and (ii) an LCDR1 amino acid sequence selected from any one of the LCDR1 amino acid sequences variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460; and an LCDR2 amino acid sequence selected from any one of the LCDR1 amino acid sequences variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. and an LCDR2 amino acid sequence selected from the LCDR3 amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, where the CDR amino acid sequences are as defined by any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems (see Table 3).

[0053] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) selected from the heavy chain CDR amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, as defined by any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems. and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) selected from the light chain CDR amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, as defined by any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems.

[0054] In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain comprising the heavy chain CDR amino acid sequences (HCDR1, HCDR2, HCDR3) and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, as defined by any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems.

[0055] In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain having a VH sequence comprising the CDR sequences of the VH sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain having a VL sequence comprising the CDR sequences of the VL sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460.

[0056] Those skilled in the art will understand that a limited number of amino acid substitutions can be introduced into the CDR sequences or VH or VL sequences of a known antibody without losing the ability of the antibody to bind to its target. Candidate amino acid substitutions can be identified by computer modeling or by techniques known in the art, such as alanine scanning, and the resulting variants are tested for binding activity by standard techniques. Thus, in certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain comprising a set of CDRs (i.e., heavy chain HCDR1, HCDR2, and HCDR3, and light chain LCDR1, LCDR2, and LCDR3) having 90% or more, 95% or more, 98% or more, 99% or more, or 100% sequence identity to the set of CDRs of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, where the % sequence identity is calculated across all six CDRs, and wherein the antigen-binding domain retains the ability to bind to hNaPi2b.

[0057] In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain comprising a variant of the set of CDR sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, wherein the variant comprises 1 to 10 amino acid substitutions across the set of CDRs (i.e., the CDRs may be modified by modifying any combination of the six CDRs, resulting in up to 10 amino acid substitutions), and wherein the antigen-binding domain retains the ability to bind to hNaPi2b. In some embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain comprising a variant of the set of CDR sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, wherein the variant comprises 1-7 amino acid substitutions, 1-5 amino acid substitutions, 1-4 amino acid substitutions, 1-3 amino acid substitutions, 1-2 amino acid substitutions, or 1 amino acid substitution across the set of CDRs, and wherein the antigen-binding domain retains the ability to bind to hNaPi2b.

[0058] In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain comprising a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, wherein the antigen-binding domain retains the ability to bind to hNaPi2b. In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain comprising a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460, wherein the antigen-binding domain retains the ability to bind to hNaPi2b.

[0059] In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain comprising a VH amino acid sequence selected from the VH amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises an antigen-binding domain comprising a VL amino acid sequence selected from the VL amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460.

[0060] In certain embodiments, an anti-NaPi2b antibody construct of the disclosure comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of variant v23855, and an antigen-binding domain comprising a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of v23855, wherein the antigen-binding domain retains the ability to bind to hNaPi2b.

[0061] In certain embodiments, an anti-NaPi2b antibody construct of the disclosure comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of variant v29456, and an antigen-binding domain comprising a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of v29456, wherein the antigen-binding domain retains the ability to bind to hNaPi2b.

[0062] In certain embodiments, an anti-NaPi2b antibody construct of the disclosure comprises a VH sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence of variant v29452, and an antigen-binding domain comprising a VL sequence that is at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VL sequence of v29452, wherein the antigen-binding domain retains the ability to bind to hNaPi2b.

[0063] In some embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises the VH and VL sequences of any one of v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460. The SEQ ID NOs for the VH and VL sequences of these variants are set forth in Table 4 below. The sequences themselves are provided in Table 2.4 in the Examples.

[0064] Table 4. VH and VL sequences of parent chimeric and humanized anti-NaPi2b antibodies TIFF2025535238000007.tif96165

[0065] In some embodiments, the anti-NaPi2b antibody construct of the disclosure comprises the VH and VL sequences of v29456. In some embodiments, the anti-NaPi2b antibody construct of the ADC of the disclosure comprises the VH and VL sequences of v29452.

[0066] In certain embodiments, an anti-NaPi2b antibody construct of the present disclosure comprises: a) a VH sequence having the three HCDRs of v29456 and having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VH sequence of v29456; and b) a VL sequence having the three LCDRs of v29456 and having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VL sequence of v29456, wherein the HCDRs and LCDRs are defined by any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems.

[0067] In certain other embodiments, the anti-NaPi2b antibody construct of the present disclosure comprises: a) a VH sequence having the three HCDRs of v29452 and having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VH sequence of v29452; and b) a VL sequence having the three LCDRs of v29452 and having at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the VL sequence of v29452, wherein the HCDRs and LCDRs are defined by any one of the IMGT, Chothia, Kabat, Contact, or AbM numbering systems.

[0068] format The anti-NaPi2b antibody constructs of the present disclosure may have a variety of formats. The minimum component of an anti-NaPi2b antibody construct is an antigen-binding domain that binds to hNaPi2b. The anti-NaPi2b antibody construct may further optionally comprise one or more additional antigen-binding domains and / or scaffolds. In those embodiments in which the anti-NaPi2b antibody construct comprises two or more antigen-binding domains, each additional antigen-binding domain may bind to the same epitope within hNaPi2b, a different epitope within hNaPi2b, or a different antigen. Thus, the anti-NaPi2b antibody construct may be, for example, monospecific, biparatopic, bispecific, or multispecific.

[0069] In certain embodiments, the anti-NaPi2b antibody construct comprises at least one antigen-binding domain that binds to hNaPi2b and a scaffold, wherein the antigen-binding domain is operably linked to the scaffold. The term "operably linked," as used herein, means that the described components are in a relationship permitting them to function in their intended manner. Suitable scaffolds are described below.

[0070] In certain embodiments, the anti-NaPi2b antibody construct comprises two antigen-binding domains operably linked to an optional scaffold. In some embodiments, the anti-NaPi2b antibody construct may comprise three or four antigen-binding domains and, optionally, a scaffold. In these formats, when a scaffold is included, at least the first antigen-binding domain is operably linked to the scaffold, and the remaining antigen-binding domain(s) may each independently be operably linked to the scaffold or the first antigen-binding domain, or, if more than two antigen-binding domains are present, may be linked to another antigen-binding domain.

[0071] Scaffold-free anti-NaPi2b antibody constructs may contain a single antigen-binding domain in a suitable format, such as an sdAb, or they may contain two or more antigen-binding domains, optionally operably linked by one or more linkers. In such anti-NaPi2b antibody constructs, the antigen-binding domain may be in the form of an scFv, Fab, sdAb, or a combination thereof. For example, using scFvs as antigen-binding domains allows for the construction of formats such as tandem scFvs ((scFv)2 or taFv), in which scFvs are connected together by a flexible linker. scFvs can also be used to construct diabody formats containing two scFvs connected by a short linker (usually about 5 amino acids in length). The limited length of the linker results in dimerization of the scFvs in a head-to-tail manner. In any of the preceding formats, the scFvs can be further stabilized by including an interdomain disulfide bond. For example, a disulfide bond may be introduced between the VL and VH by substituting a non-cysteine ​​residue in each chain with a cysteine ​​residue (e.g., at position 44 in the VH and position 100 in the VL) (see, e.g., Fitzgerald et al., 1997, Protein Engineering, 10:1221-1225), or a disulfide bond may be introduced between two VHs to provide a construct with a DART format (see, e.g., Johnson et al., 2010, J Mol. Biol., 399:436-449).

[0072] Similarly, in some embodiments, formats comprising two sdAbs, such as VH or VHH, connected together via a suitable linker may be used. Other examples of anti-NaPi2b antibody construct formats lacking a scaffold include those based on Fab fragments, e.g., Fab2 and F(ab')2 formats, where the Fab fragments are connected via a linker or IgG hinge region.

[0073] Combinations of different forms of antigen-binding domains can also be used to create alternative scaffold-less formats, for example, an scFv or sdAb can be fused to the C-terminus of either or both the light and heavy chains of a Fab fragment, resulting in a bivalent (Fab-scFv / sdAb) construct.

[0074] In certain embodiments, the anti-NaPi2b antibody construct may be in an immunoglobulin (Ig)-based antibody format. This type of format is referred to herein as full-size antibody format (FSA) or Mab format, and includes anti-NaPi2b antibody constructs comprising two Ig heavy chains and two Ig light chains. In certain embodiments, the anti-NaPi2b antibody construct may be based on an IgG class immunoglobulin, e.g., an IgG1, IgG2, IgG3, or IgG4 immunoglobulin. In some embodiments, the anti-NaPi2b antibody construct may be based on an IgG1 immunoglobulin. In the context of the present disclosure, when an anti-NaPi2b antibody construct is based on a particular immunoglobulin isotype, it means that the anti-NaPi2b antibody construct comprises all or a portion of the constant region of the particular immunoglobulin isotype. For example, an anti-NaPi2b antibody construct based on a given Ig isotype can comprise at least one antigen-binding domain operably linked to an Ig scaffold, the scaffold comprising an Fc region from the given isotype and, optionally, an Ig hinge region from the same or a different isotype. It should be understood that in some embodiments, the anti-NaPi2b antibody construct can also comprise isotype and / or subclass hybrids. It should also be understood that the Fc region and / or hinge region can be optionally modified to confer one or more desirable functional properties known in the art. Thus, in certain embodiments, the anti-NaPi2b antibody construct comprises a VH amino acid sequence (i.e., CH1, hinge, CH2, CH3 amino acid sequences) fused to an IgG1 constant domain amino acid sequence and a VL amino acid sequence (i.e., CL amino acid sequence) fused to a kappa or lambda constant amino acid domain. Exemplary amino acid sequences are provided in the Examples and Sequence Listing.

[0075] In some embodiments, the anti-NaPi2b antibody construct may be derived from two or more immunoglobulins from different species, e.g., the anti-NaPi2b antibody construct may be a chimeric antibody or a humanized antibody. The terms "chimeric antibody" and "humanized antibody" both generally refer to antibodies that combine immunoglobulin regions or domains from multiple species.

[0076] A "chimeric antibody" typically comprises at least one variable domain from a non-human antibody, such as a rabbit or rodent (e.g., murine) antibody, and at least one constant domain from a human antibody. The human constant domain of a chimeric antibody need not be of the same isotype as the non-human constant domain it replaces. Chimeric antibodies are discussed, for example, in Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-55, and U.S. Pat. No. 4,816,567.

[0077] A "humanized antibody" is a type of chimeric antibody that contains minimal sequence derived from a non-human antibody. Generally, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit, or non-human primate having the desired specificity and affinity for the target antigen. This technique for producing humanized antibodies is often referred to as "CDR grafting."

[0078] In some cases, additional modifications are made to further refine antibody performance. For example, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues, or humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. Generally, the variable domains of humanized antibodies contain all or substantially all of the hypervariable regions from the non-human immunoglobulin and all or substantially all of the FRs from the human immunoglobulin sequence. Humanized antibodies are described in further detail in, for example, Jones, et al., 1986, Nature, 321:522-525; Riechmann, et al., 1988, Nature, 332:323-329; and Presta, 1992, Curr. Op. Struct. Biol., 2:593-596.

[0079] Several approaches for selecting the most appropriate human framework for grafting nonhuman CDRs are known in the art. Early approaches used a limited subset of well-characterized human antibodies, regardless of their sequence identity with the nonhuman antibody providing the CDRs (the "fixed framework" approach). More recent approaches use variable regions with high amino acid sequence identity with the variable regions of the nonhuman antibody providing the CDRs (the "homology matching" or "best-fit" approach). An alternative approach is to select fragments of framework sequences within each light or heavy chain variable region from several different human antibodies. CDR grafting can, in some cases, partially or completely lose the affinity of the grafted molecule for its target antigen. In such cases, affinity can be restored by backmutating some of the human-derived residues to the corresponding nonhuman-derived residues. Methods for preparing humanized antibodies by these approaches are well known in the art (see, for example, Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA); Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; Presta et al., 1997, Cancer Res, 57(20):4593-4599).

[0080] Alternatively or in addition to these traditional approaches, more recent techniques may be used to further reduce the immunogenicity of CDR-grafted humanized antibodies. For example, a framework based on a human germline sequence or consensus sequence may be used as the acceptor human framework, rather than a human framework with somatic mutation(s). Another technique aimed at reducing the potential immunogenicity of non-human CDRs is to graft only specificity-determining residues (SDRs). In this approach, only the minimum CDR residues ("SDRs") required for antigen-binding activity are grafted onto a human germline framework. This method can help improve the "humanness" of humanized antibodies (i.e., similarity to human germline sequences), thus reducing the risk of immunogenicity of the variable regions. These techniques are described in various publications (see, e.g., Almagro & Fransson, 2008, Front Biosci, 13:1619-1633; Tan, et al., 2002, J Immunol, 169:1119-1125; Hwang, et al., 2005, Methods, 36:35-42; Pelat, et al., 2008, J Mol Biol, 384:1400-1407; Tamura, et al., 2000, J Immunol, 164:1432-1441; Gonzales, et al., 2004, Mol Immunol, 1:863-872; and Kashmiri, et al., 2005, Methods, 36:25-34).

[0081] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise humanized antibody sequences, e.g., one or more humanized variable domains. In some embodiments, the anti-NaPi2b antibody constructs can be humanized antibodies. Non-limiting examples of humanized antibodies based on anti-NaPi2b antibody v23855 are described herein (see the Examples and Sequence Listing for v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460).

[0082] scaffold In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise one or more antigen-binding domains operably linked to a scaffold. The antigen-binding domain(s) may be in one or a combination of the above forms (e.g., scFv, Fab, and / or sdAb). Examples of suitable scaffolds are described in more detail below and include, but are not limited to, immunoglobulin Fc regions, albumin, albumin analogs and derivatives, heterodimerizing peptides (e.g., leucine zippers, heterodimerizing "zipper" peptides derived from Jun and Fos, IgG CH1 and CL domains, or barnase barstar toxin), cytokines, chemokines, or growth factors. Other examples include antibodies based on DOCK-AND-LOCK™ (DNL™) technology developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. (see, e.g., Chang, et al., 2007, Clin. Cancer Res., 13:5586s-5591s).

[0083] The scaffold can be a peptide, polypeptide, polymer, nanoparticle, or other chemical substance.When the scaffold is a polypeptide, each antigen-binding domain of the anti-NaPi2b antibody construct can be linked to either the N-terminus or C-terminus of the polypeptide scaffold.In certain embodiments, the anti-NaPi2b antibody construct also includes a polypeptide scaffold in which one or more antigen-binding polypeptide constructs are linked to a region other than the N-terminus or C-terminus, for example, via an amino acid side chain with or without a linker.

[0084] In embodiments in which the anti-NaPi2b antibody construct comprises a peptide or polypeptide scaffold, the antigen-binding domain(s) may be linked to the scaffold by genetic fusion or chemical conjugation. Typically, when the scaffold is a peptide or polypeptide, the antigen-binding domain(s) are linked to the scaffold by genetic fusion. In some embodiments, when the scaffold is a polymer or nanoparticle, the antigen-binding domain(s) may be linked to the scaffold by chemical conjugation.

[0085] Several protein domains containing selective pairs of two different polypeptides are known in the art and can be used to form scaffolds. One example is the selectively paired leucine zipper domains, such as Fos and Jun (Kostelny, et al., J Immunol, 148:1547-53 (1992); Wranik, et al., J. Biol. Chem., 287:43331-43339 (2012)). Other selectively paired molecular pairs include, for example, the barnase barnase pair (Deyev, et al., Nat Biotechnol, 21:1486-1492 (2003)), DNA strand pairs (Chaudri, et al., FEBS Letters, 450(1-2):23-26 (1999)), and split fluorescent protein pairs (International Patent Application Publication No. WO2011 / 135040).

[0086] Other examples of protein scaffolds include immunoglobulin Fc regions, albumin, albumin analogs and derivatives, toxins, cytokines, chemokines, and growth factors. The use of protein scaffolds in combination with antigen-binding moieties has been described (see, for example, Mueller et al., 2007, J. Biol. Chem., 282:12650-12660; McDonaugh et al., 2012, Mol. Cancer Ther., 11:582-593; Vallera et al., 2005, Clin. Cancer Res., 11:3879-3888; Song et al., 2006, Biotech. Appl. Biochem., 45:147-154, and U.S. Patent Application Publication No. 2009 / 0285816).

[0087] For example, fusing an antigen-binding moiety, such as an scFv, diabody, or single-chain diabody, to albumin has been shown to improve the serum half-life of the antigen-binding moiety (Mueller et al., ibid.). The antigen-binding moiety may be fused to the N-terminus and / or C-terminus of albumin, optionally via a linker.

[0088] Derivatives of albumin have been described in the form of heteromultimers containing two transporter polypeptides obtained by segmenting the albumin protein so that the transporter polypeptides self-assemble to form quasi-native albumin (see International Patent Applications WO 2012 / 116453 and WO 2014 / 012082). As a result of the segmentation of albumin, the heteromultimer contains four termini and can therefore be fused, optionally via linkers, to up to four different antigen-binding moieties.

[0089] In certain embodiments, the anti-NaPi2b antibody construct may comprise a protein scaffold. In some embodiments, the anti-NaPi2b antibody construct may comprise a protein scaffold based on an immunoglobulin Fc region, albumin, or an albumin analog or derivative. In some embodiments, the anti-NaPi2b antibody construct may comprise a protein scaffold based on an immunoglobulin Fc region, for example, an IgG Fc region.

[0090] Fc area As used herein, the terms "Fc region," "Fc," or "Fc domain" refer to the C-terminal region of an immunoglobulin heavy chain comprising at least a portion of the constant region. This term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, the numbering of amino acid residues in an Fc region or constant region is according to the EU numbering system, also known as the EU index, as described in Kabat, et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0091] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure may comprise a scaffold that is based on an immunoglobulin Fc region. The Fc region may be dimeric and comprised of two Fc polypeptides, or alternatively, the Fc region may be comprised of a single polypeptide.

[0092] An "Fc polypeptide" in the context of a dimeric Fc refers to one of the two polypeptides that form the dimeric Fc domain, i.e., a polypeptide comprising one or more C-terminal constant regions of an immunoglobulin heavy chain capable of stable self-association. When referring to a dimeric Fc region, the terms "first Fc polypeptide" and "second Fc polypeptide" may be used interchangeably, provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.

[0093] An Fc region may comprise a CH3 domain, or it may comprise both a CH3 and a CH2 domain. For example, in certain embodiments, the Fc polypeptides of a dimeric IgG Fc region may comprise an IgG CH2 domain sequence and an IgG CH3 domain sequence. In such embodiments, the CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the dimeric Fc region, and the CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the dimeric Fc region.

[0094] In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on an IgG Fc region. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on a human IgG Fc region. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on an IgG1 Fc region. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on a human IgG1 Fc region.

[0095] In certain embodiments, an anti-NaPi2b antibody construct may comprise an IgG Fc region-based scaffold that is a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence and optionally a CH2 sequence, wherein the first and second Fc polypeptides are different. In some embodiments, an anti-NaPi2b antibody construct may comprise an Fc region-based scaffold comprising two CH3 sequences, at least one of which comprises one or more amino acid modifications. In some embodiments, an anti-NaPi2b antibody construct may comprise an Fc region-based scaffold comprising two CH3 sequences and two CH2 sequences, at least one of which comprises one or more amino acid modifications.

[0096] In some embodiments, an anti-NaPi2b antibody construct may comprise a heterodimeric Fc region comprising a modified CH3 domain, the modified CH3 domain being an asymmetrically modified CH3 domain comprising one or more asymmetric amino acid modifications. As used herein, "asymmetric amino acid modification" refers to a modification, such as a substitution or insertion, in which an amino acid at a particular position on a first CH3 or CH2 sequence is different from the amino acid at the same position on a second CH3 or CH2 sequence. These asymmetric amino acid modifications may result in only one of the two amino acids at the same respective amino acid position in each sequence being modified, or in both amino acids at the same respective position on each of the first and second CH3 or CH2 sequences being differently modified. Each of the first and second CH3 or CH2 sequences of the heterodimeric Fc may comprise one or more asymmetric amino acid modifications.

[0097] In some embodiments, the anti-NaPi2b antibody construct may comprise a heterodimeric Fc comprising a modified CH3 domain, the modified CH3 domain comprising one or more amino acid modifications that promote the formation of a heterodimeric Fc over the formation of a homodimeric Fc, in some embodiments, one or more of the amino acid modifications are asymmetric amino acid modifications.

[0098] Amino acid modifications that can be made to the CH3 domain of an Fc to promote heterodimeric Fc formation are known in the art and include, for example, those described in WO 96 / 027011 ("knobs into holes"), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 ("electrostatic steering"), Davis et al., 2010, Prot Eng Des Sel, 23(4):195-202 (strand exchange engineered domain (SEED) technology), and Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13):5145-50 (Fab arm exchange). Other examples include approaches that combine positive and negative design strategies to produce stable asymmetrically modified Fc regions, such as those described in International Publication Nos. WO2012 / 058768 and WO2013 / 063702. In certain embodiments, anti-NaPi2b antibody constructs may comprise scaffolds based on modified Fc regions such as those described in International Publication Nos. WO2012 / 058768 or WO2013 / 063702.

[0099] Table 5 provides the amino acid sequence of the human IgG1 Fc sequence (SEQ ID NO: 16), which corresponds to amino acids 231 to 447 of the full-length human IgG1 heavy chain. The CH3 sequence includes amino acids 341 to 447 of the full-length human IgG1 heavy chain. Table 5 also lists CH3 domain amino acid modifications that promote heterodimeric Fc formation, as described in International Patent Applications WO2012 / 058768 and WO2013 / 063702.

[0100] In certain embodiments, the anti-NaPi2b antibody construct may comprise a heterodimeric Fc scaffold having a modified CH3 domain comprising any one of variant 1, variant 2, variant 3, variant 4, or variant 5 modifications, as shown in Table 5.

[0101] Table 5: Human IgG1 Fc sequence 1, and CH3 domain amino acid modifications that promote heterodimer formation TIFF2025535238000008.tif130165

[0102] In some embodiments, an anti-NaPi2b antibody construct can comprise an Fc region-based scaffold comprising two CH3 sequences and two CH2 sequences, at least one of which comprises one or more amino acid modifications. Modifications in the CH2 domain can affect binding to Fc of Fc receptors (FcR), such as receptors of the FcγRI, FcγRII, and FcγRIII subclasses.

[0103] In some embodiments, the anti-NaPi2b antibody construct comprises an IgG Fc-based scaffold with a modified CH2 domain, where modification of the CH2 domain results in altered binding to one or more of the FcγRI, FcγRII, and FcγRIII receptors.

[0104] Many amino acid modifications to the CH2 domain that selectively alter the affinity of Fc for various Fcγ receptors are known in the art. Amino acid modifications that increase binding and those that decrease binding can each be useful in specific indications. For example, increasing the binding affinity of Fc to FcγRIIIa (an activating receptor) can result in increased antibody-dependent cell-mediated cytotoxicity (ADCC), which in turn leads to increased lysis of target cells. Similarly, decreasing binding to FcγRIIb (an inhibitory receptor) can be beneficial in some situations. Reducing or eliminating ADCC and complement-mediated cytotoxicity (CDC) may be desirable in certain indications. In such cases, modified CH2 domains containing amino acid modifications that increase binding to FcγRIIb or that reduce or eliminate binding of the Fc region to all Fcγ receptors ("knockout" variants) can be useful.

[0105] Examples of amino acid modifications to the CH2 domain that alter Fc binding via the Fcγ receptor include S298A / E333A / K334A and S298A / E333A / K334A / K326A (improved affinity for FcγRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1-2):132-41), F243L / R292P / Y300L / V305I / P396L (improved affinity for FcγRIIIa) (Stavenhagen, et al., 2007, Cancer Res, 67(18):8882-90), and F243L / R292P / Y300L / L235V / P396L (improved affinity for FcγRIIIa) (Nordstrom JL, et al. al., 2011, Breast Cancer Res, 13(6):R123), F243L (improved affinity for FcγRIIIa) (Stewart, et al., 2011, Protein Eng Des Sel., 24(9):671-8), S298A / E333A / K334A (improved affinity for FcγRIIIa) (Shields, et al., 2001, J Biol Chem, 276(9):6591-604), S239D / I332E / A330L and S239D / I332E (improved affinity for FcγRIIIa) (Lazar, et al., 2006, Proc Natl Acad Sci USA, 103(11):4005-10), and S239D / S267E and S267E / L328F (improved affinity for FcγRIIb) (Chu, et al., 2008, Mol Immunol, 45(15):3926-33). Various amino acid modifications to the CH2 domain that alter Fc binding by FcγRIIb are described in International Publication No. WO2021 / 232162. Additional modifications that affect Fc binding to Fcγ receptors are described in Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No. 11, ISBN 1 907568 37 9, October 2012, page 283).

[0106] In certain embodiments, the anti-NaPi2b antibody construct comprises an IgG Fc-based scaffold with a modified CH2 domain, which contains one or more amino acid modifications that result in reduced or eliminated binding of the entire Fc region to Fcγ receptors (i.e., a "knockout" variant).

[0107] Various publications describe strategies used to engineer antibodies to generate "knockout" variants (see, e.g., Strohl, 2009, Curr Opin Biotech 20:685-691, and Strohl & Strohl, "Antibody Fc engineering for optimal antibody performance" In Therapeutic Antibody Engineering, Cambridge: Woodhead Publishing, 2012, pp 225-249). These strategies include modifying glycosylation, using an IgG2 / IgG4 scaffold, or reducing effector function by introducing mutations in the hinge or CH2 domain of the Fc (see also U.S. Patent Publication No. 2011 / 0212087, International Publication No. WO2006 / 105338, U.S. Patent Publication No. 2012 / 0225058, U.S. Patent Publication No. 2012 / 0251531, and Strop et al., 2012, J. Mol. Biol., 420:204-219).

[0108] Examples of mutations that can be introduced into the hinge or CH2 domain to generate a "knockout" variant include the amino acid modifications L234A / L235A and L234A / L235A / D265S.

[0109] In certain embodiments, the anti-NaPi2b antibody constructs described herein may comprise a scaffold based on an IgG Fc with modified native glycosylation. As is known in the art, glycosylation of Fc can be modified to increase or decrease effector function. For example, mutation of the conserved asparagine residue at position 297 to alanine, glutamine, lysine, or histidine (i.e., N297A, Q, K, or H) results in a deglycosylated Fc lacking all effector function (Bolt et al., 1993, Eur. J. Immunol., 23:403-411; Tao & Morrison, 1989, J. Immunol., 143:2595-2601).

[0110] Conversely, removal of fucose from the oligosaccharide attached to heavy chain N297 has been shown to enhance ADCC due to improved binding to FcγRIIIa (see, e.g., Shields et al., 2002, J. Biol. Chem., 277:26733-26740, and Niwa et al., 2005, J. Immunol. Methods, 306:151-160). Such low-fucose antibodies can be produced, for example, in knockout Chinese hamster ovary (CHO) cells lacking fucosyltransferase (FUT8) (Yamane-Ohnuki et al., 2004, Biotechnol. Bioeng., 87:614-622), in the variant CHO cell line Lec13 with reduced ability to attach fucose to the carbohydrate attached to N297 (International Publication No. WO 03 / 035835), or in other cells that produce defucosylated antibodies (see, e.g., Li et al., 2006, Nat Biotechnol, 24:210-215; Shields et al., 2002 (ibid.); and Shinkawa et al., 2003, J. Biol. Chem., 278:3466-3473). Additionally, WO2009 / 135181 describes the addition of fucose analogs to the culture medium during antibody production to inhibit the incorporation of fucose into the carbohydrate on the antibody.

[0111] Other methods for producing antibodies with little or no fucose on the Fc glycosylation site (N297) are known in the art, e.g., GlymaX® technology (ProBioGen AG) (see von Horsten et al., 2010, Glycobiology, 20(12):1607-1618 and U.S. Patent No. 8,409,572).

[0112] Other glycosylation variants include those with bisected oligosaccharides, for example, variants in which biantennary oligosaccharides attached to the Fc region of an antibody are bisected by N-acetylglucosamine (GlcNAc). Such glycosylation variants may have reduced fucosylation and / or improved ADCC function (see, e.g., International Publication No. WO2003 / 011878, U.S. Patent No. 6,602,684, and U.S. Patent Application Publication No. US2005 / 0123546). Useful glycosylation variants also include those with at least one galactose residue in the oligosaccharide attached to the Fc region, which may have improved CDC function (see, e.g., International Publication Nos. WO1997 / 030087, WO1998 / 58964, and WO1999 / 22764).

[0113] Preparation of anti-NaPi2b antibody constructs The anti-NaPi2b antibody constructs described herein may be produced using standard recombinant methods known in the art (see, e.g., U.S. Pat. No. 4,816,567 and “Antibodies: A Laboratory Manual,” 2nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).

[0114] Typically, for recombinant production of an antibody construct, a polynucleotide or set of polynucleotides encoding the anti-NaPi2b antibody construct is generated and inserted into one or more vectors for further cloning and / or expression in a host cell. Polynucleotide(s) encoding the anti-NaPi2b antibody construct may be produced by standard methods known in the art (see, for example, Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 & update, and "Antibodies: A Laboratory Manual," 2nd Edition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014). As will be understood by those skilled in the art, the number of polynucleotides required for expression of an anti-NaPi2b antibody construct will depend on the format of the construct, including whether the antibody construct includes a scaffold. For example, if the anti-NaPi2b antibody construct is in a monospecific mAb or FSA format, two polynucleotides, each encoding one polypeptide chain, will be required. If multiple polynucleotides are required, they may be incorporated into one vector or into multiple vectors.

[0115] Generally, for expression, a polynucleotide or set of polynucleotides is incorporated into an expression vector(s) along with one or more control elements, such as transcriptional elements required for efficient transcription of the polynucleotide. Examples of such control elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. Those skilled in the art will understand that the selection of control elements will depend on the host cell selected for expression of the antibody construct and that such control elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. The expression vector may optionally further comprise a heterologous nucleic acid sequence that facilitates expression or purification of the expressed protein. Examples include, but are not limited to, signal peptides and affinity tags such as metal affinity tags, histidine tags, avidin / streptavidin coding sequences, glutathione-S-transferase (GST) coding sequences, and biotin coding sequences. The expression vector may be an extrachromosomal or integrating vector.

[0116] Suitable host cells for cloning or expressing anti-NaPi2b antibody constructs include various prokaryotic or eukaryotic cells known in the art. Eukaryotic host cells include, for example, mammalian cells, plant cells, insect cells, and yeast cells (such as Saccharomyces cells or Pichia cells). Prokaryotic host cells include, for example, E. coli cells, A. salmonicida cells, or B. subtilis cells.

[0117] In certain embodiments, anti-NaPi2b antibody constructs can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required, as described, for example, in U.S. Pat. Nos. 5,648,237, 5,789,199, and 5,840,523, and Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254, BKC Lo, ed. Humana Press, Totowa, NJ, 2003.

[0118] Eukaryotic microbes such as filamentous fungi or yeast may be suitable expression host cells in certain embodiments, particularly fungal and yeast strains in which the glycosylation pathway has been "humanized," resulting in the production of antibody constructs with partially or fully human glycosylation patterns (see, e.g., Gerngross, 2004, Nat. Biotech. 22:1409-1414, and Li et al., 2006, Nat. Biotech. 24:210-215).

[0119] Suitable host cells for the expression of glycosylated anti-NaPi2b antibody constructs are usually eukaryotic cells. For example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 describe the PLANTIBODIES™ technology for producing antigen-binding constructs in transgenic plants. Mammalian cell lines adapted to grow in suspension can be particularly useful for the expression of antibody constructs. Examples include SV40-transformed monkey kidney line CV1 (COS-7), human embryonic kidney (HEK) line 293 or 293 cells (see, e.g., Graham et al., 1977, J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse Sertoli TM4 cells (see, e.g., Mather, 1980, Biol Reprod., 23:243-251); monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma (HeLa) cells, canine kidney cells (MDCK), buffalo rat liver cells (BRL3A), human lung cells (W138), human liver cells (HepG2), mouse mammary tumor (MMT060562), TRI cells (see, e.g., Mather et al., 1982, Annals of NY Acad. Sci, 383: 44-68), MRC5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (DHFR - Exemplary mammalian host cell lines suitable for the production of antibody constructs include, but are not limited to, CHO cells; see Urlaub et al., 1980, Proc Natl Acad Sci USA, 77:4216), and myeloma cell lines (such as Y0, NS0, and Sp2 / 0). Exemplary mammalian host cell lines suitable for the production of antibody constructs are reviewed in Yazaki & Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (BKC Lo, ed. Humana Press, Totowa, NJ, 2003).

[0120] In certain embodiments, the host cell may be a transient or stable higher eukaryotic cell line, such as a mammalian cell line. In some embodiments, the host cell may be a mammalian HEK293T, CHO, HeLa, NS0, or COS cell line, or a cell line derived from any one of these cell lines. In some embodiments, the host cell may be a stable cell line that allows for mature glycosylation of the antibody construct.

[0121] Host cells containing expression vector(s) encoding the anti-NaPi2b antibody constructs can be cultured using conventional methods to produce the anti-NaPi2b antibody constructs. Alternatively, in some embodiments, host cells containing expression vector(s) encoding the anti-NaPi2b antibody constructs can be used therapeutically or prophylactically to deliver the anti-NaPi2b antibody constructs to a subject, or the polynucleotide or expression vector can be administered ex vivo to cells from a subject, which can then be returned to the subject's body.

[0122] Typically, anti-NaPi2b antibody constructs are purified after expression. Proteins may be isolated or purified in a variety of ways known to those skilled in the art (see, for example, Protein Purification: Principles and Practice, 3rd Ed., Scopes, Springer-Verlag, NY, 1994). Standard purification methods include chromatographic techniques (including ion exchange, hydrophobic interaction, affinity, sizing, or gel filtration, and reverse-phase chromatography) performed at atmospheric or elevated pressure using systems such as FPLC and HPLC. Additional purification methods include electrophoretic, immunological, precipitation, dialysis, and chromatofocusing techniques. Ultrafiltration and diafiltration techniques combined with protein concentration are also useful. As is well known in the art, various natural proteins bind to Fc and antibodies, and these proteins can be used to purify specific antibody constructs. For example, bacterial proteins A and G bind to the Fc region. Similarly, bacterial protein L binds to the Fab region of some antibodies. Purification can also be enabled by specific fusion partners. For example, antibodies can be purified by binding to glutathione resins when a GST fusion is used, or Ni when a His tag is used. +2 Purification may be performed using affinity chromatography or, if a flag tag is used, using an immobilized anti-flag antibody. The degree of purification required varies depending on the use of the anti-NaPi2b antibody construct. In some cases, no purification may be required.

[0123] In certain embodiments, the anti-NaPi2b antibody construct is substantially pure. The term "substantially pure" (or "substantially purified"), when used with reference to the anti-NaPi2b antibody constructs described herein, means that the antibody construct is substantially or essentially free from components that normally accompany or interact with the protein as found in its naturally occurring environment, such as in natural cells or, in the case of recombinantly produced constructs, in host cells. In certain embodiments, a substantially pure anti-NaPi2b antibody construct is a protein preparation having less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5% (by dry weight) of contaminating protein.

[0124] Certain embodiments of the present disclosure relate to methods for producing anti-NaPi2b antibody constructs, comprising culturing host cells into which one or more polynucleotides encoding the anti-NaPi2b antibody constructs or one or more expression vectors encoding the anti-NaPi2b antibody constructs have been introduced under conditions suitable for expression of the anti-NaPi2b antibody constructs, and optionally recovering the anti-NaPi2b antibody constructs from the host cells (or host cell culture medium).

[0125] Post-translational modifications In certain embodiments, the anti-NaPi2b antibody constructs described herein may contain one or more post-translational modifications. Such post-translational modifications may occur in vivo or may be performed in vitro after isolation of the anti-NaPi2b antibody construct from a host cell.

[0126] Post-translational modifications include various modifications known in the art (see, e.g., Proteins—Structure and Molecular Properties, 2nd Ed., TECreighton, W.H. Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, B.C. Johnson, Ed., Academic Press, New York, pp. 1-12, 1983; Seifter et al., 1990, Meth. Enzymol., 182:626-646, and Rattan et al., 1992, Ann. N.Y. Acad. Sci., 663:48-62). In embodiments in which an anti-NaPi2b antibody construct contains one or more post-translational modifications, the construct may contain the same type of modification at one or more sites, or may contain different modifications at different sites.

[0127] Examples of post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization with known protecting / blocking groups, formylation, oxidation, reduction, proteolytic cleavage or specific chemical cleavage (by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease or NaBH4).

[0128] Other examples of post-translational modifications include, for example, the addition or removal of N- or O-linked glycans, chemical modification of N- or O-linked glycans, N- or C-terminal processing, attachment of chemical moieties to the amino acid backbone, and the addition or deletion of N-terminal methionine residues resulting from expression in prokaryotic host cells. Post-translational modifications can also include modification with a detectable label, such as an enzyme label, a fluorescent label, a luminescent label, an isotopic label, or an affinity label, to enable detection and isolation of the protein. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and acetylcholinesterase. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, and phycoerythrin. Examples of luminescent materials include luminol and bioluminescent materials such as luciferase, luciferin, and aequorin. Examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon, and fluorine.

[0129] Additional examples of post-translational modifications include acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, pegylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA-mediated addition of amino acids to proteins such as arginylation, and ubiquitination.

[0130] Polynucleotides, Nucleotides and Host Cells Certain embodiments of the present disclosure relate to an isolated polynucleotide or set of polynucleotides that encodes an anti-NaPi2b antibody construct described herein. A polynucleotide in this context may encode all or part of an anti-NaPi2b antibody construct.

[0131] The terms "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Non-limiting examples of polynucleotides include genes, gene fragments, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0132] A polynucleotide "encoding" a given polypeptide is one that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence.

[0133] Certain embodiments of the present disclosure relate to vectors (such as expression vectors) that contain one or more polynucleotides encoding the anti-NaPi2b antibody constructs described herein. The polynucleotide(s) may be contained in a single vector or multiple vectors. In some embodiments, the polynucleotides are contained in a multicistronic vector.

[0134] Certain embodiments of the present disclosure relate to host cells comprising a polynucleotide(s) encoding an anti-NaPi2b antibody construct described herein, or one or more vectors comprising a polynucleotide(s). In some embodiments, the host cell is a eukaryotic organism, such as a Chinese hamster ovary (CHO) cell, a human embryonic kidney (HEK) cell, or a lymphoid cell (e.g., a Y0, NS0, or Sp20 cell).

[0135] Antibody-drug conjugates Certain embodiments of the present disclosure relate to antibody-drug conjugates (ADCs) comprising anti-NaPi2b antibody constructs conjugated to one or more drug moieties, such as a cytotoxin or an immunomodulator.

[0136] Typically, in ADCs, the anti-NaPi2b antibody construct is conjugated to a drug moiety via a linker, which can be cleavable or non-cleavable. The anti-NaPi2b antibody construct may be conjugated to a single drug molecule, or it may be conjugated to multiple drug molecules. The number of drug molecules conjugated to a single anti-NaPi2b antibody construct is defined by the drug-to-antibody ratio (DAR). In certain embodiments, the DAR in the ADCs of the present disclosure is in the range of about 1 to about 12, or about 2 to about 12, or about 2 to about 8.

[0137] In certain embodiments, the ADC comprising an anti-NaPi2b antibody construct has the general formula I: A-(L-(D) m ) n (I) and wherein A is an anti-NaPi2b antibody construct described herein; L is a linker; D is a drug moiety; m is an integer from 1 to about 8; and n is from 1 to about 12.

[0138] In certain embodiments in Formula I, m is 1 to 6. In some embodiments, m is 1 or 2. In some embodiments, n is from about 1 to about 8, e.g., from about 2 to about 8.

[0139] Various compounds known to be useful as cytotoxic or immunomodulatory ADC payloads may be used as drug moieties in ADCs containing anti-NaPi2b antibody constructs. Examples include, but are not limited to, maytansinoids and maytansinoid analogs, benzodiazepines and pyrrolobenzodiazepines, duocarmycins such as CC-1065 and analogs thereof, calicheamicins and calicheamicin analogs, auristatins and auristatin analogs, hemiasterlins and hemiasterlin analogs, tubulysins and tubulysin analogs, amatoxins and amatoxin analogs, camptothecins and camptothecin analogs, eribulin, TLR agonists (such as agonists of TLR7 and / or TLR8), and STING agonists.

[0140] In certain embodiments, the drug moiety included in an ADC of the disclosure is an auristatin or an auristatin analog, hemiasterin or a hemiasterin analog, camptothecin or a camptothecin analog, or eribulin.

[0141] Typically, in the ADCs of the present disclosure, the drug moiety is linked to the anti-NaPi2b antibody construct by a linker. The linker is a bifunctional or polyfunctional moiety that can link one or more drug molecules to the antibody construct. In some embodiments, the linker can be bifunctional (or monovalent) such that the linker links a single drug molecule to a single site on the antibody construct. In some embodiments, the linker can be polyfunctional (or multivalent) such that the linker links multiple drug molecules to a single site on the antibody construct. In some embodiments, a polyfunctional linker can also be used to link one drug molecule to multiple sites on the antibody construct.

[0142] Attachment of the linker to the anti-NaPi2b antibody construct can be achieved in a variety of ways, including reductive coupling to surface lysines, oxidized carbohydrates, or cysteine ​​residues liberated by reduction of interchain disulfide bonds. Alternatively, the attachment of the linker to the anti-NaPi2b antibody construct can be accomplished by adding an additional cysteine ​​residue (see, e.g., U.S. Patent Nos. 7,521,541, 8,455,622, and 9,000,130) or an unnatural amino acid that provides a reactive handle, such as selenomethionine, p-acetylphenylalanine, formylglycine, or p-azidomethyl-L-phenylalanine (see, e.g., Hofer et al., 2009, Biochemistry, 48:12047-12057; Axup et al., 2012, PNAS, 109:16101-16106; Wu et al., 2009, PNAS, 106:3000-3005; Zimmerman et al., 2012 ... This can be achieved by modifying the antibody construct to contain a linker (see, e.g., [PubMed], [Web of Science ® ...

[0143] The linker typically contains a functional group capable of reacting with a targeting group(s) on the antigen-binding construct and one or more functional groups capable of reacting with a targeting group on the drug moiety. Suitable functional groups are known in the art, including, for example, those described in Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press). Non-limiting examples of functional groups for reacting with free cysteine ​​or thiols include maleimides, haloacetamides, haloacetyls, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. Also useful in this context are "self-stabilizing" maleimides, such as those described in Lyon et al., 2014, Nat. Biotechnol., 32:1059-1062. Non-limiting examples of functional groups for reacting with surface lysines and amines include activated esters such as N-hydroxysuccinamide (NHS) esters or sulfo-NHS esters, imidoesters such as Traut's reagent, isothiocyanates, aldehydes, and acid anhydrides such as diethylenetriaminepentaacetic anhydride (DTPA). Other examples include succinimide-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP). Non-limiting examples of functional groups that can react with electrophilic groups (such as aldehyde or ketone carbonyl groups) on antibody constructs or drug moieties include hydrazides, oximes, aminos, hydrazines, thiosemicarbazones, hydrazine carboxylates, and arylhydrazides.

[0144] In certain embodiments, linkers containing functional groups that allow for cross-linking of two interchain cysteines on the antibody-binding construct may be used, such as the ThioBridge™ linker (Badescu et al., 2014, Bioconjug. Chem. 25:1124-1136), dithiomaleimide (DTM) linker (Behrens et al., 2015, Mol. Pharm. 12:3986-3998), dithioaryl (TCEP) pyridazinedione-based linker (Lee et al., 2016, Chem. Sci., 7:799-802), or dibromopyridazinedione-based linker (Maruani et al., 2015, Nat. Commun., 6:6645).

[0145] Various linkers for linking drugs to antibodies are known in the art, including hydrazone-, disulfide-, and peptide-based linkers. Linkers can be cleavable or non-cleavable. Cleavable linkers are typically susceptible to cleavage under intracellular conditions, for example, through lysosomal processes. Examples include protease-, acid-, or reduction-sensitive linkers. In contrast, non-cleavable linkers rely on antibody degradation in cells, which typically results in the release of the amino acid-linker-drug moiety.

[0146] Examples of cleavable linkers that may be useful in certain embodiments are peptide-containing linkers that are cleavable by intracellular proteases, such as lysosomal or endosomal proteases. Examples include dipeptide-containing linkers, such as the dipeptides Val-Cit, Phe-Lys, Val-Lys, Ala-Lys, Phe-Lys, Val-Cit, Phe-Cit, Leu-Cit, Ile-Cit, Trp-Cit, Phe-Arg, Ala-Phe, Val-Ala, Met-Lys, Asn-Lys, Ile-Pro, Ile-Val, Asp-Val, His-Val, Met-(D)Lys, Asn-(D)Lys, Val-(D)Asp, NorVal-(D)Asp, Ala-(D)Asp, Me3Lys, These include those containing s-Pro, phenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys, or Met-(D)Lys; tripeptide-containing linkers, such as those containing the tripeptides Met-Cit-Val, Gly-Cit-Val, (D)Phe-Phe-Lys, or (D)Ala-Phe-Lys; and tetrapeptide-containing linkers, such as those containing the tetrapeptides Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly, or Ala-Leu-Ala-Leu.

[0147] Additional useful cleavable linkers include linkers that are hydrolyzable at a specific pH or within a pH range, such as disulfide-containing linkers and hydrazone linkers. Examples of disulfide-containing linkers include, but are not limited to, N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB) and N-succinimidyl-4-(2-pyridyldithio)-2-sulfobutanoate (sulfo-SPDB). Optionally, disulfide-containing linkers may contain additional groups adjacent to the disulfide bond to provide steric hindrance (e.g., incorporation of geminal dimethyl groups) to improve the extracellular stability of the linker. Linkers containing a combination of these functional groups may also be useful; for example, linkers containing both hydrazones and disulfides are known in the art.

[0148] Further examples of cleavable linkers include β-glucuronide-containing linkers that can be cleaved by β-glucuronidase, an enzyme present in lysosomes and tumor stroma (see, e.g., De Graaf et al., 2002, Curr. Pharm. Des., 8:1391-1403).

[0149] The cleavable linker may optionally further comprise one or more additional functional groups, such as a self-immolative / self-leaving group, a stretcher, or a hydrophilic moiety.

[0150] Self-immolative / self-leaving groups utilized in linkers include, for example, p-aminobenzyloxycarbonyl (PABC) and p-aminobenzyl ether (PABE) groups, as well as methylated ethylenediamine (MED). Other examples of self-immolative groups include, but are not limited to, aromatic compounds electronically similar to PABC or PABE groups, such as heterocyclic derivatives, for example, 2-aminoimidazole-5-methanol derivatives, as described in U.S. Patent No. 7,375,078. Other examples include groups that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amide (Rodrigues et al., 1995, Chemistry Biology 2:223-227) and 2-aminophenylpropionic acid amide (Amsberry, et al., 1990, J. Org. Chem. 55:5867-5877). Self-immolative / self-leaving groups, alone or in combination, are often included in peptide-based linkers, but can also be included in other types of linkers. In some embodiments, a linker can include one or more self-immolative / self-leaving groups, such as a PABC group, a PABE group, or a combination of a PABC or PABE group with a MED.

[0151] Stretchers that have found use in linkers for ADCs include, for example, alkylene groups and stretchers based on fatty acids, diacids, amines, or diamines, such as diglycolate, malonate, caproate, and caproamide. Other stretchers include, for example, glycine-based stretchers and polyethylene glycol (PEG) or monomethoxypolyethylene glycol (mPEG) stretchers. PEG and mPEG stretchers can also function as hydrophilic moieties and may be particularly useful with hydrophobic drugs, although their use in linkers with other drugs is also contemplated in some embodiments.

[0152] ADCs comprising anti-NaPi2b antibody constructs can be prepared by one of several routes known in the art using standard organic chemistry reactions, conditions, and reagents (see, for example, Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press)). For example, conjugation can be achieved by (1) reacting a functional group of the antibody construct with a bivalent linker reagent to form an antibody-linker intermediate Ab-L via a covalent bond, followed by reaction with an activated drug moiety D, or (2) reacting a functional group of the drug moiety with a linker reagent to form a drug-linker intermediate DL via a covalent bond, followed by reaction with a functional group of the antibody construct. Conjugation methods (1) and (2) may be used with a variety of antibody constructs, drug moieties, and linkers to prepare the ADCs described herein.

[0153] A variety of prepared linkers, linker components, and drugs are commercially available or can be prepared using standard synthetic organic chemistry techniques (see, e.g., March's Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki et al., 2002, J. Org. Chem. 67:1866-1872; Frisch et al., 1997, Bioconj. Chem., 7:180-186; Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press); and Antibody-Drug Conjugates: Methods in Molecular Biology (Ducry (Ed.), 2013, Springer)). Additionally, several preformed drug-linkers suitable for reaction with a selected antibody construct are also commercially available; for example, drug-linkers containing DM1, DM4, MMAE, MMAF, or duocarmycin SA are available from Creative Biosciences. Antibody-drug conjugation services are available from BioLabs (Shirley, NY). Various antibody-drug conjugation services are also commercially available from companies such as Lonza Inc. (Allendale, NJ), Abzena PLC (Cambridge, UK), ADC Biotechnology (St. Asaph, UK), Baxter BioPharma Solutions (Baxter Healthcare Corporation, Deerfield, IL), and Piramal Pharma Solutions (Grangemouth, UK).

[0154] Once prepared, the ADCs can be purified by standard techniques such as chromatography (e.g., HPLC, size exclusion, adsorption, ion exchange and / or affinity capture), dialysis and / or tangential flow filtration.

[0155] How to use Certain aspects of the present disclosure relate to therapeutic or diagnostic uses of anti-NaPi2b antibody constructs and ADCs. NaPi2b is overexpressed in a variety of cancers, and therefore, certain embodiments of the present disclosure relate to methods of using anti-NaPi2b antibody constructs and ADCs in the treatment or diagnosis of NaPi2b-positive cancers.

[0156] Examples of cancers that may be treated in certain embodiments include carcinomas, including adenocarcinomas and squamous cell carcinomas, melanomas, and sarcomas. Carcinomas and sarcomas are also often referred to as "solid tumors." Examples of commonly occurring solid tumors that may be treated in certain embodiments include, but are not limited to, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, renal cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, gastric cancer, uterine cancer, non-small cell lung cancer (NSCLC), and colorectal cancer. Various forms of lymphoma can also lead to the formation of solid tumors and therefore may also be considered solid tumors in certain circumstances. Typically, the cancer to be treated is a NaPi2b-expressing cancer.

[0157] Certain embodiments relate to methods for inhibiting the growth of NaPi2b-positive tumor cells, comprising contacting the cells with an anti-NaPi2b antibody construct or ADC described herein. The cells may be in vitro or in vivo. In certain embodiments, the anti-NaPi2b antibody construct or ADC may be used in methods for treating NaPi2b-positive cancers or tumors in a subject.

[0158] Cancers that overexpress NaPi2b are typically solid tumors, including, but not limited to, ovarian cancer, endometrial cancer, and lung cancer (such as non-small cell lung cancer (NSCLC)).

[0159] Treating a NaPi2b-positive cancer may result in one or more of the following: alleviation of symptoms, reduction in tumor size, inhibition of tumor growth, reduction in one or more direct or indirect pathological consequences of the disease, prevention of metastasis, slowing of disease progression, improvement or palliation of disease symptoms, improved survival, prolonged progression-free survival, remission, and / or improved prognosis.

[0160] In certain embodiments, when used in the treatment of cancer, anti-NaPi2b antibody constructs and ADCs may be administered systemically to the subject being treated, for example, by bolus injection or continuous infusion into the subject's bloodstream. In certain embodiments, when used in the treatment of cancer, anti-NaPi2b antibody constructs and ADCs may be administered locally to the subject at the site to be treated.

[0161] It is contemplated that anti-NaPi2b antibody constructs and ADCs can be used alone or in combination with one or more known chemotherapeutic or immunotherapeutic agents typically used in cancer treatment. Combining an anti-NaPi2b antibody construct or ADC with a standard chemotherapeutic or immunotherapeutic agent can improve the efficacy of the chemotherapeutic or immunotherapeutic agent, thus improving standard cancer therapy. This application can be important in the treatment of drug-resistant cancers that do not respond to standard treatments. When used in combination with one or more known chemotherapeutic or immunotherapeutic agents, the anti-NaPi2b antibody construct or ADC can be administered before or after the administration of the chemotherapeutic or immunotherapeutic agent, or they can be administered simultaneously.

[0162] The dosage of the anti-NaPi2b antibody construct or ADC administered will be a therapeutically effective amount, without being subject to defined limitations. A "therapeutically effective amount" refers to an amount of the anti-NaPi2b antibody construct or ADC described herein that is sufficient to treat a particular indication when administered to a subject. A therapeutically effective amount of an anti-NaPi2b antibody construct or ADC for cancer therapy may, for example, have one or more of the following effects: a reduction in the number of cancer cells, a reduction in tumor size, inhibition of cancer cell invasion into peripheral organs, inhibition of tumor metastasis, inhibition of tumor growth, increased survival, and / or some alleviation of one or more symptoms associated with cancer. With respect to cancer therapy, efficacy can alternatively be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0163] Certain embodiments relate to methods of detecting the presence of NaPi2b in a biological sample, such as a sample containing cells or tissue, using the anti-NaPi2b antibody constructs described herein. In some embodiments, the biological sample may be obtained from a patient, for example, a patient known or suspected to have cancer. Some embodiments relate to methods of detecting the presence of NaPi2b in a biological sample, comprising contacting the sample with an anti-NaPi2b antibody construct described herein.

[0164] Certain embodiments relate to methods for diagnosing disorders associated with increased expression of NaPi2b, such as cancer, using the anti-NaPi2b antibody constructs described herein. The diagnostic method can be an in vivo method in which the anti-NaPi2b antibody construct is administered to a subject, or an in vitro method in which a sample taken from a subject is contacted with the anti-NaPi2b antibody construct. In the case of in vivo methods, administration can be systemic or local.

[0165] In methods for detecting the presence of NaPi2b or diagnosing disorders associated with increased expression of NaPi2b, the anti-NaPi2b antibody construct may be labeled with a detectable label, such as a fluorescent, luminescent, colorimetric, chemiluminescent, radioactive or enzymatic label, as known in the art.

[0166] Pharmaceutical Composition For therapeutic use, the anti-NaPi2b antibody constructs and ADCs can be provided in the form of pharmaceutical compositions comprising the anti-NaPi2b antibody construct or ADC and a pharmaceutically acceptable carrier or diluent. The compositions can be prepared by known procedures using well-known and readily available ingredients.

[0167] Pharmaceutical compositions can be formulated for administration to a subject, for example, by parenteral, oral (e.g., buccal or sublingual), topical, rectal, or vaginal routes, or by inhalation or spray. As used herein, the term "parenteral" includes subcutaneous injection, and intradermal, intraarticular, intravenous, intramuscular, intravascular, intrasternal, or intrathecal injection or infusion. Pharmaceutical compositions will generally be formulated in a manner suitable for administration to a subject, for example, as syrups, elixirs, tablets, troches, lozenges, hard capsules, soft capsules, pills, suppositories, oily suspensions, aqueous suspensions, dispersible powders, dispersible granules, emulsions, injections, or solutions. Pharmaceutical compositions may be provided as unit-dosage formulations.

[0168] In certain embodiments, a pharmaceutical composition comprising an anti-NaPi2b antibody construct or ADC may be formulated for parenteral administration, for example, as a lyophilized preparation or aqueous solution, by injection, or in a unit dosage injectable form.

[0169] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. Examples of such carriers include buffers such as phosphate, citric acid, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkylparabens (such as methyl or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol; low molecular weight (less than about 10 residues) polypeptides; serum albumin or These include, but are not limited to, proteins such as gelatin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates such as glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes such as Zn-protein complexes, and non-ionic surfactants such as polyethylene glycol (PEG).

[0170] In certain embodiments, pharmaceutical compositions containing anti-NaPi2b antibody constructs or ADCs may be in the form of sterile injectable aqueous or oily solutions or suspensions. Such suspensions may be formulated using suitable dispersing or wetting agents and / or suspending agents known in the art. Sterile injectable solutions or suspensions may contain anti-NaPi2b antibody constructs or ADCs in an orally acceptable, non-toxic diluent or solvent. Acceptable diluents and solvents that may be employed include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution. In addition, sterile, fixed oils may be used as a solvent or suspending medium. For this purpose, various brands of fixed oils, including synthetic mono- or diglycerides, may be employed. In addition, fatty acids such as oleic acid are also used in the preparation of injectable solutions. Adjuvants such as local anesthetics, preservatives, and / or buffers may also be included in the injectable solution or suspension.

[0171] In certain embodiments, pharmaceutical compositions containing anti-NaPi2b antibody constructs or ADCs can be formulated for intravenous administration to a subject, e.g., a human. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the compositions may also include a solubilizing agent and / or a local anesthetic, such as lignocaine, to ease pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in a unit dosage form, e.g., as a dry lyophilized powder or water-free concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of active agent. When the composition is to be administered by inhalation, it can be dispensed via an inhalation bottle containing pharmaceutical-grade sterile water or saline. When the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients can be mixed prior to administration.

[0172] Other pharmaceutical compositions and methods for preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remington's Pharmaceutical Sciences"), Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0173] Medicine Kit Certain embodiments relate to pharmaceutical kits comprising the anti-NaPi2b antibody constructs or ADCs described herein.

[0174] The kit typically includes a container holding the anti-NaPi2b antibody construct or ADC and a label and / or package insert on or associated with the container. The label or package insert includes instructions customarily included in commercial packaging for therapeutic products, such as those providing information or instructions regarding the indications, use, dosage, administration, contraindications, and / or warnings for use of the therapeutic product. The label or package insert may further include a notice in a form prescribed by a government agency regulating the manufacture, use, or sale of pharmaceutical or biological products, reflecting approval by that agency for manufacture, use, or sale for human or animal administration. In some embodiments, the container may have a sterile access port. For example, the container may be an intravenous solution bag or vial having a stopper that can be pierced by a hypodermic needle.

[0175] In addition to the container holding the anti-NaPi2b antibody construct or ADC, the kit may optionally include one or more additional containers containing other components of the kit, such as a pharmaceutically acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, or dextrose solution), other buffers, or diluents.

[0176] Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, etc. The containers may be formed from a variety of materials, such as glass or plastic. Where appropriate, one or more components of the kit may be lyophilized or provided in a dried form, such as a powder or granules, and the kit may additionally include a suitable solvent for the reconstitution of the lyophilized or dried component(s).

[0177] Kits may further include other materials desirable from a commercial or user standpoint, including filters, needles, and syringes.

[0178] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. [Example]

[0179] Example 1: Preparation of anti-NaPi2b antibodies An antibody construct that specifically binds to human NaPi2b was generated by immunizing mice with human cells overexpressing NaPi2b, as summarized below.

[0180] HEK293-6E cells (National Research Council of Canada) were transiently transfected with a pTT5-based expression plasmid encoding human NaPi2b (National Research Council of Canada, pTT5-huNaPi2b, expressing the NaPi2b sequence shown in SEQ ID NO: 1) using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. Ten B6x129 mice were subcutaneously immunized with the transfected HEK293-6E cells for 63 days, after which they were bled and their spleens were harvested.

[0181] Anti-human NaPi2b antibody titers were determined by flow cytometry using CHO-S cells expressing human NaPi2b. All 10 mice showed significant responses to human NaPi2b.

[0182] Splenocytes from all mice were then pooled and used for hybridoma generation. P3X63Ag8.653 cells (ATCC catalog no. CRL-1580) were mixed with IgG+ B cells isolated from the spleens and fused using an ECM2001 electrofusion apparatus (BTX, Harvard Bioscience) with optimized settings. After overnight recovery, hybridomas were diluted and plated in selection medium containing the following final concentrations of substituents: 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine. After 14 days of selection, hybridomas were diluted to an average of one cell per well and plated in 96-well plates. Cell supernatants containing secreted antibodies were evaluated for binding to CHO-S cells transfected with the same plasmid (pTT5-huNaPi2b) used to transfect HEK293-6E cells. Hybridoma cells were harvested from wells containing supernatants with antibodies that bind to NaPi2b and sequenced.

[0183] The mouse VH and VL sequences of one of the identified anti-NaPi2b antibodies were used to prepare a mouse-human chimeric IgG1 / kappa antibody construct, v23855, as follows. The coding sequences for the antibody variable regions were cloned in frame into a human IgG1 expression vector (with the human IgG1 constant region starting at alanine 118 according to Kabat numbering) or a human Ckappa expression vector (with the human Ckappa constant region starting at arginine 108 according to Kabat numbering). Both expression vectors are based on pTT5. The activity of the resulting recombinant chimeric antibody construct was confirmed in specificity binding assays and found to be comparable to that of the parent antibody (data not shown).

[0184] Example 2: Humanization of anti-NaPi2b antibody constructs A chimeric anti-human NaPi2b antibody construct, variant v23855, generated as described in Example 1, was humanized. The CDR sequences of v23855 are provided in Table 2.1 and the murine VH and VL sequences are provided in Table 2.2. Humanization was performed as described below.

[0185] Table 2.1. CDR sequences of anti-NaPi2b antibody construct v23855 TIFF2025535238000009.tif166165

[0186] Table 2.2. VH and VL sequences of anti-NaPi2b antibody construct v23855 TIFF2025535238000010.tif62165

[0187] 2.1 Humanization Sequence alignment of the murine VH and VL sequences of v23855 to their respective human germline sequences identified IGHV1-46*03 and IGKV1D-39*01 as the closest and most frequent human germline sequences (and selected the J region germline sequences of IGHJ4*03 and IGKJ2*04, respectively). As shown in Figures 1A and 1B, AbM-defined CDR sequences (see Table 2.1) were grafted onto the frameworks of these selected human germline sequences. Backmutations to murine residues in the resulting sequences (NaPi2b) at positions deemed likely to be important for retaining binding affinity to the antigen were included to generate several humanized sequences, where the resulting sequences were built on the previous sequences and the first humanized sequence contained no backmutations. None of the variants modified the CDRs of the parent antibody, as defined by the AbM method.

[0188] This process resulted in four humanized variable heavy chain sequences and three humanized variable light chain sequences. A complete heavy chain sequence containing a humanized heavy chain variable domain (VH) and a hIgG1 heavy chain constant domain (CH1, hinge, CH2, CH3), and a complete light chain sequence containing a humanized light chain variable domain (VL) and a human kappa light chain constant domain (kappaCL), were assembled. Monoclonal antibody (mAb) variants were then assembled, with each humanized heavy chain paired with each humanized light chain, providing 12 humanized variants that were experimentally evaluated.

[0189] 2.2 Production of humanized antibodies Each of the 12 humanized constructs, as well as the parental v23855 construct, was produced in full-size antibody (FSA) format and contained two identical full-length heavy chains and two identical kappa light chains.

[0190] The full-length heavy chain contained the human CH1-hinge-CH2-CH3 domain sequence of IGHG1*01 (SEQ ID NO: 33, see Table 2.3). The light chain contained the human kappa CL sequence of IGKC*01 (SEQ ID NO: 34, see Table 2.3).

[0191] Table 2.3: Constant heavy and light chain sequences TIFF2025535238000011.tif102165

[0192] Each of the mouse VH domain sequences, in addition to the humanized VH domain sequence, was appended to the human CH1-hinge-CH2-CH3 domain sequence of IGHG1*01 to provide four humanized complete heavy chain sequences and one parental mouse-human chimeric complete heavy chain sequence. Each of the humanized VL domain sequences or mouse VL domain sequences was appended to the human kappa CL sequence of IGKC*01 to provide three humanized light chain sequences and one parental mouse-human chimeric light chain sequence. All sequences were reverse translated into DNA, codon-optimized for mammalian expression, and genes were synthesized.

[0193] The heavy chain vector insert containing the signal peptide (artificially designed sequence: MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 35) (Barash et al., (2002), Biochem and Biophys Res. Comm., 294:835-842)) and the heavy chain clone ending at residue G446 (EU numbering) of the CH3 domain was ligated into the pTT5 vector to generate the heavy chain expression vector. The light chain vector insert containing the same signal peptide was ligated into the pTT5 vector to generate the light chain expression vector. The resulting heavy and light chain expression vectors were sequenced to confirm the correct reading frame and sequence of the encoding DNA. The sequences of the humanized VH and VL sequences are provided below in Table 2.4.

[0194] Table 2.4: Amino acid sequences of humanized VH and VL sequences TIFF2025535238000012.tif180165

[0195] The heavy and light chains of each humanized antibody variant and the parental mouse-human chimeric antibody variant were expressed in 300 mL cultures of CHO-3E7 cells. Briefly, 1.7-2 × 10 6 CHO-3E7 cells at a density of 100 cells / mL with >95% viability were cultured in FreeStyle™ F17 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 4 mM glutamine (Hyclone SH30034.01) and 0.1% Pluronic® F-68 (Gibco / Thermo Fisher Scientific, Waltham, MA) at 37°C. A total volume of 300 mL of CHO-3E7 cells plus 1× antibiotic / antimycotic (GE Life Sciences, Marlborough, MA) was transfected with a total of 300 μg of DNA (150 μg of antibody DNA and 150 μg of GFP / AKT / stuffer DNA) using PEI-MAX® (Polyscience, Inc., Philadelphia, PA) at a DNA:PEI ratio of 1:4 (w / w). 24 hours after addition of the DNA-PEI mixture, 0.5 mM valproic acid (final concentration) + 1% (w / v) tryptone (final concentration) was added to the cells, which were then transferred to 32°C and incubated for a further 6 days before harvesting.

[0196] Protein-A purification was performed using a 1 mL HiTrap™ MabSelect™ SuRe™ column (Cytiva, Marlborough, MA). The clarified supernatant sample was loaded onto a column equilibrated with Dulbecco's PBS (DPBS). The column was washed with DPBS. Protein was eluted with 100 mM sodium citrate buffer (pH 3.0). The pH of the eluted fraction was adjusted by adding 10% (v / v) 1 M HEPES (pH approximately 10.6-10.7) to a final pH of 6-7. The sample was buffer exchanged into DPBS using a 5 mL Zeba™ Spin column (Thermo Scientific). Protein was quantified based on absorbance at 280 nm (A280nm).

[0197] After purification, sample purity was assessed by SDS-PAGE under non-reducing and reducing conditions. Protein samples were mixed with NuPAGE® LDS sample buffer and NuPAGE® sample reducing agent (reducing conditions only) according to the manufacturer's protocol, and then the samples were heated at 70°C for 15 minutes. The treated protein samples, containing 1.5 μg of protein and MW Precision Plus Protein™ Dual Color (Bio-Rad) standards for molecular weight (MW) estimation, were loaded onto NuPAGE 4-12% Bis-Tris gels (15 wells). Gel electrophoresis was performed at 200 V for 50 minutes using a Life Technologies (Thermo Fisher Scientific) XCell SureLock® Mini-Cell system and NuPAGE® MOPS SDS running buffer. The gel was stained with BioSafe Coomassie solution, and gel images were captured using a ChemiDoc™ MP imaging system (Bio-Rad).

[0198] The yields for each of the 12 humanized antibody variants were similar, ranging from approximately 23 to 30 mg (or approximately 77 to 100 mg / L of culture), approximately twice as high as the parent mouse-human chimeric antibody v23855 (14 mg yield). SDS-PAGE results for these antibody samples are shown in Figure 2A (non-reduced) and Figure 2B (reduced). As can be seen in these figures, non-reduced (NR) and reduced (R) SDS-PAGE reflected a single species corresponding to the full-size antibody and intact heavy and light chains.

[0199] 2.3 Quality assessment of humanized antibodies Species homogeneity of the humanized antibody variants was assessed by UPLC-SEC after Protein A purification.

[0200] UPLC-SEC was performed using a Waters Acquity BEH200 SEC column (2.5 mL, 4.6 x 150 mm, stainless steel, 1.7 μm particles) (Waters LTD, Mississauga, ON) set at 30 °C and installed in a Waters Acquity UPLC™ H-Class Biosystem equipped with a photodiode array (PDA) detector. The mobile phase was Dulbecco's phosphate-buffered saline (DPBS) with 0.02% Tween 20, pH 7.4, and the flow rate was 0.4 mL / min. The total run time for each injection was 7 min, and the total mobile phase volume was 2.8 mL. Elution was monitored by UV absorbance in the range of 210–500 nm, and chromatograms were extracted at 280 nm. Peak integration was performed using Waters Empower® 3 software employing Apex Track™ to detect shoulder features.

[0201] Figures 2C and 2D show the UPLC-SEC profiles of the parental mouse-human chimeric antibody v23855 and a representative humanized antibody v29456 sample. The UPLC-SEC profile of the representative humanized antibody sample reflected high species homogeneity comparable to that of the parental mouse-human chimeric antibody sample. Samples from the remaining humanized antibody variants had profiles similar to those shown for the representative humanized antibody sample.

[0202] 2.4 Purity assessment of humanized antibodies The apparent purity of the humanized antibody variants was assessed using mass spectrometry and native deglycosylation.

[0203] 10 μg of each sample was incubated with 1 μg of deglycosylation mix (NEB, P6044) for 1 hour at room temperature and then transferred to a 37° C. incubator for 16 hours.

[0204] After deglycosylation, 5 μL of the eluted sample was transferred to a glass insert in an LC-MS vial. For LC-MS analysis, an Agilent 1290 Infinity II LC system coupled to an Agilent 6545 QTOF equipped with a Dual Jet Stream electrospray ionization source was used. A column temperature of 70°C and a flow rate of 0.3 mL / min were used with an Agilent PLRP-S column (1000 Å, 2.1 × 50 mm, 8 μm). The mobile phase consisted of A: LC-MS-grade water containing 0.1% v / v formic acid, 0.025 v / v trifluoroacetic acid, and 10% v / v isopropyl alcohol; and B: acetonitrile containing 0.1% v / v formic acid and 10% v / v isopropyl alcohol. The column was pre-equilibrated with 20% mobile phase B before injection. A gradient of 20 to 40% mobile phase B in 20 min was then applied, followed by a gradient of 27 to 90% mobile phase B in 2 min, with a 2 min column wash at 99% mobile phase B.

[0205] Example 3: Characterization of humanized anti-NaPi2b antibody constructs - Assessment of thermal stability The thermal stability of the humanized antibody variants was assessed by differential scanning calorimetry (DSC) as described below.

[0206] 400 μL of purified sample, primarily at a concentration of 0.4 mg / mL in PBS, was used for DSC analysis using a VP-Capillary DSC (Malvern Panalytical Inc., Westborough, MA). At the beginning of each DSC run, five buffer blank injections were performed to stabilize the baseline, and a buffer injection was set up before each sample injection for reference. Each sample was scanned from 20 to 100 °C at a rate of 60 °C / h using low feedback, an 8-second filter, a 3-minute pre-scan thermostat, and 70 psi nitrogen pressure. The resulting thermograms were referenced and analyzed using Origin 7 software (OriginLab Corporation, Northampton, MA) to determine the melting temperature (Tm) as an indicator of thermal stability.

[0207] The Fab Tm values ​​determined for the humanized variants are shown in Table 3.1. All humanized variants showed increased thermal stability compared to the parent antibody v23855 (Fab Tm of approximately 72.4°C), with Fab Tm values ​​ranging from approximately 78 to 83°C.

[0208] Table 3.1. Thermostability of humanized variants TIFF2025535238000013.tif91165

[0209] Example 4: Functional characterization of anti-NaPi2b antibody constructs—Competitive binding (epitope binning) To characterize the binding of the parent chimeric anti-NaPi2b antibody v23855 to NaPi2b, competitive binding or epitope binning assays were performed against the anti-NaPi2b reference antibodies MX-35 (v18992) and rifastuzumab (v18993). Binding was assessed by flow cytometry using HEK293-6e cells as described below.

[0210] Anti-NaPi2b detection antibodies: v23855, v18992, v18993, and palivizumab (anti-RSV, v16955) were conjugated with the AF647 fluorophore using the Zenon Human IgG Labeling Kit (ThermoFisher Scientific Corporation, Waltham, MA, catalog number Z25408, lot number 1937175). HEK293-6e cells were transfected for approximately 24 hours to transiently express human NaPi2b (pTT5-NaPi2b at 1 μg per million cells) or GFP (ATUM, Menlo Park, CA, pD2610-v23, also at 1 μg per million cells). After transfection, human NaPi2b-expressing HEK296-6e cells were mixed with transfected GFP-expressing HEK296-6e cells at a 4:1 ratio. 100,000 cells of this mixture were seeded into each well of a V-bottom 96-well plate and incubated with 100 μg / mL of unlabeled competing anti-NaPi2b antibody on ice for 1 hour. After incubation, the cells were washed and stained with 1 μg / mL of AF647-conjugated anti-NaPi2b detection antibody on ice for 1 hour. After staining and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ), collecting a minimum of 10,000 events per well. AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-human AF647 binding) was calculated for the FITC / GFP-negative live cell population using FlowJo™ version 10.8.1 (BD Biosciences, Falklin Lake, NJ). Percent inhibition was calculated using the following formula: TIFF2025535238000014.tif22165

[0211] Competitive binding results of the parent chimeric anti-NaPi2b antibody v23855 against v18992 (MX35) and v18993 (rifastuzumab) are shown in Table 4.1.

[0212] (Table 4.1) Competitive binding. Percent inhibition (relative to negative control antibody). GFP- population TIFF2025535238000015.tif81165

[0213] Each of the anti-NaPi2b antibodies tested competed with itself as expected (>95% inhibition, see bolded data). The chimeric anti-NaPi2b antibody v23855 competed for binding to v18992 and v18993, as demonstrated by comparable % inhibition (>94%) relative to itself. As expected, no competitive binding was observed with the negative control palivizumab (v16955).

[0214] Example 5: Functional characterization of humanized anti-NaPi2b antibody constructs - Cynomolgus monkey and mouse NaPi2b binding The binding cross-reactivity of humanized antibody variant v29456 to human, cynomolgus monkey, and mouse NaPi2b was assessed by flow cytometry using HEK293-6e transfected cells. Reference anti-NaPi2b antibodies MX-35 (v18992) and rifastuzumab (v18993) were included as comparisons, and the anti-RSV antibody palivizumab (v22277) was included as a negative control.

[0215] Briefly, HEK293-6e cells were transfected for approximately 24 hours to transiently express human NaPi2b, cynomolgus monkey, or mouse NaPi2b at 1 μg of DNA per million cells. After transfection, 50,000 cells per well were seeded into a V-bottom 96-well plate and treated with 200 nM primary antibody for 18–24 hours at 4°C to prevent internalization. After incubation, cells were washed and stained with anti-human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA, catalog number 109-605-098, lot number 124868) for 1 hour at 4°C. After staining and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ), collecting a minimum of 10,000 events per well. AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-human AF647 binding) was calculated for a single live cell population for each primary antibody using FlowJo™ v8 software (BD Biosciences, Franklin Lake, NJ). GraphPad Prism version 9 (GraphPad Software, San Diego, CA) was used to calculate the Bmax and Kd for each primary antibody.

[0216] The binding results for humanized antibody variants v29456, MX-35 (v18992) and rifastuzumab (v18993) are shown in Table 5.1 and in Figure 3A (human NaPi2b), Figure 3B (cynomolgus monkey NaPi2b), and Figure 3C (mouse NaPi2b).

[0217] Table 5.1. Cross-reactivity of v29456 to cynomolgus monkey and mouse NaPi2b TIFF2025535238000016.tif86165

[0218] v29456 and v18992 showed binding to human, cynomolgus monkey, and mouse NaPi2b on transfected HEK296-6e cells. Rifastuzumab (v18993) showed binding to human and cynomolgus monkey NaPi2b, with minimal binding to mouse NaPi2b-transfected HEK293-6e cells.

[0219] v29456 had apparent Kd values ​​comparable to v18992 and v18993 for human NaPi2b binding and showed the greatest binding to cynomolgus monkey NaPi2b, resulting in apparent Kds 10- and 2-fold lower than v18992 (MX35) and v18993 (rifastuzumab), respectively. The negative control palivizumab (v22277), as expected, did not bind to any species tested.

[0220] Example 6: Functional characterization of anti-NaPi2b antibody constructs - Cell binding of monovalent antibodies by Kinexa The binding affinity of anti-NaPi2b antibody constructs was assessed by Kinexa in the endogenously NaPi2b-expressing cell line IGROV-1. The affinity of the antibody constructs was assessed in a monovalent format to reduce the effects of avidity and internalization, and compared directly to the parent chimeric antibody variants, also in a monovalent format. v29814 (monovalent format of parent chimeric variant 23855), v36123 (monovalent format of humanized antibody variant 29452), and v36124 (monovalent format of humanized antibody variant 29456) were evaluated. Experiments were performed as described below.

[0221] IGROV-1 cell preparation: IGROV-1 cells were cultured in ATCC-modified RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA) in T175 culture flasks (Corning, Corning, NY) and incubated at 37°C and 5% CO2 until 80% confluency was achieved. Cells were detached from the culture vessel by incubation with Cell Dissociation Buffer (Invitrogen, Waltham, MA) for 30–60 min at 37°C and 5% CO2. Cells were harvested by neutralizing the Cell Dissociation Buffer with at least five volumes of ATCC-modified RPMI 1640 medium supplemented with 10% FBS and kept on ice until use. Cells were counted using a Vi-Cell™ XR viable cell analyzer (Beckman Coulter, Brea, CA).

[0222] KinExA preparation: The solid phase was prepared by coating one vial of PMMA (polymethyl methacrylate) beads (Sapidyne, Boise, Idaho) with 1 mL of 20 μg / mL BSA-biotin (Sigma-Aldrich, St. Louis, Missouri) in PBS pH 7.4. The beads were incubated for 2 hours at room temperature with gentle rotation. The beads were allowed to settle, the supernatant removed, and the beads rinsed five times with PBS pH 7.4. The beads were then coated with 1 mL of 100 μg / mL streptavidin (Jackson ImmunoResearch, West Grove, PA) in PBS pH 7.4 containing 10 mg / mL BSA (Sigma-Aldrich, St. Louis, Missouri) for 1 hour at room temperature with rotation. The beads were allowed to settle, the supernatant removed, and the beads rinsed five times with PBS pH 7.4. The final step in solid phase preparation was coating with 30 μg / mL biotin goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) for 1 hour at room temperature with rotation.

[0223] Cell binding assay: Cell binding assays were set up using antibody constructs or variants as stationary binding partners at two different concentrations: 50 pM and 500 pM. For titration curves with antibody variants fixed at 50 pM, at least 10 million IGROV-1 cells were used as titrants. For titration curves with antibody variants fixed at 500 pM, at least 5 million cells were used as titrants. Antibody variants and cells were mixed in PBS pH 7.4, 1 mg / mL BSA, 0.2% NaN3 and incubated at 4°C for 7 days with gentle rotation to reach equilibrium. After incubation, the antibody variant and cell mixture was centrifuged to separate the cells from unbound free antibody variants. Free antibody variants were loaded onto a KinExA 3200 (Sapidyne, Boise, Idaho) using biotinylated anti-human IgG PMMA as the solid phase and 0.5 μg / mL Alexa 647 goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) as the detection antibody.

[0224] Results for the parent chimeric monovalent antibody variant (v29814) and two humanized monovalent antibody variants are shown in Table 6.1. N-curve analysis was used to calculate affinity and receptor expression levels using the antibody variant concentrations as reference points. Narrow 95% confidence intervals were obtained for both affinity and receptor expression levels, with percent fit errors less than 1.5%. N-curve analyses are shown in Figure 4A (v29814), Figure 4B (v36123), and Figure 4C (v36124). For each panel, the right curve shows data for 500 pM constant binding partner, and the left curve shows data for 50 pM constant binding partner.

[0225] Table 6.1. Binding of anti-NaPi2b antibody constructs to IGROV-1 cells TIFF2025535238000017.tif90165

[0226] All humanized anti-NaPi2b antibody variants exhibited similar binding profiles and approximately two-fold lower binding affinities compared to the chimeric parent antibody construct. Calculated receptor expression levels ranged from 0.9 to 1.3 million per cell.

[0227] Example 7: Functional characterization of anti-NaPi2b antibody constructs - Internalization Internalization of the chimeric parent anti-NaPi2b antibody v23855 and a representative humanized variant v29456 (H1L2) in NaPi2b-expressing cell lines (HCC-78 and NCI-H441) was determined by flow cytometry as described below. The NaPi2b-targeting antibodies rifastuzumab (v18993) and MX35 (v18992) were used as positive controls, and the anti-RSV antibody palivizumab (anti-RSV) (v22277) was used as a negative control.

[0228] Briefly, antibodies were fluorescently labeled by conjugation to anti-human IgG Fc-targeting Fab fragment AF488 conjugates (Jackson Immuno Research Labs, West Grove, PA; catalog number 109-547-008) at a 1:1 molar ratio in PBS pH 7.4 (Thermo Fisher Scientific, Waltham, MA; catalog number 10010-023) for 24 hours at 4°C. Cells were seeded at 50,000 cells / well in RPMI 1640, ATCC modified (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) in 48-well plates and incubated overnight under standard culture conditions (37°C / 5% CO2) to allow binding. The next day, conjugated antibodies were added to the cells at 10 nM and incubated under standard culture conditions for 5-24 hours to allow internalization. After incubation, cells were dissociated and washed, and surface AF488 fluorescence was quenched using 100 nM anti-AF488 antibody (Life Technologies, Carlsbad, CA, catalog no. A-11094) for 30 minutes at 4°C. Quenched AF488 fluorescence (internalized fluorescence) was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ), collecting a minimum of 1,000 events per well. AF488 / FITC-A GeoMean (fluorescence signal geometric mean, proportional to anti-human Fab AF488 labeling) was calculated for live single-cell populations using FlowJo™ version 10.8.1 (BD Biosciences, Franklin Lake, NJ) and plotted using GraphPad Prism version 9 (GraphPad Software, San Diego, CA).

[0229] The results are shown in Figure 5A (HCC-78) and Figure 5B (NCI-H441) and in Table 7.1 below.

[0230] Table 7.1. Internalization of antibody constructs TIFF2025535238000018.tif82165

[0231] The chimeric parent antibody v23855 and humanized antibody variant v29456 exhibited comparable levels of internalization to the humanized antibody MX35 (v18992) at all time points (5 and 24 hours) in both HCC-78 and NCI-H441 cells at 10 nM antibody treatment, and significantly higher levels of internalization compared to the humanized antibody rifastuzumab (v18993). For example, after 5 hours of incubation in HCC-78 cells, v23855 and v29456 exhibited a 21.9-fold and 25.3-fold increase in internalization fluorescence, respectively, compared to the negative control palivizumab. Similarly, after 24 hours of incubation in HCC-78 cells, v23855 and v29456 exhibited a 50.9-fold and 59.9-fold increase in internalization fluorescence, respectively, compared to the negative control palivizumab.

[0232] Example 8: Evaluation of the developability of anti-NaPi2b antibodies The isoelectric points, self-aggregation tendency, and nonspecific binding of anti-NaPi2b antibodies v23855 (parent chimera), v29452 (H1L3), and v29456 (H1L2) were determined to assess their potential for development. Isoelectric points were measured by capillary isoelectric focusing (cIEF), self-aggregation tendency was measured by affinity capture self-interacting nanoparticle spectroscopy (AC-SINS), and nonspecific binding was measured by NS-ELISA, as described below.

[0233] Capillary isoelectric focusing (cIEF) cIEF was performed using the Maurice C. (ProteinSimple®) system, system adaptation kit, and method development kit. System suitability standards, fluorescence calibration standards, cartridges, and samples were prepared according to the vendor's recommendations. The capillaries were autocalibrated using pre-prepared fluorescence standards with the Maurice cIEF system adaptation kit to ensure proper capillary function. Antibody samples were diluted to a concentration of 0.5 mg / mL in a final volume of 40 μL in Gibco® distilled water and mixed with the Maurice cIEF method development kit sample. The samples were then vortexed, centrifuged, and the supernatants pipetted into individual wells of a 96-well plate. All electropherograms were detected by UV absorbance at 280 nm. All data analysis was performed using the vendor software Compass for iCE (ProteinSimple®). The Compass software aligns each electropherogram using a pI marker so that the x-axis is displayed as the normalized pI for each injection.

[0234] AC-SINS assay The AC-SINS method was performed in a 384-well plate format (Corning® #3702). First, 20 nm gold nanoparticles (Ted Pella, Inc., #15705), washed with 0.22 μm-filtered Gibco™ distilled water, were buffer-exchanged into 20 mM sodium acetate, pH 4.3, and coated with a mixture of capture antibody 80% AffiniPure goat anti-human IgG (H+L) (Jackson ImmunoResearch Laboratories® #109-005-088) and non-capture antibody 20% ChromPure goat IgG whole molecule (Jackson ImmunoResearch Laboratories® #005-000-003), diluted to 0.4 mg / mL. The mixture of gold nanoparticles, capture antibody, and non-capture antibody was incubated for 18 hours at room temperature in the dark. Unoccupied sites on the gold nanoparticles were blocked with 1 μM thiolated polyethylene glycol (2 kDa) in 20 mM sodium acetate (pH 4.3) to a final concentration of 0.1 μM, followed by incubation at room temperature for 1 hour. The coated nanoparticles were then concentrated by centrifugation at 21,000 x g for 7 minutes at 8°C. 95% of the supernatant was removed, and the gold pellet was resuspended in the remaining buffer. 5 μL of concentrated nanoparticles was added to 45 μL of 0.05 mg / mL antibody in Gibco™ PBS pH 7.4 in a 384-well plate. The coated nanoparticles were incubated with the antibody of interest in the dark at room temperature for 4 hours. Absorbance readings were taken every 1 nm from 450 to 700 nm, and a Microsoft Excel macro was used to identify the absorbance maximum, smooth the data, and fit the data using a second-order polynomial equation. The Δlambda (nm) was calculated based on the smoothed maximum absorbance of the average blank (PBS alone) subtracted from the smoothed maximum absorbance of the antibody sample to determine the antibody's AC-SINS score. Antibody-antibody interactions directly correlate with the shift in the maximum absorbance wavelength of gold nanoparticles coated with the antibody of interest. A Δlambda cutoff of 10 nm was set as a high tendency for antibodies to self-aggregate.

[0235] NS-ELISA NS-ELISA was used to measure the tendency of antibodies to bind to various biomolecules to emulate unwanted non-specific interactions to biological matrices in vivo, as described below.

[0236] NS-ELISA was performed in Corning® 96-well EIA / RIA Easy Wash™ Clear Flat Bottom Polystyrene High-Binding Microplates coated overnight at 4°C with 50 mL of heparin (Sigma, H3149) diluted in 50 mM sodium carbonate, pH 9.6, to a final concentration of 250 μg / mL. The plates were incubated at room temperature for 2 days, and the heparin-coated wells were allowed to air dry uncovered. Insulin (Sigma-Aldrich®, I9278) and KLH (Sigma-Aldrich®, H8283) were each diluted to a final concentration of 5 μg / mL in 50 mM sodium carbonate, pH 9.6. ssDNA (Sigma-Aldrich®, D8899) and dsDNA (Sigma-Aldrich®, D4553) were diluted to a final concentration of 10 μg / mL in Gibco™ PBS, pH 7.4. 50 μL each of insulin, KLH, dsDNA, and ssDNA was added to a 96-well plate, followed by incubation at 37° C. for 2 hours. The coating material was removed, and the plate was blocked with 200 μL of Gibco™ PBS pH 7.4, 0.1% Tween® 20 and incubated at room temperature for 1 hour with shaking at 200 rpm. The plate was washed three times with Gibco™ PBS pH 7.4, 0.1% Tween® 20. 50 μL each of mAb at 100 nM (15 mg / mL) in Gibco™ PBS pH 7.4, 0.1% Tween® 20 was added to duplicate wells and incubated at room temperature for 1 hour with shaking at 200 rpm. The plate was washed three times with Gibco™ PBS pH 7.4, 0.1% Tween 20, and 50 μL of 50 ng / mL anti-human IgG HRP (Thermofisher Scientific®, H10307) was added to each well. The plate was incubated for 1 hour at room temperature with shaking at 200 rpm. The plate was washed three times with Gibco™ PBS pH 7.4, 0.1% Tween 20, and 100 μL of TMB substrate (Cell Signaling Technology®, 7004P6) was added to each well.After approximately 10 minutes, 100 μL of 1 M HCl was added to each well to stop the reaction, and absorbance was read at 450 nm. Binding scores were calculated as the ratio of the antibody ELISA signal (antibody-treated) to the signal from wells containing buffer instead of primary antibody (untreated). The cutoffs considered for each binding molecule (ssDNA, KLH, insulin, dsDNA, and heparin) were calculated internally based on the average of antibodies from Zymeworks Inc. and published antibody benchmarks.

[0237] The results of all these assays are shown in Table 8.1. For these assays, scores higher than the cutoff were considered to indicate potentially less desirable biophysical properties.

[0238] Table 8.1: Evaluation results of the development feasibility of anti-NaPi2b antibodies TIFF2025535238000019.tif63165

[0239] The pI values ​​determined for the major isoforms of variants v23855, v29452, and v29456 were 8.35, 8.53, and 8.53, respectively, all of which fall within the typical range for therapeutic antibodies. ΔLambda analysis indicated no potential issues with AC-SINS for all variants. Furthermore, no potential issues were identified by NS-ELISA.

[0240] Example 9: Stability of humanized antibody variants in mouse plasma or PBS The objective of this experiment was to assess whether fragmentation of antibody variant v29456 and reference antibody MX35 (v18992) occurs over time following incubation at 37°C in mouse plasma or PBS pH 7.4.

[0241] Briefly, v29456 or v18992 were diluted to a final concentration of 0.5 mg / ml in either PBS or mouse plasma, respectively, and incubated at 37°C. Samples were removed after 0, 7, and 14 days and stored at -80°C until characterization. For characterization, samples were thawed at room temperature, and 50 μg was incubated with 5 μg of recombinant EndoS endoglycosidase for 1 hour at room temperature. Immunoprecipitation slurries were generated by incubating 95 μL / sample of magnetic Sepharose streptavidin-coated beads with 15 μg / sample of biotinylated goat anti-human IgG Fc capture antibody for 45 minutes, followed by four washes with PBS pH 7.4 using a DynaMag™-2 magnet (Invitrogen™).

[0242] After deglycosylation, the plasma samples were incubated with 95 μL of immunoprecipitation slurry at room temperature for 1.5 hours. The slurry was then washed six times with PBS pH 7.4 and twice with LC-MS grade water using a DynaMag™-2 magnet (Invitrogen™). A final wash with PBS pH 7.4 was performed before elution. Protein was eluted by incubating the beads with 35 μL of LC-MS grade water containing 20% ​​acetonitrile and 0.1% formic acid at room temperature for 1 hour. PBS samples were not immunoprecipitated.

[0243] Five microliters of the eluted sample was transferred to a glass insert in an LC-MS vial. For LC-MS analysis, 1 μL of the sample was injected onto a Waters™ BioSuite Phenyl column (1000 Å, 10 μm, 4.6 mm × 75 mm) using a Waters™ ACQUITY™ UPLC I Class HPLC system coupled to a Waters™ Synapt™ G2-Si HDMS at a column temperature of 70 °C and a flow rate of 0.3 mL / min. The mobile phase consisted of A: LC-MS grade water containing 0.1% v / v formic acid, 0.025% v / v trifluoroacetic acid, and 10% v / v isopropyl alcohol, and B: acetonitrile containing 0.1% v / v formic acid and 10% v / v isopropyl alcohol. The column was pre-equilibrated with 10% mobile phase B before injection. A gradient of 10 to 27% mobile phase B was then applied in 20 min, followed by a gradient of 27 to 90% mobile phase B in 2 min, with a 2 min column wash at 99% mobile phase B.

[0244] Between runs, the column was re-equilibrated with 10% mobile phase B for 2 min. ESI was performed in positive mode with a capillary voltage of 3 kV, a source temperature of 120 °C, a sampling cone voltage of 100 V, a source offset of 80 V, a source gas flow rate of 0 ml / min, a desolvation temperature of 500 °C, a cone gas flow rate of 0 L / h, a desolvation gas flow rate of 800 L / h, and a nebulizer gas flow rate of 6.5 bar. The data format was continuous, the analyzer was set in sensitivity mode, and the m / z range was 500–7000.

[0245] Peak integration, MS deconvolution, and mass assignment were performed with Protein Metrics Byos® v4.0 using a deconvolution window of 60,000–160,000 Da and an m / z range of 1,000–4,000. At all time points, the most intense deconvoluted mass was assigned as the reference mass of v29456. The reference mass was defined as the average mass of v29456 or v18992, and included the formation of two 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)] stubs, 16 disulfide bonds, and, where applicable, pyroglutamic acid at the N-terminus resulting from EndoS activity on N-glycans. A mass tolerance of ±10 Da was applied to mass assignment. Other assigned mAb proteoforms were the mAb reference mass with a phosphate adduct, the mAb reference mass missing one fucose unit, and the mAb reference mass with an additional hexose unit. A pentasaccharide adduct (presumably pentamannose) was also identified compared to the v29456 reference mass without 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-[alpha-L-fucopyranose-(1-6)]. Apparent purity was calculated as the ratio of the deconvoluted peak intensity of all v29456 or v18992 mAb proteoforms divided by the total observed deconvoluted peak intensity. Mouse plasma proteins present in the day 0 mouse plasma control but not observed in the PBS control were not considered in the apparent purity calculation.

[0246] The data are provided in Table 9.1 and show that there was no evidence of fragmentation of v29456 incubated in mouse plasma or PBS pH 7.4 at 37°C for 7 and 14 days, with apparent purity after 7 and 14 days being similar to the day 0 control. v18992 showed fragmentation after 14 days in PBS based on the appearance of low molecular weight species and a greater than 10% decrease in apparent purity compared to the day 0 control. No fragmentation of v18992 was observed in plasma.

[0247] Table 9.1: Stability of anti-NaPi2b antibodies in mouse plasma (apparent % of desired proteoform) TIFF2025535238000020.tif40165

[0248] Example 10: Quantification of surface NaPi2b on tumor cells Surface NaPi2b protein was measured in tumor cell lines by quantitative flow cytometry using a set of beads with known levels of antibody binding capacity (ABC), as described below. Tumor cells and beads were fluorescently labeled using the reference humanized antibody MX35 (v18992) conjugated to Alexa Fluor® AF647 and the control anti-RSV antibody palivizumab (v21995) conjugated to Alexa Fluor® AF647. Variant 22277 differs from the anti-RSV antibody v21995 used in the previous example in that it has a heterodimeric Fc, which does not affect the function of the antibody. Representative cell lines evaluated were OVCAR-3, IGROV-1, HCC-78, TOV-21G, NCI-H441, HCT116, and EBC-1.

[0249] Conjugation of v18992 and v21995 to Alexa Fluor® AF647 was performed as follows: v18992 and v21995 were each reacted with 8 equivalents of NHS-AF647 (Thermo Fisher #A20006, 10 mM) in PBS. The reactions were allowed to proceed for 200 and 150 minutes, respectively, at room temperature, protected from light. After incubation, the reactions were purified through four and two rounds of purification, respectively, using a 40 kDa Zeba column (Thermo Fisher, Waltham, MA) pre-equilibrated with PBS, pH 7.4. Confirmation of conjugation and quantification of unconjugated NHS-AF647 were measured by SEC chromatography (Ex: 650 nm, Em: 665 nm).

[0250] Cells were detached from the culture vessel using Cell Dissociation Buffer (Invitrogen, Waltham, MA) and seeded in triplicate at 50,000 cells / well in a conical-bottom 96-well plate. Cells and anti-human QSC® beads (Bangs Laboratories, Inc., Fishers, IN) were stained with v18992-AF647 containing a given excess level of conjugated antibody or the same concentration of negative control v21995-AF647 and incubated for 30 minutes at 4°C. After incubation, cells and beads were washed in FACS buffer and analyzed on a BD™ Fortessa HTS using FlowJo™ v8 software (BD Biosciences, Falklin Lake, NJ).

[0251] The median AF647 fluorescence intensity for all bead populations was plotted against the associated ABC values ​​using the Bangs Laboratories QuickCal v2.3 calibration line template for QSC® Anti-Human IgG Lot No. 14490.

[0252] Surface protein expression of cell lines was calculated based on a monovalent binding model and therefore corresponds to background-subtracted ABC (SABC). The median fluorescence intensity of v21995-AF647-stained cells from each of the respective cell lines was used as the background value for determining SABC.

[0253] The results are shown in Table 10.1. The reported NaPi2b protein per cell is the average of at least two biological replicates. Tumor cell lines were designated as high, medium, low, or negative expression of the target. If the average number of NaPi2b proteins detected was greater than 900,000 per cell, the cell line was designated as a "high" expresser. If the number was between 40,000 and 900,000 per cell, it was designated as "medium." If the number was between 500 and 40,000 per cell, it was designated as "low." If the number was negative (below the limit of quantitation of the calibration beads), it was designated as "negative."

[0254] Table 10.1. Surface NaPi2b quantification on tumor cell lines TIFF2025535238000021.tif80165

[0255] Example 11: Preparation of antibody-drug conjugates The antibody-drug conjugates (ADCs) shown in Table 11.1 were prepared. Exemplary protocols for preparing these ADCs are provided below, followed by drug-linker (DL) descriptions and payload structures in Table 11.2.

[0256] Table 11.1 Antibody Drug Conjugates and DAR TIFF2025535238000022.tif97165

[0257] Exemplary Protocol v29456-MC-GGFG-AM-DXd1 DAR8:A solution (2.47 mL) of humanized variant v29456 (54 mg) in PBS, pH 7.4 was diluted with PBS, pH 7.4 (1.00 mL) and reduced by the addition of 5 mM diethylenetriaminepentaacetic acid (DTPA) (0.90 mL in PBS, pH adjusted to 7.4) and 25 mM aqueous tris(2-carboxyethyl)phosphine (TCEP) (134 μL, 9.0 equiv.). After 4 hours at 37°C, the reduced antibody was purified by passage through a Zeba™ spin desalting column (40 KDa MWCO, Thermo Scientific™) pre-equilibrated with 1 mM DTPA in PBS, pH 7.4. An aliquot (13.5 mg, 1.32 mL) of the reduced antibody solution was diluted with 1 mM DTPA (33.5 μL in PBS, pH adjusted to 7.4). To the antibody solution was added 38.3 μL of DMSO and excess MC-GGFG-AM-DXd1 (111.7 μL; 12 equivalents) from a 10 mM DMSO stock solution. The conjugation reaction was allowed to proceed at room temperature for 120 minutes with mixing, at which point additional drug-linker (14 μL, 1.5 equivalents) was added. The conjugation reaction was allowed to proceed at room temperature for an additional 60 minutes with mixing. The conjugation reaction was quenched by adding an excess of 10 mM N-acetyl-L-cysteine ​​solution (76.8 μL, 8 equivalents).

[0258] v29456-MT-VC-Compound 1 DAR4: A solution (43.9 μL) of humanized variant v29456 (0.5 mg) in PBS, pH 7.4, was diluted to 44.3 μL with PBS, pH 7.4, and reduced by the addition of 5 mM diethylenetriaminepentaacetic acid (DTPA) (24 μL in PBS, pH adjusted to 7.4) and 1 mM aqueous tris(2-carboxyethyl)phosphine (TCEP) (7.76 μL, 2.25 equiv.). After 2 h at 37 °C, the reduced antibody was cooled to 0-4 °C. Excess MT-VC-Compound 1 (1.72 μL, 10 equiv.) from a 20 mM stock solution was added. The conjugation reaction was allowed to proceed for 90 min at 0-4 °C.

[0259] v23855-MT-VC-Compound 1 and v23855-MC-VC-PABC-MMAE:A solution (2.52 mL) of chimeric variant v23855 (15 mg) in PBS, pH 7.4, was diluted to 457 μL of PBS, pH 7.4, and reduced by the addition of 5 mM diethylenetriaminepentaacetic acid (DTPA) (750 μL in PBS, pH adjusted to 7.4) and 10 mM aqueous tris(2-carboxyethyl)phosphine (TCEP) (26.0 μL, 2.6 equiv.). After 2 h at 37 °C, the reduced antibody was cooled to 0-4 °C. Excess MT-VC-compound 1 or MC-VC-PABC-MMAE (50.0 μL, 10 equiv.) from a 20 mM stock solution was added. The conjugation reaction was allowed to proceed for 60 min at 0-4 °C. The conjugation reaction was quenched by adding an excess of 10 mM N-acetyl-L-cysteine ​​solution (26.7 μL, 8 equivalents). The quenching reaction was allowed to proceed for 30 minutes at 0-4°C.

[0260] v18992-MC-GGFG-AM-DXd1 DAR8: A solution (120.8 μL) of humanized variant v18992 (1 mg) in PBS, pH 7.4 was diluted with PBS, pH 7.4 (29.6 μL) and reduced by the addition of 5 mM diethylenetriaminepentaacetic acid (DTPA) (40.0 μL in PBS, pH adjusted to 7.4) and 10 mM aqueous tris(2-carboxyethyl)phosphine (TCEP) (9.62 μL, 14.0 equivalents). After 3 hours at 37 °C, the reduced antibody was purified by passage through a Zeba™ spin desalting column (40 KDa MWCO, Thermo Scientific™) pre-equilibrated with 10 mM NaOAc, pH 4.5. To the reduced antibody solution, 20.0 μL of DMSO and excess MC-GGFG-AM-DXd1 (10.31 μL; 15 equivalents) from a 10 mM DMSO stock solution were added. The conjugation reaction was allowed to proceed for 120 minutes at room temperature with mixing.

[0261] v18992-MT-VC-Compound 1 DAR3:A solution (4.23 mL) of humanized variant v18992 (35 mg) in PBS, pH 7.4, was diluted with PBS, pH 7.4 (2.2 mL) and reduced by the addition of 5 mM diethylenetriaminepentaacetic acid (DTPA) (1.75 mL in PBS, pH adjusted to 6.7) and 10 mM aqueous tris(2-carboxyethyl)phosphine (TCEP) (50.8 μL, 2.1 equiv.). After 2 h at 37 °C, the reduced antibody was cooled to 0-4 °C. Excess MT-VC-Compound 1 (120.9 μL, 10 equiv.) from a 20 mM stock solution was added. The conjugation reaction was allowed to proceed for 60 min at 0-4 °C. The conjugation reaction was quenched by the addition of excess 10 mM N-acetyl-L-cysteine ​​solution (64.5 μL, 8 equiv.). The quench reaction was allowed to proceed at 0-4°C for 30 minutes.

[0262] v18993-MC-GGFG-AM-DXd1 DAR8: A solution (156.5 μL) of humanized variant v18993 (1 mg) in PBS, pH 7.4 was diluted with PBS, pH 7.4 (1.96 μL) and reduced by the addition of 5 mM diethylenetriaminepentaacetic acid (DTPA) (42.0 μL in PBS, pH adjusted to 7.4) and 10 mM aqueous tris(2-carboxyethyl)phosphine (TCEP) (9.55 μL, 14.0 equivalents). After 3 hours at 37 °C, the reduced antibody was purified by passage through a Zeba™ spin desalting column (40 KDa MWCO, Thermo Scientific™) pre-equilibrated with 10 mM NaOAc, pH 4.5. To the reduced antibody solution, 20.0 μL of DMSO and excess MC-GGFG-AM-DXd1 (10.23 μL; 15 equivalents) from a 10 mM DMSO stock solution were added. The conjugation reaction was allowed to proceed for 120 minutes at room temperature with mixing.

[0263] v18993-MT-VC-Compound 1 DAR4:A solution (78.3 μL) of humanized variant v18993 (0.5 mg) in PBS, pH 7.4, was diluted with PBS, pH 7.4 (10.25 μL) and reduced by the addition of 5 mM diethylenetriaminepentaacetic acid (DTPA) (24.0 μL in PBS, pH adjusted to 7.4) and 1 mM aqueous tris(2-carboxyethyl)phosphine (TCEP) (7.50 μL, 2.2 equiv.). After 2 h at 37 °C, the reduced antibody was cooled to 0-4 °C. Excess MT-VC-Compound 1 (1.71 μL, 10 equiv.) from a 20 mM stock solution was added. The conjugation reaction was allowed to proceed at room temperature for 60 min.

[0264] v18993-MC-VC-PABC-MMAE DAR4: A solution (5.84 mL) of humanized variant v18993 (30 mg) in PBS, pH 7.4, was diluted with 0.60 mL of PBS, pH 7.4, and reduced by the addition of 5 mM diethylenetriaminepentaacetic acid (DTPA) (1.62 mL in PBS, pH adjusted to 7.4) and 10 mM aqueous tris(2-carboxyethyl)phosphine (TCEP) (55.3 μL, 2.7 equiv.). After 2.5 h at 37 °C, the reduced antibody was cooled to 0-4 °C. PBS, pH 7.4 (0.24 mL) was added, followed by the addition of excess MC-VC-PABC-MMAE (225 μL, 11 equiv.) from a 10 mM stock solution. The conjugation reaction was allowed to proceed for 16 h at 0-4 °C. The conjugation reaction was quenched by adding an excess of 10 mM N-acetyl-L-cysteine ​​solution (0.90 mL, 44 equivalents). The quenched reaction was allowed to proceed at room temperature for 60 minutes.

[0265] v22277-MC-GGFG-AM-DXd1 DAR8:A solution (2.18 mL) of control variant v22277 (10 mg) in PBS, pH 7.4, was diluted with PBS, pH 7.4 (4.1 μL) and reduced by the addition of 5 mM diethylenetriaminepentaacetic acid (DTPA) (563 μL in PBS, pH adjusted to 6.7) and 10 mM aqueous tris(2-carboxyethyl)phosphine (TCEP) (68.9 μL, 10.0 equivalents). After 3 hours at 37°C, the reduced antibody was purified by passage through a Zeba™ spin desalting column (40 KDa MWCO, Thermo Scientific™) pre-equilibrated with 10 mM NaOAc, pH 5.5. To the reduced antibody solution, 230.0 μL of DMSO and excess MC-GGFG-AM-DXd1 (51.7 μL; 15 equivalents) from a 20 mM DMSO stock solution were added. The conjugation reaction was allowed to proceed for 60 minutes at room temperature with mixing. The conjugation reaction was quenched by adding an excess of 20 mM N-acetyl-L-cysteine ​​solution (51.7 μL, 15 equivalents). The quenched reaction was allowed to proceed for 30 minutes at 0-4°C.

[0266] v22277-MT-VC-Compound 1 DAR4, v22277-MC-VC-PABC-MMAE DAR4: A solution (4.36 mL) of control variant v22277 (20 mg) in PBS, pH 7.4, was reduced by the addition of 5 mM diethylenetriaminepentaacetic acid (DTPA) (1.10 mL in PBS, pH adjusted to 7.4) and 10 mM aqueous tris(2-carboxyethyl)phosphine (TCEP) (35.1 μL, 2.55 equiv.). After 1.5 h at 37 °C, the reduced antibody was cooled to 0-4 °C. Excess MT-VC-compound 1 or MC-VC-PABC-MMAE (82.7 μL, 12 equiv.) from a 20 mM stock solution was added. The conjugation reaction was allowed to proceed for 60 min at room temperature or 0-4 °C.

[0267] v22277-AD-VC-Compound 1 DAR4: A solution (4.31 mL) of variant v22277 (20 mg) was diluted with 0.69 mL of PBS, pH 7.4. Excess TFP-AD-VC-Compound 1 (65.4 μL, 9.5 equiv.) from a 20 mM DMSO stock solution was added, and the conjugation was allowed to proceed overnight at room temperature.

[0268] v22277-MC-VC-PABC-MMAE DAR8: A solution (1.08 mL) of control variant v22277 (5 mg) in PBS, pH 7.4 was diluted to 81.1 μL of PBS, pH 7.4, and reduced by the addition of 5 mM diethylenetriaminepentaacetic acid (DTPA) (300 μL in PBS, pH adjusted to 6.7) and 10 mM aqueous tris(2-carboxyethyl)phosphine (TCEP) (41.3 μL, 10.0 equiv.). After 3 h at 37 °C, the reduced antibody was purified by passage through a Zeba™ spin desalting column (40 KDa MWCO, Thermo Scientific™) pre-equilibrated with PBS, pH 7.4. Excess MC-VC-PABC-MMAE (20.7 μL; 12 equiv.) from a 20 mM DMSO stock solution was added to the reduced antibody solution. The conjugation reaction was allowed to proceed for 60 min at room temperature with mixing.

[0269] Table 11.2: Drug-linkers used in the preparation of ADCs TIFF2025535238000023.tif39165

[0270] structure: TIFF2025535238000024.tif232165TIFF2025535238000025.tif180165

[0271] Example 12: Purification and characterization of antibody-drug conjugates ADCs prepared by cysteine ​​conjugation chemistry as described in Example 11 were purified by one to two passes over a Zeba™ Spin desalting column (40 KDa MWCO, Thermo Scientific™) pre-equilibrated with 10 mM NaOAc, pH 4.5 or pH 5.5, or PBS, pH 7.4.

[0272] ADCs prepared on a large scale as described in Example 11 were purified on an AKTA™ pure chromatography system (Cytiva Life Sciences, Marlborough, MA) using a 53 mL HiPrep 26 / 10 Desalting column (Cytiva Life Sciences, Marlborough, MA) and a mobile phase consisting of 10 mM NaOAc, pH 4.5 with 150 mM NaCl, at a flow rate of 10 mL / min. The purified ADCs were then sterile filtered (0.2 μm).

[0273] ADCs prepared by lysine conjugation chemistry as described in Example 11 were purified by a single pass through a Zeba™ Spin desalting column (40 KDa MWCO, Thermo Scientific™) pre-equilibrated with PBS, pH 7.4.

[0274] After purification, the ADC prepared for in vivo use was then sterile filtered (0.2 mm).

[0275] After purification, the concentration of the ADC was determined by BCA assay with reference to a standard curve generated using the humanized variant v29456. Alternatively, the concentration was estimated by measuring the absorbance at 280 nm using extinction coefficients obtained from the literature (EP 3342785 for DL1) or experimentally determined (for the remaining drug linker). The ADC was also characterized by hydrophobic interaction chromatography (HIC), reversed-phase ultra-performance liquid chromatography mass spectrometry (RP-UPLC-MS), and size-exclusion chromatography (SEC), as described below.

[0276] Hydrophobic interaction chromatography The antibody and ADC were analyzed by HIC to estimate the drug-to-antibody ratio (DAR). Chromatography was performed using a TSKgel® Butyl-NPR column (2.5 μm, 4.6 × 35 mm; TOSOH Bioscience GmbH, Griesheim, Germany) with a gradient of 95 / 5% MPA / MPB to 5 / 95% MPA / MPB (MPA = 1.5 M (NH4)2SO4, 25 mM NaCl) over a 12-minute period at a flow rate of 0.5 mL / min. x PO4 (pH 7) and MPB = 75% 25 mM Na x Analysis was performed on an Agilent Infinity II 1290 HPLC (Agilent Technologies, Santa Clara, CA) using a gradient of 25% HCl (pH 7, 25% isopropanol). Detection was by absorbance at 280 nm.

[0277] Reversed-phase ultra-high performance liquid chromatography mass spectrometry: Alternatively, the ADC may be analyzed by RP-UPLC-MS to determine the average drug-to-antibody ratio (DAR).

[0278] DAR determination of lysine-conjugated ADCs by RP-UPLC-MS The ADC samples were deglycosylated using Endo S for 1 hour at room temperature and injected onto an Agilent 1290 Infinity II LC coupled to an Agilent 6545 quadrupole time-of-flight (Q-TOF) mass spectrometer (Agilent Technologies, Santa Clara, CA). A PLRP-S column (1000 Å, 8 μM, 50 × 2.1 mm) was used to separate protein species at a flow rate of 0.3 ml / min using the gradient shown in Table 12.1. Mobile phase A: 0.1% formic acid (FA), 0.025% trifluoroacetic acid (TFA), and 10% isopropyl alcohol (IPA) in water. Mobile phase B: 0.1% FA and 10% IPA in acetonitrile (ACN).

[0279] Table 12.1: RP-HPLC-MS gradients TIFF2025535238000026.tif40165

[0280] The MS source conditions are shown in Table 12.2 and the acquisition parameters were as follows: Mode: MS, mass range: 500-7000 m / z, acquisition speed: 1 spectrum / s and 1000 ms / spectrum, 3354 transients / spectrum.

[0281] (Table 12.2) MS source conditions TIFF2025535238000027.tif34165

[0282] Qualitative analysis using MassHunter software (Agilent Technologies, Santa Clara, CA) was used for deconvolution and data analysis. Deconvolution parameters were as follows:

[0283] Deconvolution algorithm: Maximum entropy, mass range: 70,000-160,000, mass step: 1.0, limited m / z range to use: 1,000-7,000, baseline subtraction: 7.0, adducts: protons, isotope width: automatic, height filter: peak signal-to-noise ≥ 30.0, maximum number of peaks: limited by height 100.

[0284] The average DAR was calculated from the deconvoluted spectra using the following formula: TIFF2025535238000028.tif13165

[0285] DAR determination of cysteine-conjugated ADCs by RP-UPLC-MS The ADC samples were deglycosylated using Endo S for 1 hour at room temperature, reduced and denatured by TCEP incubation at 70°C for 1 hour, and injected onto an Agilent 1290 Infinity II LC coupled to an Agilent 6545 quadrupole time-of-flight (Q-TOF) mass spectrometer (Agilent Technologies, Santa Clara, CA). A PLRP-S column (1000Å, 8 μM, 50 × 2.1 mm) was used to separate protein species at a flow rate of 0.3 ml / min using the gradient shown in Table 12.3. Mobile phase A: 0.1% formic acid (FA), 0.025% trifluoroacetic acid (TFA), and 10% isopropyl alcohol (IPA) in water. Mobile phase B: 0.1% FA and 10% IPA in acetonitrile (ACN).

[0286] Table 12.3: RP-HPLC-MS gradients TIFF2025535238000029.tif40165

[0287] The MS source conditions are shown in Table 12.4 and the acquisition parameters were as follows: Mode: MS, Mass Range: 500-7000 m / z, Acquisition Speed: 1 spectrum / s and 1000 ms / spectrum, 3354 transients / spectrum.

[0288] (Table 12.4) MS source conditions TIFF2025535238000030.tif34165

[0289] Qualitative analysis using MassHunter software (Agilent Technologies, Santa Clara, CA) was used for deconvolution and data analysis. For a typical IgG1 ADC, reduction of the sample will separate the different species, with the light chain (LC) eluting first (DAR0, followed by DAR1, 2) and the heavy chain (HC) eluting later (DAR0, followed by DAR1, 2, 3). The TIC is integrated in two regions corresponding to the light and heavy chains and then deconvoluted. The deconvolution parameters were as follows:

[0290] Deconvolution algorithm: maximum entropy, mass range: 20,000-60,000, mass step: 1.0, limited m / z range to use: 700-3,000, baseline subtraction: 7.0, adducts: proton, isotope width: automatic, height filter: peak signal-to-noise ≥ 30.0, maximum number of peaks: limited by height 100.

[0291] The average DAR was calculated from the deconvoluted spectra using the following formula: TIFF2025535238000031.tif20165

[0292] Size exclusion chromatography The extent of antibody and ADC aggregation (approximately 15-150 μg, 5 μL injection volume) was assessed by SEC on an Agilent Infinity II 1260 HPLC (Agilent Technologies, Santa Clara, CA) using an AdvanceBio SEC column (300 Å, 2.7 μm, 7.8 × 150 mm) (Agilent, Santa Clara, California) and a mobile phase consisting of 150 mM phosphate, pH 6.95, and a flow rate of 1 mL / min. Detection was by absorbance at 280 nm.

[0293] result When determining DAR by HIC, the individual contributions of the DAR0, DAR2, DAR4, DAR6, and DAR8 species to the average DAR of the purified ADCs were assessed by integration of the HPLC-HIC chromatograms. The average drug-to-antibody ratio (DAR) for each ADC was determined by the weighted average of each DAR species. Regardless of the method, when rounded to the nearest integer, the average DAR for each ADC was the same as the target DAR shown in Table 12.1 below.

[0294] The degree of aggregation and monomer content were assessed by integration of HPLC-SEC chromatograms. The monomer peak for each ADC was identified as the peak with the same retention time as the unconjugated antibody from which each ADC was derived. All peaks with a shorter retention time than the monomeric species were determined to be aggregated species. The percent monomeric species determined for each ADC is shown in Table 12.1.

[0295] Table 12.1. ADC Characterization TIFF2025535238000032.tif97165

[0296] Example 13: Functional characterization of anti-NaPi2b antibody constructs - Cell binding of bivalent antibodies by flow cytometry The ability of the parent chimeric antibody construct v23855 and the humanized antibody variants described in Examples 2 and 3 to bind to NaPi2b expressed on cells was assessed by flow cytometry in the endogenous NaPi2b-expressing cell line IGROV-1, which expresses high levels of endogenous NaPi2b.

[0297] Briefly, cells were seeded at 50,000 cells / well in conical-bottom 96-well plates and treated with test antibodies for 24 hours at 4°C to prevent internalization. Palivizumab (anti-RSV antibody, v22277) was included as a negative control. Reference anti-NaPi2b antibodies rifastuzumab (v18993) and MX35 (v18992) conjugated to a maleimide-functionalized auristatin drug linker (DL2) were included for comparison. Conjugation of this drug linker showed no effect on antibody binding capacity (data not shown). After incubation, cells were washed and stained with anti-human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; catalog number 109-605-098) for 30 minutes at 4°C. After incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ), collecting a minimum of 1,000 events per well. AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-human AF647 binding) was calculated for the viable cell population using FlowJo™ version 10.8.1 (BD Biosciences, Franklin Lake, NJ) and plotted for each test antibody using GraphPad Prism version 9 (GraphPad Software, San Diego, CA).

[0298] The results for the parent chimeric construct (v23855) and all humanized antibody variants are tabulated in Table 13.1. Full dose-response binding curves for the parent chimeric antibody (v23855) and two representative humanized antibody variants (v29452, v29456) are shown in Figure 6.

[0299] Table 13.1. Binding of parental chimeric and humanized antibody variants to IGROV-1 cells TIFF2025535238000033.tif138165

[0300] All humanized antibody variants bound similarly to IGROV-1 cells, yielding apparent Kd values ​​within 2-fold and comparable Bmax values. The reference antibody MX35-DL2 ADC showed comparable binding to the chimeric v23855 and humanized antibodies. The humanized antibody rifastuzumab-DL2 ADC showed reduced binding, a lower Bmax value, and a higher apparent Kd value compared to all other targeted antibodies. The negative control palivizumab (v22277), as expected, showed no cell binding (NB).

[0301] Example 14: Functional Characterization of Anti-NaPi2b ADCs—Cell Binding by Flow Cytometry The ability of the ADCs to bind to cells expressing NaPi2b was evaluated. ADCs of a representative humanized variant antibody v29456 (H1L2) were prepared by conjugation to DL2 and DL1 as described in Example 11. Binding to the endogenously high NaPi2b-expressing cell lines IGROV-1 and HCC-78 was assessed by flow cytometry according to the method described in Example 5.

[0302] The results are shown in Table 14.1 and plotted in Figure 7A for IGROV-1 cells and Figure 7B for HCC-78 cells.

[0303] Table 14.1. Cell Binding of Antibodies and Antibody-Drug Conjugates TIFF2025535238000034.tif81165

[0304] Both antibody-drug conjugates yielded similar apparent Kd and Bmax values ​​compared to the unconjugated parent antibody v29456 in both the high NaPi2b-expressing cell lines IGROV-1 and HCC-78. The negative control palivizumab (v22277), as expected, did not bind to either cell line.

[0305] These results indicate that the ability of representative antibody variant v29456 to bind to cells expressing NaPi2b was not affected by conjugation to a drug-linker.

[0306] Example 15: Functional characterization of anti-NaPi2b ADCs—in vitro cytotoxicity in 2D monolayer cultures The cytotoxicity, as measured by cell growth inhibition, of humanized variant v29456 (H1L2) conjugated to various drug-linkers was evaluated in a panel of NaPi2b-expressing cell lines as described below. The cell lines used were OVCAR-3 (ovarian cancer), IGROV-1 (ovarian cancer), and HCC-78 (lung cancer). An ADC containing the antibody palivizumab (anti-RSV) (v22277) was used as a non-targeting control.

[0307] OVCAR-3 cells were cultured in ATCC-modified RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 20% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA) and 0.01 μg / mL human insulin (Sigma-Aldrich, Oakville, ON). IGROV-1 and HCC-78 cells were cultured in ATCC-modified RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS (Thermo Fisher Scientific, Waltham, MA). Briefly, cells were seeded in 384-well plates in 50 μL per well of standard culture medium and treated with 20 μL per well of a titration of test article prepared in ATCC-modified RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS. Cells were incubated under standard culture conditions for 4 days. After incubation, CellTiter-Glo® reagent (Promega Corporation, Madison, WI) was added to all wells, and luminescence corresponding to the ATP present in each well was measured using a Synergy™ H1 plate reader (BioTek Instruments, Winooski, VT). Percent cytotoxicity values ​​were calculated using the following formula: (1 - (luminescence of treated cells / mean luminescence of untreated cells)) x 100 and plotted against test article concentration using GraphPad Prism 9 software (GraphPad Software, San Diego, CA). EC50 values ​​were calculated using GraphPad Prism 9 based on nonlinear regression log(agonist) vs. response, variable slope (4 parameters).

[0308] The results are shown in Table 15.1 and representative curves are plotted in Figure 8A (OVCAR-3), Figure 8B (IGROV-1), and Figure 8C (HCC-78).

[0309] Table 15.1: In vitro cytotoxicity - 2D monolayers TIFF2025535238000035.tif83165

[0310] When comparing conjugates containing the same drug linker (DL2 and MC-GGFG-DXd) against all three NaPi2b-expressing cell lines, the humanized variant v29456 and humanized antibody MX35 (v18992) showed comparable potency and higher potency than the humanized antibody rifastuzumab (v18993). EC50 values ​​for NaPi2b target killing could not be calculated for the DXd ADC in OVCAR-3 cells (incomplete curve). Chimeric antibody v23855 conjugated to DL3 demonstrated subnanomolar NaPi2b target killing against all three cell lines. As expected, the palivizumab control ADC demonstrated log-fold higher EC50 values ​​than the NaPi2b-targeting ADCs against all three NaPi2b-expressing cell lines.

[0311] Example 16: In vivo activity of chimeric antibody drug conjugates The in vivo antitumor activity of the chimeric v23855 ADC was evaluated in the OVCAR3 xenograft model of ovarian cancer, which expresses high levels of NaPi2b. The antitumor activity of ADCs based on the reference antibodies rifastuzumab (v18993) and MX35 (v18992) was also evaluated for comparison. The study was performed as described below.

[0312] These data demonstrate that v23855-based ADCs are active against NaPi2b-expressing tumors in vivo.

[0313] In the high Napi2b-expressing OVCAR3 ovarian cancer model, tumor fragments (approximately 1 mm) derived from stock mice were 3 ) were subcutaneously implanted into female CB.17 SCID mice. The average tumor volume was approximately 100–150 mm. 3Upon reaching 100 mg / kg / day, animals were assigned to groups (n=8 per group) and administered a single IV dose of test article on Study Day 1. Tumor volumes and body weights were measured twice weekly for the 39-49 day study period. For statistical analysis, linear mixed-effects models were fitted to log-transformed tumor volumes, followed by F-tests and post-hoc pairwise comparisons for the null hypothesis of equal mean growth rates. Two studies were conducted with treatment groups listed below in Tables 16.1 and 16.2.

[0314] Table 16.1: Treatment Groups in OVCAR3 Study 1 TIFF2025535238000036.tif40165

[0315] Table 16.2: Treatment Groups in OVCAR3 Study 2 TIFF2025535238000037.tif46165

[0316] The results of OVCAR3 Study 1 are shown in Figure 9A and demonstrate that the parent chimeric antibody v23855-DL2 at doses of 6 and 18 mg / kg resulted in significant inhibition of tumor growth compared to vehicle control (p<0.05). v23855-DL2 inhibited tumor growth comparable to the reference antibody v18992-DL2 at either the 6 or 18 mg / kg dose level.

[0317] The results of OVCAR3 Study 2 are shown in Figure 9B and demonstrate that a 6 mg / kg dose of chimeric v23855-DL2 resulted in significant inhibition of tumor growth compared to the vehicle control (p<0.05). At 6 mg / kg, v23855-DL2 resulted in greater inhibition of tumor growth than the reference antibody v18993-DL2. At 6 mg / kg, v23855-DL3 resulted in greater inhibition of tumor growth than the reference antibody v18993-DL3.

[0318] Taken together, these studies demonstrate that ADCs containing the parent chimeric v23855, when conjugated to either DL2 or DL3, exhibit superior antitumor activity compared to ADCs containing the reference antibody v18993. When conjugated to DL2, v23855 exhibited non-inferior activity to the reference antibody v18992.

[0319] Example 17: In vivo activity of humanized antibody drug conjugates The in vivo antitumor activity of an ADC of humanized antibody variant v29456 conjugated to DXd1 was evaluated in the OVCAR3 xenograft model of ovarian cancer and the NCI-H441 xenograft model of lung cancer, both of which express high levels of NaPi2b.

[0320] In the high NaPi2b-expressing OVCAR3 ovarian cancer model, tumor fragments (approximately 1 mm 3 ) were subcutaneously implanted into female CB.17 SCID mice. The average tumor volume was approximately 100–150 mm. 3 Once this was reached, animals were assigned to groups (n=8 per group) and administered a single IV dose of test article on Study Day 1. Tumor volumes and body weights were measured twice weekly for the 60-day study period. Treatment groups are listed in Table 17.1.

[0321] Table 17.1: Treatment Groups in the OVCAR3 Trial TIFF2025535238000038.tif34165

[0322] In the NCI-H441 model of lung cancer, 5 × 10 cells in 0.1 ml of 1:1 PBS:Matrigel were implanted subcutaneously into male NU-Foxn1 mice. The mean tumor volume was approximately 145 mm. 3Upon reaching 17.5 mg / kg / day, animals were assigned to groups (n=6 per group) and administered a single IV dose of test article on study day 0. Tumor volumes and body weights were measured twice weekly for the 28-35 day study period. For statistical analysis, linear mixed-effects models were fitted to log-transformed tumor volumes, followed by F-tests and post-hoc pairwise comparisons for the null hypothesis of equal mean growth rates. Two studies were conducted in the NCI-H441 model with treatment groups described in Table 17.2 and Table 17.3.

[0323] Table 17.2: Treatment Groups in NCI-H441 Study 1 TIFF2025535238000039.tif34165

[0324] Table 17.3: Treatment Groups in NCI-H441 Study 2 TIFF2025535238000040.tif34165

[0325] Results from the OVCAR3 model are shown in Figure 10 and demonstrate that v29456-DL1 produced potent tumor growth inhibition at 1, 3 and 10 mg / kg.

[0326] The results of NCI-H441 Model Study 1 are shown in Figure 11A and demonstrate that v29456-DL1 produced potent tumor growth inhibition at 1, 3 and 10 mg / kg.

[0327] Results from NCI-H441 Model Study 2 are shown in Figure 11B and demonstrate that v29456-DL1 produced moderate inhibition of tumor growth at 0.3 mg / kg and potent tumor growth inhibition at 1 mg / kg. At 1 mg / kg, the activity of the non-targeting control v21995 (palivizumab)-DL1 was lower than that of v29456-DL1, demonstrating the target-dependent activity of the v29456 ADC.

[0328] Example 18: Evaluation of the specificity of anti-NaPi2b antibodies A Membrane Proteome Array™ (Integral Molecular, Philadelphia, PA, USA) was used to screen for specific off-target binding interactions of the antibody humanized v38591 anti-NaPi2b (SLC34A2) variant. This anti-NaPi2b humanized antibody variant has essentially the same amino acid sequence as v29456. The DNA sequence encoding the heavy chain of v38591 is identical to that encoding v29456, except that the heavy chain contains a C-terminal lysine, which is cleaved from most antibody products upon secretion from the cells in which they are produced.

[0329] Briefly, the study consisted of three phases: Phase (1) determination of assay screening conditions, Phase (2) membrane proteome array (library) screening, and Phase (3) protein target validation. In Phase (1), we determined appropriate conditions for detecting v38591 binding by high-throughput flow cytometry, including optimal antibody concentrations and cell types (two cell types were tested: HEK293T and avian QT6). In Phase (2), using the optimal conditions determined in Phase (1), v38591 was screened against a library of over 6,000 human membrane proteins (individually expressed in unfixed HEK293T cells), including 94% of all single-pass, multi-pass, and GPI-anchored proteins, including GPCRs, ion channels, and transporters. In Phase (3), each protein target hit from the screening stage (potential off-target interactions) was evaluated in a titration experiment using flow cytometry.

[0330] In Phase (1), the HEK293T cell type and an antibody concentration of 20 μg / mL were determined to be optimal for library screening. As shown in Figure 12A, library screening resulted in validated protein target hits for NaPi2b's primary target and FcgR1A, which binds to the Fc portion of the antibody. Another validated protein target hit was CLDN3. CLDN3 validation data showed that it was a very weak binder to humanized v38591 in the validation assay (Figure 12B), as indicated by the low binding signal to v38591 across a range of concentrations (MFI approximately 60-275) compared to the strong binding signal for NaPi2b (MFI approximately 3500-7000). In general, this data demonstrates the high specificity of humanized v38591 for its primary target, NaPi2b.

[0331] Example 19: Functional characterization of anti-NAPI2B antibodies - Cell binding of anti-NAPI2B antibodies by flow cytometry The ability of humanized antibody variants v38591 and v29456 to bind to cell-expressed NaPi2b was assessed by flow cytometry in the endogenously NaPi2b-expressing tumor cell lines IGROV-1 (ovarian adenocarcinoma) and TOV-21G (ovarian carcinoma). IGROV-1 and TOV-21G cells express endogenous NaPi2b at high and moderate levels, respectively, as described in Example 10.

[0332] Cell binding was performed according to the methods described in Example 13. The reference anti-NaPi2b antibody rifastuzumab (v18993) and the anti-RSV antibody palivizumab (v22277) were included as positive and negative controls, respectively.

[0333] The results are shown in Table 19.1 and plotted in Figure 13. Both v38591 and v29456 showed comparable cell binding to IGROV-1 and TOV-21G cell lines, indicating that the addition of a C-terminal lysine to the antibody heavy chain had minimal impact on the antibody's cell binding ability. The anti-NaPi2b antibody control v18993 showed slightly weaker binding compared to v38591 in IGROV-1 and TOV-21G, with approximately 1.2- and 1.4-fold decreases in the upper Bmax, respectively. The negative control palivizumab (v22277), as expected, showed no cell binding.

[0334] Table 19.1 Cell binding of anti-NaPi2b antibodies TIFF2025535238000041.tif80165

[0335] Example 20: Functional characterization of anti-NaPi2b antibodies—NaPi2b specificity The binding cross-reactivity of humanized antibody variant v38591 to human NaPi2b, NaPi2a, and NaPi2c was assessed by flow cytometry using HEK293-6e transfected cells. Reference anti-NaPi2b antibodies MX35 (v18992) and rifastuzumab (v18993) were included as positive controls. Reference anti-NaPi2a (polyclonal rabbit anti-human SLC34A1; Atlas Biotechnologies Inc, Edmonton, AB; catalog number HPA051255) and anti-NaPi2c (polyclonal rabbit anti-human SLC34A3; Thermo Fisher Scientific, Waltham, MA; catalog number PA5-50762) antibodies were included. Palivizumab (anti-RSV antibody, v22277) was included as a negative control.

[0336] HEK293-6e cells were maintained under standard culture conditions (37°C / 5% CO2) with shaking at 110 rpm for suspension and cultured in FreeStyle™ 293 Expression Medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 1% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) and 1X penicillin-streptomycin (Thermo Fisher Scientific, Waltham, MA). Cells were transfected with human NaPi2b (pTT5-huNaPi2b) (CL_#13432), human NaPi2a (CL_#13435), or human NaPi2c (CL_#13436) (all from GenScript Biotech, Piscataway, NJ) at 1 μg DNA per million cells using 293Fectin™ Transfection Reagent (Thermo Fisher Scientific, Waltham, MA) and Opti-MEM™ I Reduced Serum Medium (Thermo Fisher Scientific, Waltham, MA) and incubated for 24 hours under standard culture conditions (37°C / 5% CO2 / 110 rpm). After transfection, cells were seeded at 50,000 cells / well in conical-bottom 96-well plates and treated with test antibodies for 24 hours at 4°C to prevent internalization. After incubation, cells were washed and stained with anti-human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA, catalog number 109-605-098) or anti-rabbit IgG F(ab")2 AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA, catalog number 111-605-047). After incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) collecting a minimum of 1,000 events per well.AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-human AF647 binding) was calculated for the live cell population using FlowJo™ version 10.8.1 (BD Biosciences, Falklin Lake, NJ) and plotted for each test antibody using GraphPad Prism version 9 (GraphPad Software, San Diego, CA).

[0337] The results are shown in Table 20.1. Antibody variant v38591 and reference antibodies v18993 and v18992 showed comparable binding to each other on NaPi2b-transfected HEK293-6e cells and minimal binding to NaPi2a- and NaPi2c-transfected HEK293-6e cells. The positive NaPi2a-binding control antibody from Atlas Bio showed binding on NaPi2a-transfected cells, some binding to NaPi2c-transfected cells, and no binding to NaPi2b-transfected cells. The positive NaPi2c-binding control antibody from Thermo Fisher Scientific showed some binding to NaPi2c-transfected cells, but no binding to NaPi2a- or NaPi2b-transfected cells, as expected. The negative control palivizumab (v22277) showed no cell binding, as expected.

[0338] Table 20.1: Cross-reactivity of v38591 ADC, parental antibody, and control to cynomolgus monkey and mouse NaPi2b TIFF2025535238000042.tif150165

[0339] Example 21: Pharmacokinetic evaluation of anti-NaPi2b antibodies in TG32 mice The pharmacokinetics (PK) of the v29456 antibody and v18993 (rifastuzumab)-MCvcPABC-MMAE DAR4 were evaluated in humanized FcRn Tg32 mice, a mouse model that is predictive of human drug pharmacokinetics (see Avery et al. (2016) Utility of a human FcRn transgenic mouse model in drug discovery for early assessment and prediction of human pharmacokinetics of monoclonal antibodies, mAbs, 8:6, 1064-1078).

[0340] As shown in Table 26.1, all test articles were administered at 5 mg / kg by intravenous injection to hFcRn Tg32 mice (The Jackson Laboratory, Sacramento, CA; stock number 014565). For each test article, blood was collected from n=4 animals by retro-orbital plexus bleeding at 1, 3, and 6 hours and 1, 3, 7, 10, 14, and 21 days post-dose. Blood was processed to serum and stored frozen at -80°C in 96-well storage plates prior to pharmacokinetic analysis.

[0341] Table 26.1: Test substances and doses for PK evaluation TIFF2025535238000043.tif33165

[0342] Mouse serum containing v29456 antibody and v18993 (rifastuzumab)-MCvcPABC-MMAE DAR4 was captured onto a 384-well plate coated with goat anti-human IgG Fc antibody (Jackson 109-005-098). Total antibodies were detected with goat anti-human IgG Fab biotin antibody (Jackson 109-065-097) followed by streptavidin SULFO-TAG conjugate (Mesoscale). After addition of MSD GOLD Read Buffer A, the electrochemiluminescence (ECL) signal from the SULFO-TAG label was measured using a plate reader (Mesoscale).

[0343] The resulting PK profiles are shown in Figure 14. The anti-NaPi2b antibody v29456 exhibited a typical antibody PK profile that was largely comparable between the test article and the rifastuzumab-MMAE comparator.

[0344] The disclosures of all patents, patent applications, publications, and database entries mentioned in this specification are specifically incorporated by reference in their entirety into this specification to the same extent as if each individual patent, patent application, publication, and database entry was specifically and individually indicated to be incorporated by reference.

[0345] Modifications of the specific embodiments described herein which will be obvious to those skilled in the art are intended to be included within the scope of the following claims.

[0346] Table of Additional Sequences (SEQ ID NOs: 36-59) (Table A) Variant clone numbers TIFF2025535238000044.tif112165 (Table B) Clone amino acid sequence (full chain) TIFF2025535238000045.tif70165TIFF2025535238000046.tif208165TIFF2025535238000047.tif213165TIFF2025535238000048.tif123165(Table C) Clone DNA sequences (VH and VL of v29449-29460) TIFF2025535238000049.tif81165TIFF2025535238000050.tif174165

Claims

1. 1. An antibody construct comprising an antigen-binding domain that binds to human NaPi2b (sodium-dependent phosphate transport protein 2B), wherein the antigen-binding domain comprises: (a) a heavy chain CDR1 (HCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 7, a heavy chain CDR2 (HCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 8, and a heavy chain CDR3 (HCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO: 9; and (b) a light chain CDR1 (LCDR1) amino acid sequence comprising the sequence set forth in SEQ ID NO: 19, a light chain CDR2 (LCDR2) amino acid sequence comprising the sequence set forth in SEQ ID NO: 20, and a light chain CDR3 (LCDR3) amino acid sequence comprising the sequence set forth in SEQ ID NO:

18. The antibody construct comprising:

2. the antigen-binding domain (a) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 24, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 29; (b) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 24, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 30; (c) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 26, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 30; (d) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 25, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 30; (e) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 27, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 30; (f) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 27, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 29; (g) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 26, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 29; (h) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 25, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 29; (i) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 27, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 28; (j) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 26, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 28; (k) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 25, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 28; (l) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 24, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 28; or (m) a VH domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO: 31, and a VL domain having at least 90% sequence identity to the sequence set forth in SEQ ID NO:

32.

2. The antibody-drug construct of claim 1, comprising:

3. 3. The antibody construct of claim 1 or 2, further comprising a scaffold, wherein the antigen-binding domain is operably linked to the scaffold.

4. The antibody construct of claim 3 , wherein the scaffold comprises an IgG Fc region.

5. The antibody construct of any one of claims 1 to 4, further comprising a second antigen-binding domain.

6. The antibody construct of claim 5, wherein the second antigen-binding domain binds to NaPi2b.

7. The antibody construct of claim 6, wherein the second antigen-binding domain is the same as the first antigen-binding domain.

8. An antibody construct according to any one of claims 4 to 7, further comprising one or more additional antigen binding domains.

9. A polynucleotide or set of polynucleotides encoding the antibody construct of any one of claims 1 to 8.

10. An expression vector or a set of expression vectors comprising the polynucleotide or set of polynucleotides of claim 9.

11. A host cell comprising the expression vector or set of expression vectors according to claim 10.

12. An antibody-drug conjugate comprising the antibody construct of any one of claims 1 to 8 conjugated to one or more drug moieties.

13. 13. The antibody-drug conjugate of claim 12, wherein the antibody conjugate is conjugated to 1 to about 8 drug moieties.

14. General formula I: A-(L-(D) ｍ ) ｎ (I) and During the ceremony, A is the antibody construct; L is a linker, D is a drug moiety; m is an integer from 1 to about 8; n is 1 to about 12; The antibody-drug conjugate of claim 13.

15. The antibody-drug conjugate of claim 14, wherein m is 1 or 2.

16. The antibody-drug conjugate of claim 14 or 15, wherein n is from about 2 to about 8.

17. 17. The antibody-drug conjugate of any one of claims 13 to 16, wherein the drug moiety is a maytansinoid, a maytansinoid analog, benzodiazepine, pyrrolobenzodiazepine, duocarmycin, calicheamicin, a calicheamicin analog, auristatin, an auristatin analog, hemiasterin, a hemiasterin analog, tublysin, a tublysin analog, amatoxin, an amatoxin analog, camptothecin, a camptothecin analog, eribulin, a TLR agonist, or a STING agonist.

18. The antibody-drug conjugate of any one of claims 13 to 17, wherein the drug moiety is an auristatin, an auristatin analog, hemiasterin, a hemiasterin analog, camptothecin, a camptothecin analog, or eribulin.

19. A pharmaceutical composition comprising the antibody construct of any one of claims 1 to 8 or the antibody-drug conjugate of any one of claims 12 to 18, and a pharmaceutically acceptable carrier or diluent.

20. An antibody construct according to any one of claims 1 to 8 or an antibody-drug conjugate according to any one of claims 12 to 18 for use in therapy.

21. 21. The antibody construct or antibody-drug conjugate for use according to claim 20, wherein said therapy comprises the treatment of cancer.

22. Use of the antibody construct of any one of claims 1 to 8 or the antibody-drug conjugate of any one of claims 12 to 18 in the manufacture of a medicament for the treatment of cancer.

23. 19. A method of inhibiting the proliferation of NaPi2b-positive tumor cells, the method comprising contacting said cells with the antibody construct of any one of claims 1 to 8 or the antibody-drug conjugate of any one of claims 12 to 18.

24. 19. A method of treating a subject having cancer, comprising administering to the subject an effective amount of the antibody construct of any one of claims 1 to 8 or the antibody-drug conjugate of any one of claims 12 to 18.

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