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 an anti-NaPi2b antibody construct conjugated to a drug, such as a cytotoxin or an immunomodulator, and their use as therapeutic or diagnostic agents, for example in the treatment or diagnosis of cancer.

Description

Technical Field

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

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

[0003] Given the overexpression of NaPi2b in certain types of cancer, NaPi2b-targeting agents have been investigated in clinical trials for the treatment of cancer, but the results have been mixed. Mersana Therapeutics conducted a Phase I / II clinical trial to investigate the efficacy of upifitamab rilsodotin (an antibody-drug conjugate (ADC) of the NaPi2b-targeting antibody MX-35 and the auristatin-F payload (Dolaflexin platform)) in patients with platinum-resistant ovarian cancer or non-small cell lung cancer (NSCLC). The NSCLC cohort of the study was discontinued due to lack of efficacy, while upifitamab rilsodotin was granted Fast Track Designation for the treatment of patients with platinum-resistant ovarian cancer who had received three to four prior therapies. 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 the auristatin-F payload using its Dolasynthen platform. However, the development of this ADC has been discontinued. Lifastuzumab vedotin (an ADC of lifastuzumab with an MMAE payload) was investigated in a clinical trial of patients with ovarian cancer or NSCLC sponsored by Genentech, but the trial has since been discontinued.

[0004] The purpose of providing this background information is to make the applicant believe that the known information may be relevant to the present disclosure. It is not necessarily intended to admit, nor should it be construed, that any of the foregoing information constitutes prior art against the claimed invention. Summary of the Invention

[0005] This text describes anti-NaPi2b antibodies and methods of use. One aspect of the present disclosure relates to antibody constructs that comprise an antigen-binding domain that binds to human NaPi2b (sodium-dependent phosphate transporter 2B), the antigen-binding domain comprising: a heavy chain CDR1 (HCDR1) amino acid sequence containing the sequence shown in SEQ ID NO:7, a heavy chain CDR2 (HCDR2) amino acid sequence containing the sequence shown in SEQ ID NO:8, and a heavy chain CDR3 (HCDR3) amino acid sequence containing the sequence shown in SEQ ID NO:9, as well as a light chain CDR1 (LCDR1) amino acid sequence containing the sequence shown in SEQ ID NO:19, a light chain CDR2 (LCDR2) amino acid sequence containing the sequence shown in SEQ ID NO:20, and a light chain CDR3 (LCDR3) amino acid sequence containing the sequence shown in SEQ ID NO:18.

[0006] Another aspect of the present disclosure relates to a polynucleotide or a set of polynucleotides encoding the anti-NaPi2b antibody construct as described herein.

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

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

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

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

[0011] Another aspect of the present disclosure relates to the use of the anti-NaPi2b antibody construct as described herein or the antibody-drug conjugate as described herein in the preparation 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, the method comprising contacting the cells with the anti-NaPi2b antibody construct as described herein or the antibody-drug conjugate as described herein.

[0013] Another aspect of the present disclosure relates to a method of treating a subject having cancer, the method comprising administering to the subject an effective amount of an anti-NaPi2b antibody construct as described herein or an antibody-drug conjugate as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A shows the sequences of the CDRs of the murine heavy chain variable domain of the chimeric anti-NaPi2b antibody v23855 grafted onto the human VH germline (IGHV1-46*03), and Figure 1 B shows the sequences of the CDRs of the murine light chain variable domain of the chimeric antibody v23855 grafted onto the human VL framework (IGKVID-39*01). The CDRs are designated by AbM definition and are marked in bold and underlined.

[0015] Figure 2A Shows the non-reducing (NR) SDS-PAGE profiles of all humanized variants and the parental chimeric variant 23855. Figure 2B Shows the reducing (R) SDS-PAGE profiles of all humanized variants and the parental chimeric variant 23855. Figure 2C Shows the UPLC-SEC profile of the parental mouse-human chimeric antibody v23855. Figure 2D Shows the UPLC-SEC profile of the representative humanized antibody v29456.

[0016] Figure 3A Depicts the binding of the humanized antibody variants v29456, MX-35 (v18992), and rituximab (v18993) to human NaPi2b. Figure 3B Depicts the binding of the humanized antibody variants v29456, MX-35 (v18992), and rituximab (v18993) to cynomolgus monkey NaPi2b. Figure 3C Depicts the binding of the humanized antibody variants v29456, MX-35 (v18992), and rituximab (v18993) to mouse NaPi2b.

[0017] Figure 4A Depicts the N-curve analysis of the binding of v29814 to NaPi2b expressed on IGROV-1 cells. Figure 4B Depicts the N-curve analysis of the binding of v36123 to NaPi2b expressed on IGROV-1 cells. Figure 4C Depicts the N-curve analysis of the binding of v36124 to NaPi2b expressed on IGROV-1 cells. For each figure, the right curve shows the data for the 500 pM constant binding partner, and the left curve shows the data for the 50 pM constant binding partner.

[0018] Figure 5A Comparison of the ability of v23855 (parental chimeric), v29456 (H1L2), v18992 (MX35), and v18993 (rituximab) to internalize in HCC-78 cells. Figure 5B Comparison of the ability of v23855 (parental chimeric), v29456 (H1L2), v18992 (MX35), and v18993 (rituximab) to internalize in NCI-H441 cells.

[0019] Figure 6 Depicts the binding of the parental chimeric antibody (v23855), humanized antibody variants v29452 and v29456 to IGROV-1 cells in addition to the binding of MX35 and rituximab ADCs to these cells.

[0020] Figure 7A Depicts the ability of humanized antibody variant v29456 and v29456 conjugated to DL1 or DL2 to bind IGROV-1 cells. Figure 7B Depicts the ability of v29456 and v29456 conjugated to DL1 or DL2 to bind HCC-78 cells.

[0021] Figure 8A Shows the cytotoxicity of the ADC of humanized antibody variant v29456 and the ADCs of reference antibodies MX35 and rituximab in OVCAR-3 cells. Figure 8B Shows the cytotoxicity of the ADC of v29456 and the ADCs of reference antibodies MX35 and rituximab in IGROV-1 cells. Figure 8C Shows the cytotoxicity of the ADC of v29456 and the ADCs of reference antibodies MX35 and rituximab in HCC-78 cells.

[0022] Figure 9A Depicts the in vivo efficacy of the ADC of parental chimeric v23855 compared to the ADC of reference antibody v18992 (MX35) in an OVCAR-3 xenograft model of ovarian cancer. Figure 9B Depicts the in vivo efficacy of the ADC of parental chimeric v23855 compared to the ADC of reference antibody v18993 (rituximab) in an OVCAR-3 xenograft model of ovarian cancer.

[0023] Figure 10 Depicts the in vivo efficacy of the ADC of v29456 at 1, 3, and 10 mg / kg in an OVCAR-3 xenograft model of ovarian cancer.

[0024] Figure 11AShows the in vivo efficacy of ADCs of v29456 at 1, 3, and 10 mg / kg in the NCI-H441 xenograft model of lung cancer. Figure 11B Shows the in vivo efficacy of ADCs of v29456 at 0.3 and 1 mg / kg in the NCI-H441 xenograft model of lung cancer.

[0025] Figure 12A Depicts the results measured using the Membrane Proteome Array of v38591 TM Measured results. Figure 12B Depicts the validation data of CLDN3, showing weak binding to CLDN3.

[0026] Figure 13 Depicts the binding of humanized antibody variants v38591 and v29456 to IGROV-1 cells and TOV-21G cells compared to the binding of rituximab.

[0027] Figure 14 Shows the PK curves of v29456 and v18993 (rituximab)-MC-VC-PABC-MMAE (DL3) in Tg32 mice. Detailed Description

[0028] 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 are also capable of binding to cynomolgus monkey NaPi2b.

[0029] The present disclosure also relates to antibody-drug conjugates (ADCs) that comprise an anti-NaPi2b antibody construct as described herein conjugated to a drug (such as a cytotoxin or an immunomodulator). The anti-NaPi2b antibody constructs and ADCs of the present disclosure can be used as, for example, therapeutic or diagnostic agents. Certain aspects of the present disclosure relate to methods of treatment and uses of the anti-NaPi2b antibody constructs and ADCs, for example, in the treatment of cancer. Some aspects relate to methods of diagnosis and uses of the anti-NaPi2b antibody constructs and ADCs, for example, in the diagnosis or analysis of cancer.

[0030] Definitions

[0031] 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.

[0032] As used herein, the term "about" refers to a variation of approximately + / - 10% from a given value. It should be understood that such variations are always included in any given value provided herein, whether or not specifically mentioned.

[0033] As used herein, when the term "comprising" is used in combination, the use of the word "a" or "an" can mean "one", but it also conforms to the meanings of "one or more", "at least one", and "one or more than one".

[0034] As used herein, the terms "comprising", "having", "including", and "containing" and their grammatical variants are inclusive or open-ended and do not exclude additional, unrecited elements and / or method steps. When used in combination with a composition, use, or method herein, the term "consisting essentially of" means that additional elements and / or method steps may be present, but these additions do not materially affect the manner in which the recited composition, method, or use functions. The term "consisting of" when used in combination with a composition, use, or method herein does not include the presence of additional elements and / or method steps. Compositions, uses, or methods described herein as comprising certain 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 these embodiments are specifically recited.

[0035] "Complementary determining region" or "CDR" is an amino acid sequence that contributes to antigen-binding specificity and affinity. The "framework" region (FR) can help maintain the correct conformation of the CDR to facilitate the binding between the antigen-binding region and the antigen. From the N-terminus to the C-terminus, the variable light chain region (VL) and variable heavy chain region (VH) of an antibody typically contain 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. The CDRs provide most of the contact residues for the binding of the antibody to the antigen or epitope. Generally, three heavy chain CDRs and three light chain CDRs are required to bind an antigen. However, in some cases, even a single variable domain can confer antigen-binding specificity. In addition, as is known in the art, in some cases, antigen binding can also occur through a combination of at least one or more CDRs (such as HCDR3) selected from the VH and / or VL domains.

[0036] Many 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 the definitions of IMGT, AbM (University of Bath), and Contact (MacCallum et al., 1996, J Mol Biol, 262(5):732-745). For example, the CDR definitions according to Kabat, Chothia, IMGT, AbM, and Contact are provided in Table 1 below. Thus, it will be apparent to those skilled in the art that the exact numbering and placement of CDRs can vary based on the numbering system employed. However, it should be understood that the disclosure herein of VH includes the disclosure of the relevant (native) heavy chain CDRs (HCDRs) as defined by any known numbering system. Similarly, the disclosure herein of VL includes the disclosure of the relevant (native) light chain CDRs (LCDRs) as defined by any known numbering system.

[0037] Table 1: Common CDR Definitions 1

[0038]

[0039] 1 The Kabat or Chothia numbering systems can be used for HCDR2, HCDR3, and light chain CDRs for all definitions except Contact, which uses Chothia numbering.

[0040] 2 Using Kabat numbering. The positions in the Kabat numbering scheme that delimit the ends of the Chothia and IMGT CDR-H1 loops vary according to the length of the loop, since Kabat places insertions outside those CDR definitions at positions 35A and 35B. However, the IMGT and Chothia CDR-H1 loops can be defined explicitly using Chothia numbering. CDR-H1 definition using Chothia numbering: Kabat H31-H35, Chothia H26-H32, AbM H26-H35, IMGT H26-H33, Contact H30-H35.

[0041] In the context of two or more polynucleotide or polypeptide sequences, the term "identical" refers to two or more identical sequences or subsequences. When sequences are compared and aligned to obtain maximum correspondence as measured over a comparison window or over a specified region using one of the commonly used sequence comparison algorithms known to those of ordinary skill in the art or by manual alignment and visual inspection, the sequences are "substantially identical" if they have a certain percentage of identical amino acid residues or nucleotides (e.g., about 80%, about 85%, about 90%, about 95%, or about 98% identity within a specified region). For sequence comparison, a test sequence is generally compared to a designated reference sequence. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are designated. Either the default program parameters can be used, or alternative parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0042] "Comparison window" refers to a segment of a sequence that includes contiguous amino acid or nucleotide positions, which may be, for example, about 10 to 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, at which a test sequence can be compared to a reference sequence at the same number of contiguous positions after the two sequences are optimally aligned. Sequence alignment methods for comparison are known to those of ordinary 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; the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443; the similarity search method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444; or computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, or TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, (Suppl. 1995), Cold Spring Harbor Laboratory Press). Examples of available algorithms suitable for determining the percentage of 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 analysis is publicly available through the website of the National Center for Biotechnology Information (NCBI).

[0043] As used herein, the term "subject" refers to an animal, which in some embodiments is a mammal, that is the object of treatment, observation, or experimentation. The animal can be a human, a non-human primate, a companion animal (e.g., dog, cat, etc.), a farm 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.

[0044] Any implementation described in this discussion is expected to be implemented by any method, use, or composition disclosed in the present invention, and vice versa.

[0045] The specific features, structures, and / or characteristics described in connection with one implementation disclosed herein can be combined with the features, structures, and / or characteristics described in connection with another implementation disclosed herein in any suitable manner to provide one or more other implementations.

[0046] It should also be understood that an affirmative statement of a feature in one implementation is the basis for excluding the feature in another implementation. For example, in the case of presenting a list of options for a given implementation or claim, it should be understood that one or more options can be removed from the list, and the shortened list can form an alternative implementation, regardless of whether such an alternative implementation is specifically mentioned.

[0047] Anti-NaPi2b antibody construct

[0048] The present disclosure relates to antibody constructs that specifically bind to human NaPi2b (hNaPi2b). In this context, the term "antibody construct" refers to a polypeptide or group of polypeptides that contains one or more antigen-binding domains, where each of the one or more antigen-binding domains specifically binds to an epitope or antigen. In the case where an antibody construct contains two or more antigen-binding domains, each antigen-binding domain can bind to the same epitope or antigen (i.e., the antibody construct is monospecific), or they can bind to different epitopes or antigens (i.e., the antibody construct is bispecific or multispecific). The antibody construct can also contain a scaffold, and the one or more antigen-binding domains can be fused or covalently linked to the scaffold, optionally via a linker.

[0049] According to the present disclosure, the anti-NaPi2b antibody construct contains at least one antigen-binding domain that specifically binds to hNaPi2b. The so-called "specifically binds" to hNaPi2b means that the antibody construct binds to hNaPi2b but does not exhibit significant binding to either human NaPi2a or NaPi2c. In certain implementations, the anti-NaPi2b antibody construct of the present disclosure is capable of binding to NaPi2b from one or more non-human species. In certain implementations, the anti-NaPi2b antibody construct of the present disclosure is capable of binding to cynomolgus monkey NaPi2b.

[0050] Human NaPi2b is also known as human "solute carrier family 34 member 2" or "SLC34A2". Protein sequences of hNaPi2b from various sources are known in the art and are readily available from publicly accessible databases such as GenBank or UniProtKB. Examples of hNaPi2b sequences include, for example, 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).

[0051] Table 2: Human, cynomolgus monkey, and mouse NaPi2b protein sequences

[0052]

[0053]

[0054]

[0055] 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 (using, for example, a BIAcore instrument) (Liljeblad et al., 2000, Glyco J, 17:323-329), flow cytometry, or traditional binding assays (Heeley, 2002, Endocr Res, 28:217-229). In certain embodiments, specific binding can be defined as, for example, a degree of binding to a non-target protein (such as hNaPi2a or hNaPi2c) measured by ELISA or flow cytometry that is less than about 5% to 10% of the binding to hNaPi2b.

[0056] As used herein, the term "dissociation constant (K D or K d )" is intended to refer to the equilibrium dissociation constant of a particular ligand-protein interaction. As used herein, ligand-protein interaction refers to, but is not limited to, protein-protein interaction or antibody-antigen interaction. K D measures the tendency of two proteins (e.g., AB) complexed together to dissociate reversibly into their constituent components (A + B), and is defined as the dissociation rate constant (also referred to as the "dissociation rate (k off)”) and the ratio of the association rate constant or “association rate (k on )”. Thus, K D equals k off / k on , and is expressed in molar concentration (M). It can be seen that the smaller the K D , the stronger the binding affinity. Therefore, a decrease in K D indicates an increase in affinity. Thus, compared with a K D of 1 nM, a K D of 1 mM indicates a weak binding affinity. Affinity is sometimes measured as K A or K a , which is the reciprocal of K D or K d . The K D between an antibody and its antigen can be determined using well-established methods in the art. One method for determining such K D is by using surface plasmon resonance (SPR), typically using a biosensor system such as system. Isothermal titration calorimetry (ITC) is another method that can be used to measure K D . The Octet TM system can also be used to measure the affinity of an antibody for a target antigen.

[0057] In certain embodiments, specific binding of the antibody construct to NaPi2b can be defined by a dissociation constant (Kd or K D ) ≤ 1 μM, such as ≤ 500 nM, ≤ 250 nM, ≤ 100 nM, ≤ 50 nM, or ≤ 10 nM. In certain embodiments, specific binding of the antibody construct to a particular antigen or epitope can be defined by a dissociation constant (K D ) of 10 -6 M or less, such as 10 -7 M or less or 10 -8 M or less. In some embodiments, specific binding of the antibody construct to a particular antigen or epitope can be defined by a dissociation constant (K D ) between 10 -6 M and 10 -9 M, such as between 10 -7 M and 10 -9 M. As is known in the art, the numerical value of the dissociation constant obtained can vary depending on the manner in which it is tested. For example, the expression level of NaPi2b in a cell line, the form of the antibody construct (i.e., monovalent or bivalent), and the type of assay (i.e., ELISA or flow cytometry) can 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.

[0058] In some embodiments, when measured by flow cytometry in cells that highly express NaPi2b, the Kd of the anti-NaPi2b antibody constructs of the present disclosure is lower than the Kd of the reference antibody, rituximab, and is comparable to the Kd of the reference antibody, MX35. Thus, in these embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise antigen-binding domains that have a greater affinity for human NaPi2b than the reference antibody rituximab and have an affinity comparable to the reference antibody MX35.

[0059] In certain embodiments, the anti-NaPi2b antibody constructs exhibit an internalization level comparable to the reference antibody MX35 and exhibit a higher internalization level compared to the reference antibody rituximab in cells that highly and moderately express NaPi2b. In some embodiments, internalization is measured 4 hours, 5 hours, or 24 hours after treatment.

[0060] Antibody internalization can be measured using methods known in the art, e.g., by direct internalization assays 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 dyes (ThermoFisher Scientific Corporation, Waltham, MA), and Fabfluor-pH antibody labeling reagents (Sartorius AG, Germany), and analytical techniques such as microscopy, FACS, high-content imaging, or other plate-based assays.

[0061] NaPi2b expression varies according to the cell type indicated throughout the present disclosure, and the level of NaPi2b expression is referred to herein as "high", "medium", "low", or "negative". These terms are used for reference to describe the general expression levels according to the names shown in Table 10.1 in Example 10 and are not intended to be limited to the specific values of the average NaPi2b protein of each cell included therein.

[0062] antigen-binding domain

[0063] The anti-NaPi2b antibody construct of the present disclosure comprises at least one antigen-binding domain capable of binding to hNaPi2b. The at least one antigen-binding domain capable of binding to hNaPi2b is generally 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 Fab (scFab), single-chain Fv (scFv), and single-domain antibodies (sdAb).

[0064] A "Fab fragment" contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CH1), as well as the variable domains of the light and heavy chains (VL and VH, respectively). A Fab′ fragment differs from a Fab fragment in that it has several amino acid residues added to the carboxyl terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. A Fab fragment can also be a single-chain Fab molecule, i.e., a Fab molecule in which the Fab light chain and the Fab heavy chain are linked by a peptide linker to form a single polypeptide chain. For example, the C-terminus of the Fab light chain can be linked to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.

[0065] A "scFv" contains the variable domain of the heavy chain (VH) and the variable domain of the light chain (VL) of an antibody in a single polypeptide chain. The scFv can optionally further contain a polypeptide linker between the VH and VL domains, thereby enabling the scFv to form the structure required for antigen binding. For example, the scFv can include a VL that is linked from its C-terminus to the N-terminus of the VH via a polypeptide linker. Alternatively, the scFv can contain a VH that is linked via its C-terminus to the N-terminus of the VL by a polypeptide linker (see the review by Pluckthun in The Pharmacology of Monoclonal Antibodies, Volume 113, edited by Rosenberg and Moore, Springer-Verlag, New York, pages 269-315 (1994)).

[0066] The "sdAb" format refers to a single immunoglobulin domain. The sdAb can be, for example, of camel origin. Camel antibodies lack a light chain, and their antigen-binding site consists of a single domain called "VHH". The sdAb contains three CDRs / hypervariable loops, CDR1, CDR2, and CDR3, that form the antigen-binding site. The sdAb is quite stable and easy to express, for example, as a fusion with the antibody Fc chain (see, for example, Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., 77(1):13-22).

[0067] 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 (such as an antigen-binding antibody fragment) or a non-immunoglobulin-based domain (such as a non-immunoglobulin antibody mimetic), or other polypeptide or small molecule capable of specifically binding its target (e.g., a natural or engineered ligand). Non-immunoglobulin antibody mimetic forms include, for example, anticalin, fynomer, affimer, alphabody, DARPin, and avimer.

[0068] The present disclosure describes herein the identification of a murine antibody that specifically binds hNaPi2b; a murine-human chimeric variant of the antibody was identified as variant 23855. The anti-NaPi2b antibody constructs of the present disclosure comprise antigen-binding domains derived from the 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 human NaPi2b having the sequence shown in SEQ ID NO: 1.

[0069] In certain embodiments, the anti-NaPi2b antibody construct competes with any one of the humanized antibody variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460 or with the parental chimeric antibody v23855 for binding to human NaPi2b. In the competition assays described below, each of variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, v29460, and v23855 is referred to as a competition reference antibody.

[0070] Competitive assays known in the art can be used to determine whether an antibody construct competes with variants v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460 or the parental chimeric antibody v23855 for binding to hNaPi2b. For example, a competitive reference antibody is first allowed to bind hNaPi2b under saturating conditions, and then the ability of the test antibody construct to bind hNaPi2b is measured. If the test antibody construct can bind hNaPi2b simultaneously with the competitive reference antibody, the test antibody construct is considered to bind a different epitope from the competitive reference antibody. Conversely, if the test antibody construct cannot bind hNaPi2b simultaneously with the competitive reference antibody, the test antibody construct is considered to bind an epitope that is the same as, overlapping with, or very close to the epitope bound by the competitive reference antibody. Such competitive assays can be performed using techniques such as ELISA, radioimmunoassay, surface plasmon resonance (SPR), biolayer interferometry, flow cytometry, etc. An "antibody that competes with a competitive reference antibody" refers to an antibody that blocks the binding of the reference antibody to its epitope by 50% or more in a competitive assay.

[0071] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise at least one antigen-binding domain that specifically binds hNaPi2b, wherein the antigen-binding domain comprises a set of CDRs based on the CDRs of the parental chimeric antibody v23855 described herein. The CDR sequences of the parental chimeric antibody v23855 and representative humanized antibody variants are shown in Table 3.

[0072] Table 3: CDR Sequences of Anti-NaPi2b Antibody Constructs

[0073]

[0074] 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) and light-chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3), wherein the heavy-chain CDR amino acid sequences comprise the sequences shown in SEQ ID NOs: 7, 8, and 9, and the light-chain CDR amino acid sequences comprise the sequences shown in SEQ ID NOs: 19, 20, and 18.

[0075] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain having:

[0076] (i) The 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; the HCDR2 amino acid sequence selected from the HCDR2 amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 or v29460; and the HCDR3 amino acid sequence selected from the HCDR3 amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 or v29460, and

[0077] (ii) The LCDR1 amino acid sequence selected from the LCDR1 amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 or v29460; the LCDR2 amino acid sequence selected from the LCDR2 amino acid sequences of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 or v29460; and the LCDR3 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, wherein the CDR amino acid sequences are defined according to any one of the IMGT, Chothia, Kabat, Contact or AbM numbering systems (see Table 3).

[0078] 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) and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3), wherein the heavy chain CDR amino acid sequences are selected from the heavy chain CDR amino acid sequences of any one of the 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 the light chain CDR amino acid sequences are selected from the light chain CDR amino acid sequences of any one of the 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.

[0079] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain comprising heavy chain CDR amino acid sequences (HCDR1, HCDR2, and HCDR3) and light chain CDR amino acid sequences (LCDR1, LCDR2, and LCDR3) of any one of the 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.

[0080] In certain embodiments, an anti-NaPi2b antibody construct of the disclosure comprises an antigen-binding domain having a VH sequence that comprises 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 disclosure comprises an antigen-binding domain having a VL sequence that comprises 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.

[0081] 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 the antibody losing its ability to bind 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 binding activity of the resulting variants can be tested by standard techniques. Thus, in certain embodiments, an anti-NaPi2b antibody construct of the disclosure comprises an antigen-binding domain that comprises a set of CDRs having 90% or higher, 95% or higher, 98% or higher, 99% or higher, or 100% sequence identity to a set of CDRs of any one of variants v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, or v29460 (i.e., heavy chain HCDR1, HCDR2, and HCDR3, and light chain LCDR1, LCDR2, and LCDR3), where the % sequence identity is calculated across all six CDRs, and where the antigen-binding domain retains the ability to bind hNaPi2b.

[0082] In certain embodiments, an anti-NaPi2b antibody construct of the disclosure comprises an antigen-binding domain that comprises a variant of a 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 from 1 to 10 amino acid substitutions in the set of CDR sequences (i.e., the CDRs may be modified by up to 10 amino acid substitutions, where any combination of the six CDRs is modified), and wherein the antigen-binding domain retains the ability to bind hNaPi2b. In some embodiments, an anti-NaPi2b antibody construct of the disclosure comprises an antigen-binding domain that comprises a variant of a 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 from 1 to 7 amino acid substitutions, from 1 to 5 amino acid substitutions, from 1 to 4 amino acid substitutions, from 1 to 3 amino acid substitutions, from 1 to 2 amino acid substitutions, or 1 amino acid substitution in the set of CDRs, and wherein the antigen-binding domain retains the ability to bind hNaPi2b.

[0083] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain that comprises a VH sequence having 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% identity 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 hNaPi2b. In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain that comprises a VL sequence having 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% identity 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 hNaPi2b.

[0084] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain that comprises 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, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain that comprises 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.

[0085] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain comprising a VH sequence having 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% identity to the VH sequence of v23855, and a VL sequence having 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% identity to the VL sequence of v23855, wherein the antigen-binding domain retains the ability to bind hNaPi2b.

[0086] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain comprising a VH sequence having 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% identity to the VH sequence of v29456, and a VL sequence having 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% identity to the VL sequence of v29456, wherein the antigen-binding domain retains the ability to bind hNaPi2b.

[0087] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise an antigen-binding domain comprising a VH sequence having 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% identity to the VH sequence of v29452, and a VL sequence having 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% identity to the VL sequence of v29452, wherein the antigen-binding domain retains the ability to bind hNaPi2b.

[0088] In some embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise VH and VL sequences of any one of v23855, v23855, v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459 or v29460. The SEQ ID NOs: of the VH and VL sequences of these variants are provided in Table 4 below. The sequences themselves are provided in Table 2.4 of the Examples.

[0089] Table 4: VH and VL Sequences of Parental Chimeric and Humanized Anti-NaPi2b Antibodies

[0090]

[0091]

[0092] In some embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise the VH sequence and the VL sequence of v29456. In some embodiments, the anti-NaPi2b antibody constructs of the ADCs of the present disclosure comprise the VH sequence and the VL sequence of v29452.

[0093] In certain embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise: a) a VH sequence having the 3 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 3 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.

[0094] In certain other embodiments, the anti-NaPi2b antibody constructs of the present disclosure comprise: a) a VH sequence having the 3 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 3 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.

[0095] Format

[0096] The anti-NaPi2b antibody constructs of the present disclosure can have various formats. The minimal component of an anti-NaPi2b antibody construct is an antigen-binding domain that binds hNaPi2b. The anti-NaPi2b antibody constructs can also 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 can bind the same epitope within hNaPi2b, can bind different epitopes within hNaPi2b, or can bind different antigens. Thus, the anti-NaPi2b antibody constructs can be, for example, monospecific, bispecific, bispecific or multispecific.

[0097] In certain embodiments, the anti-NaPi2b antibody construct comprises at least one antigen-binding domain that binds hNaPi2b and a scaffold, wherein the antigen-binding domain is operably linked to the scaffold. As used herein, the term "operably linked" means that the described components are in a relationship that permits them to function in their intended manner. Suitable scaffolds are described below.

[0098] In certain embodiments, the anti-NaPi2b antibody construct comprises two antigen-binding domains optionally operably linked to a scaffold. In some embodiments, the anti-NaPi2b antibody construct can 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 domains can each independently be operably linked to the scaffold or the first antigen-binding domain, or, when there are more than two antigen-binding domains, operably linked to another antigen-binding domain.

[0099] Anti-NaPi2b antibody constructs lacking a scaffold may comprise a single antigen binding domain in an appropriate format, such as an sdAb, or they may comprise two or more antigen binding domains optionally operably connected via one or more linkers. In such anti-NaPi2b antibody constructs, the antigen binding domain may be in the format of scFv, Fab, sdAb, or a combination thereof. For example, using scFv as the antigen binding domain, a format such as a tandem scFv ((scFv) 2 In some embodiments, the scFv is a double antibody, which is a double antibody, wherein the scFv is connected together by a flexible joint. ScFv can also be used to construct a double antibody format, which includes two scFvs connected by a short joint (usually about 5 amino acids in length). The restricted joint length causes scFv to dimerize in a head-to-tail manner. In any of the aforementioned formats, scFv can be further stabilized by comprising an interdomain disulfide bond. For example, disulfide bonds can be introduced between VL and VH by replacing the non-cysteine ​​residues in each chain with cysteine ​​residues (for example, at position 44 of VH and position 100 of VL) (see, for example, Fitzgerald et al., 1997, Protein Engineering, 10: 1221-1225), or disulfide bonds can be introduced between two VH to provide a construct with a DART format (see, for example, Johnson et al., 2010, J Mol.Biol., 399: 436-449).

[0100] Similarly, in some embodiments, a format comprising two sdAbs (such as VH or VHH) linked together by a suitable linker may be employed. Other examples of anti-NaPi2b antibody construct formats lacking a scaffold include those based on Fab fragments, such as Fab 2 and F(ab') 2 format, in which the Fab fragments are connected by a linker or IgG hinge region.

[0101] Combinations of antigen binding domains of varying sizes can also be employed to generate alternative scaffold-free formats. For example, a scFv or sdAb can be fused to the C-terminus of one or both of the light and heavy chains of a Fab fragment, thereby generating a bivalent (Fab-scFv / sdAb) construct.

[0102] In certain embodiments, the anti-NaPi2b antibody construct can be an immunoglobulin (Ig)-based antibody format. This type of format is referred to herein as a full-size antibody format (FSA) or Mab format and includes an anti-NaPi2b antibody construct containing two Ig heavy chains and two Ig light chains. In certain embodiments, the anti-NaPi2b antibody construct can be based on IgG-class immunoglobulins, such as IgG1, IgG2, IgG3, or IgG4 immunoglobulins. In some embodiments, the anti-NaPi2b antibody construct can be based on IgG1 immunoglobulin. In the context of the present disclosure, when the anti-NaPi2b antibody construct is based on a specified immunoglobulin isotype, it means that the anti-NaPi2b antibody construct contains all or a portion of the constant region of the specified immunoglobulin isotype. For example, an anti-NaPi2b antibody construct based on a given Ig isotype can contain at least one antigen-binding domain operably linked to an Ig scaffold, where the scaffold contains the 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 contain hybrids of isotypes and / or subclasses. It should also be understood that the Fc region and / or hinge region can optionally be modified to confer one or more desired functional properties as known in the art. Thus, in certain embodiments, the anti-NaPi2b antibody construct contains a VH amino acid sequence fused to the IgG1 constant domain amino acid sequence (i.e., the CH1, hinge, CH2, CH3 amino acid sequences) and a VL amino acid sequence fused to a κ or λ constant amino acid sequence domain (i.e., the CL amino acid sequence). Exemplary amino acid sequences are provided in the Examples and Sequence Listing.

[0103] In some embodiments, the anti-NaPi2b antibody construct can be derived from two or more immunoglobulins from different species. For example, the anti-NaPi2b antibody construct can be a chimeric antibody or a humanized antibody. The terms "chimeric antibody" and "humanized antibody" generally refer to antibodies that combine immunoglobulin regions or domains from more than one species.

[0104] A "chimeric antibody" generally contains 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 have 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. Patent No. 4,816,567.

[0105] "Humanized antibodies" are a class of chimeric antibodies that contain the minimal sequences derived from non-human antibodies. Typically, a humanized antibody is a human immunoglobulin (recipient antibody) in which the residues from the hypervariable regions of the recipient are replaced with residues from the hypervariable regions of a non-human species (donor antibody) that have the desired specificity and affinity for the target antigen, such non-human species as mice, rats, rabbits, or non-human primates. This technique for creating humanized antibodies is often referred to as "CDR grafting".

[0106] In some cases, additional modifications are made to further improve antibody performance. For example, residues in the framework regions (FRs) of the human immunoglobulin are replaced with corresponding non-human residues, or the humanized antibody can contain residues not found in either the recipient antibody or the donor antibody. In general, the variable domains in a humanized antibody will contain all or substantially all of the hypervariable regions from a non-human immunoglobulin and all or substantially all of the FRs from a human immunoglobulin sequence. Humanized antibodies are described in more 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.

[0107] Many methods are known in the art for selecting the most appropriate human framework in which to graft non-human CDRs. Early methods used a limited subset of well-characterized human antibodies, regardless of sequence identity with the non-human antibody providing the CDRs ("fixed framework" methods). More recent methods have employed variable regions with high amino acid sequence identity to the variable regions of the non-human antibody providing the CDRs ("homology matching" or "best fit" methods). An alternative approach is to select fragments of framework sequences from within each light or heavy chain variable region from several different human antibodies. In some cases, CDR grafting may result in partial or complete loss of affinity of the grafted molecule for its target antigen. In such cases, affinity can be restored by backmutating some human-derived residues to the corresponding non-human residues. Methods for preparing humanized antibodies by these methods are well known in the art (see, e.g., 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).

[0108] Alternatively, or in addition to these traditional methods, newer techniques can be employed to further reduce the immunogenicity of humanized antibodies for CDR grafting. For example, frameworks based on germline sequences or consensus sequences can be used as the acceptor human framework instead of a human framework with somatic mutations. Another technique aimed at reducing the potential immunogenicity of non-human CDRs is to graft only the specific determining residues (SDRs). In this approach, only the minimal number of CDR residues required for antigen-binding activity ("SDRs") are grafted into a germline framework. This method improves the "humanity" of the humanized antibody (i.e., similarity to germline sequences) and thus may help reduce the immunogenicity risk of the variable region. These techniques have been described in various publications (see, e.g., Almagro and 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).

[0109] In certain embodiments, the anti-NaPi2b antibody constructs of the disclosure comprise humanized antibody sequences, such as 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 the anti-NaPi2b antibody v23855 are described herein (see Examples and Sequence Listing, and the sequences of v29449, v29450, v29451, v29452, v29453, v29454, v29455, v29456, v29457, v29458, v29459, and v29460).

[0110] scaffold

[0111] 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 domains can be 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 (such as leucine zippers, "zipper" peptides derived from Jun and Fos that form heterodimers, IgG CH1 and CL domains, or barnase-barstar toxins), cytokines, chemokines, or growth factors. Other examples include antibodies based on the DOCK-AND-LOCK TM (DNL TM ) technology (see, e.g., Chang et al., 2007, Clin. Cancer Res., 13:5586s-5591s).

[0112] The scaffold can be a peptide, polypeptide, polymer, nanoparticle, or other chemical entity. When the scaffold is a polypeptide, each antigen-binding domain of the anti-NaPi2b antibody construct can be linked to the N-terminus or C-terminus of the polypeptide scaffold. Anti-NaPi2b antibody constructs comprising a polypeptide scaffold are also contemplated in certain embodiments, wherein one or more antigen-binding polypeptide constructs are linked, with or without a linker, to a region other than the N-terminus or C-terminus, for example, via the side chain of an amino acid.

[0113] In embodiments in which the anti-NaPi2b antibody construct comprises a scaffold that is a peptide or polypeptide, the antigen-binding domain can be linked to the scaffold by genetic fusion or chemical conjugation. Generally, when the scaffold is a peptide or polypeptide, the antigen-binding domain is linked to the scaffold by genetic fusion. In some embodiments, when the scaffold is a polymer or nanoparticle, the antigen-binding domain can be linked to the scaffold by chemical conjugation.

[0114] Many protein domains are known in the art that involve the selective pairing of two different polypeptides and can be used to form scaffolds. Examples are leucine zipper domains that pair selectively together, 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 - barstar 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. WO 2011 / 135040).

[0115] 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, Müller 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).

[0116] For example, it has been demonstrated that fusing antigen - binding moieties such as scFv, diabodies, or single - chain diabodies to albumin can improve the serum half - life of the antigen - binding moiety (Müller et al., supra). The antigen - binding moiety can optionally be fused at the N - terminus and / or C - terminus of albumin via a linker.

[0117] Albumin derivatives in heteromeric form have been described that contain two transporter polypeptides obtained by albumin fragmentation such that the transporter polypeptides self - assemble to form a native - like albumin (see International Patent Application Publication Nos. WO 2012 / 116453 and WO 2014 / 012082). Due to albumin fragmentation, the heteromer includes four termini and can thus optionally be fused to up to four different antigen - binding moieties via linkers.

[0118] 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 analogue or derivative. In some embodiments, the anti-NaPi2b antibody construct may comprise a protein scaffold based on an immunoglobulin Fc region (e.g., an IgG Fc region).

[0119] Fc region

[0120] As used herein, the terms "Fc region", "Fc", or "Fc domain" refer to the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. Unless otherwise specified herein, the numbering of amino acid residues in the 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).

[0121] In certain embodiments, the anti-NaPi2b antibody construct of the present disclosure may comprise a scaffold based on an immunoglobulin Fc region. The Fc region may be dimeric and composed of two Fc polypeptides, or alternatively, the Fc region may be composed of a single polypeptide.

[0122] In the case of a dimeric Fc, an "Fc polypeptide" refers to one of the two polypeptides that form the dimeric Fc domain, i.e., a polypeptide that contains 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 contains one first Fc polypeptide and one second Fc polypeptide.

[0123] The Fc region may comprise a CH3 domain or it may comprise both CH3 and CH2 domains. For example, in certain embodiments, the Fc polypeptide of a dimeric IgG Fc region may comprise IgG CH2 domain sequences and IgG CH3 domain sequences. In such embodiments, the CH3 domain contains two CH3 sequences, i.e., one from each of the two Fc polypeptides of the dimeric Fc region, and the CH2 domain contains two CH2 sequences, i.e., one from each of the two Fc polypeptides of the dimeric Fc region.

[0124] In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on the IgG Fc region. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on the human IgG Fc region. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on the IgG1 Fc region. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on the human IgG1 Fc region.

[0125] In certain embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on an IgG Fc region that is a heterodimeric Fc region comprising a first Fc polypeptide and a second Fc polypeptide, each of the first Fc polypeptide and the second Fc polypeptide comprising a CH3 sequence and optionally a CH2 sequence, and wherein the first Fc polypeptide and the second Fc polypeptide are different. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on an Fc region that comprises two CH3 sequences, at least one of the CH3 sequences comprising one or more amino acid modifications. In some embodiments, the anti-NaPi2b antibody construct may comprise a scaffold based on an Fc region that comprises two CH3 sequences and two CH2 sequences, at least one of the CH2 sequences comprising one or more amino acid modifications.

[0126] In some embodiments, the anti-NaPi2b antibody construct may comprise a heterodimeric Fc region containing a modified CH3 domain, wherein the modified CH3 domain is 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, wherein the amino acid at a particular position on the first CH3 or CH2 sequence is different from the amino acid at the same position on the second CH3 or CH2 sequence. These asymmetric amino acid modifications can be the result of modification of only one of the two amino acids at the same corresponding amino acid position on each sequence, or the result of different modifications of each of the two amino acids at the same corresponding position on each of the first and second CH3 or CH2 sequences. Each of the first and second CH3 or CH2 sequences of the heterodimeric Fc may comprise one or more than one asymmetric amino acid modification.

[0127] In some embodiments, the anti-NaPi2b antibody construct may comprise a heterodimeric Fc containing a modified CH3 domain, wherein the modified CH3 domain comprises one or more amino acid modifications that promote heterodimeric Fc formation relative to homodimeric Fc. In some embodiments, one or more of the amino acid modifications are asymmetric amino acid modifications.

[0128] Amino acid modifications that can be made to the CH3 domain of Fc to promote the formation of heterodimeric Fc are known in the art and include, for example, those described in International Publication No. WO 96 / 027011 ("staple - socket"), 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 methods that combine positive and negative design strategies to produce a stable asymmetric modified Fc region, as described in International Publications WO 2012 / 058768 and WO 2013 / 063702. In certain embodiments, the anti - NaPi2b antibody construct can comprise a scaffold based on a modified Fc region, as described in International Publication No. WO 2012 / 058768 or WO 2013 / 063702.

[0129] 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 comprises amino acids 341 - 447 of the full - length human IgG1 heavy chain. Also shown in Table 5 are CH3 domain amino acid modifications that promote heterodimeric Fc formation, as described in International Patent Application Publication Nos. WO 2012 / 058768 and WO 2013 / 063702.

[0130] In certain embodiments, the anti - NaPi2b antibody construct can comprise a heterodimeric Fc scaffold having a modified CH3 domain, the modified CH3 domain comprising the modification of any one of Variant 1, Variant 2, Variant 3, Variant 4, or Variant 5, as shown in Table 5.

[0131] Table 5: Human IgG1 Fc Sequences Promoting Heterodimer Formation 1 and CH3 Domain Amino Acid Modifications

[0132]

[0133]

[0134] 1 Sequence (EU numbering) at positions 231 - 447

[0135] In some embodiments, the anti-NaPi2b antibody construct can comprise an Fc region-based scaffold that includes two CH3 sequences and two CH2 sequences, with at least one of the CH2 sequences comprising one or more amino acid modifications. Modifications in the CH2 domain can affect the binding of Fc receptors (FcRs) to Fc, such as receptors of the FcγRI, FcγRII, and FcγRIII subclasses.

[0136] In some embodiments, the anti-NaPi2b antibody construct comprises a scaffold based on an IgG Fc with a modified CH2 domain, wherein the modification of the CH2 domain results in an altered binding to one or more of the FcγRI, FcγRII, and FcγRIII receptors.

[0137] A variety of amino acid modifications that selectively alter the affinity of Fc for different Fcγ receptors in the CH2 domain are known in the art. Amino acid modifications that result in increased binding and those that result in decreased binding can each be used for certain indications. For example, increasing the binding affinity of Fc for FcγRIIIa, an activating receptor, can cause an increase in antibody-dependent cell-mediated cytotoxicity (ADCC), which in turn causes an increase in the lysis of target cells. Reducing the binding to FcγRIIb, an inhibitory receptor, can also be beneficial in some cases. In certain indications, it may be desirable to reduce or eliminate ADCC and complement-mediated cytotoxicity (CDC). In such cases, a modified CH2 domain (“knockout” variant) containing an amino acid modification that results in increased binding to FcγRIIb or an amino acid modification that reduces or eliminates the binding of the Fc region to all Fcγ receptors may be useful.

[0138] Examples of amino acid modifications to the CH2 domain that alter the binding of Fcγ receptors to Fc include, but are not limited to, the following: S298A / E333A / K334A and S298A / E333A / K334A / K326A (increased affinity for FcγRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1-2):132-41); F243L / R292P / Y300L / V305I / P396L (increased affinity for FcγRIIIa) (Stavenhagen, et al., 2007, Cancer Res, 67(18):8882-90); F243L / R292P / Y300L / L235V / P396L (increased affinity for FcγRIIIa) (Nordstrom JL, et al., 2011, Breast Cancer Res, 13(6):R123); F243L (increased affinity for FcγRIIIa) (Stewart, et al., 2011, Protein Eng Des Sel., 24(9):671-8); S298A / E333A / K334A (increased affinity for FcγRIIIa) (Shields, et al., 2001, J Biol Chem, 276(9):6591-604); S239D / I332E / A330L and S239D / I332E (increased affinity for FcγRIIIa) (Lazar, et al., 2006, Proc Natl Acad Sci USA, 103(11):4005-10), and S239D / S267E and S267E / L328F (increased affinity for FcγRIIb) (Chu, et al., 2008, Mol Immunol, 45(15):3926-33). Various amino acid modifications to the CH2 domain that alter the binding of FcγRIIb to Fc are described in International Publication No. WO 2021 / 232162. Other modifications that affect the binding of Fc 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).

[0139] In certain embodiments, the anti-NaPi2b antibody construct comprises a scaffold based on an IgG Fc with a modified CH2 domain, wherein the modified CH2 domain comprises one or more amino acid modifications that result in reduced or eliminated binding of the Fc region to all Fcγ receptors (i.e., a “knockout” variant).

[0140] A variety of publications have described strategies that have been used to engineer antibodies to produce “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 reducing effector function by glycosylation modification, using IgG2 / IgG4 scaffolds, or introducing mutations in the hinge or CH2 domain of the Fc (see also U.S. Patent Publication No. 2011 / 0212087, International Publication No. WO 2006 / 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).

[0141] Examples of mutations that can be introduced into the hinge or CH2 domain to produce a “knockout” variant include the amino acid modifications L234A / L235A and L234A / L235A / D265S.

[0142] In certain embodiments, the anti-NaPi2b antibody constructs described herein may comprise a scaffold based on an IgG Fc, wherein the native glycosylation has been modified. As is known in the art, the glycosylation of the 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 non-glycosylated Fc that lacks all effector functions (Bolt et al., 1993, Eur. J. Immunol., 23:403-411; Tao & Morrison, 1989, J. Immunol., 143:2595-2601).

[0143] In contrast, removal of fucose from the N297-linked oligosaccharides of the heavy chain has been shown to enhance ADCC based on 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 afucosylated 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 Lec 13 with reduced ability to link fucose to the N297-linked carbohydrate (International Publication No. WO 03 / 035835), or in other cells that produce afucosylated 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, International Publication No. WO 2009 / 135181 describes adding fucose analogs to the culture medium during antibody production to inhibit fucose incorporation into the carbohydrates on the antibody.

[0144] Other methods for generating antibodies with little or no fucose at the Fc glycosylation site (N297) are well known in the art. For example, techniques (ProBioGen AG) (see von Horsten et al., 2010, Glycobiology, 20(12):1607-1618 and U.S. Patent No. 8,409,572).

[0145] Other glycosylation variants include those with bisected oligosaccharides, e.g., variants in which the biantennary oligosaccharide linked to the Fc region of the antibody is bisected by N-acetylglucosamine (GlcNAc). Such glycosylation variants can have reduced fucosylation and / or improved ADCC function (see, e.g., International Publication No. WO 2003 / 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 linked to the Fc region, which can have improved CDC function (see, e.g., International Publication No. WO 1997 / 030087, WO1998 / 58964, and WO 1999 / 22764).

[0146] Preparation of Anti-NaPi2b Antibody Constructs

[0147] The anti-NaPi2b antibody constructs described herein can be produced using standard recombinant methods known in the art (see, for example, U.S. Patent No. 4,816,567 and “Antibodies: A Laboratory Manual”, 2nd edition, edited by Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).

[0148] Generally, for recombinant production of antibody constructs, a polynucleotide or a 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. The polynucleotide encoding the anti-NaPi2b antibody construct can 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 and updates, and “Antibodies: A Laboratory Manual”, 2nd edition, edited by 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 the anti-NaPi2b antibody construct will depend on the format of the construct, including whether the antibody construct contains a scaffold. For example, when the anti-NaPi2b antibody construct is in a monospecific mAb or FSA format, two polynucleotides each encoding a polypeptide chain will be required. When multiple polynucleotides are needed, they can be incorporated into one vector or more than one vector.

[0149] Generally, for expression, the polynucleotide or set of polynucleotides is incorporated into one or more expression vectors together with one or more regulatory elements, such as transcriptional elements, which are required for efficient transcription of the polynucleotide. Examples of such regulatory elements include, but are not limited to, promoters, enhancers, terminators, and polyadenylation signals. Those skilled in the art will understand that the choice of regulatory elements depends on the host cell selected for expression of the antibody construct, and such regulatory elements can be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. The expression vector can optionally further contain heterologous nucleic acid sequences that facilitate 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 can be an extrachromosomal vector or an integrating vector.

[0150] Suitable host cells for cloning or expressing an anti-NaPi2b antibody construct include a variety of 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 or Pichia cells). Prokaryotic host cells include, for example, Escherichia coli, Aeromonas salmonicida, or Bacillus subtilis cells.

[0151] In certain embodiments, the anti-NaPi2b antibody construct can be produced in bacteria, especially when glycosylation and Fc effector functions are not required, as described, for example, in U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523, and Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254, edited by B.K.C. Lo, Humana Press, Totowa, N.J., 2003.

[0152] In certain embodiments, eukaryotic microorganisms such as filamentous fungi or yeast can be suitable expression host cells, especially fungal and yeast strains whose glycosylation pathways have been "humanized" resulting in the production of antibody constructs with a partially or fully human glycosylation pattern (see, for example, Gerngross, 2004, Nat. Biotech. 22:1409-1414, and Li et al., 2006, Nat. Biotech. 24:210-215).

[0153] Suitable host cells for expressing a glycosylated anti-NaPi2b antibody construct are generally 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 PLANTIBODIES for producing antigen-binding constructs in transgenic plants. TMTechniques. Mammalian cell lines suitable for growth in suspension are particularly useful for expressing antibody constructs. Examples include, but are not limited to, monkey kidney CV1 line transformed by SV40 (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, dog kidney cells (MDCK), buffalo rat hepatocytes (BRL 3A), human lung cells (W138), human hepatocytes (Hep G2), mouse mammary tumor (MMT 060562), TRI cells (see, e.g., Mather et al., 1982, Annals N.Y. Acad Sci, 383:44-68), MRC 5 cells, FS4 cells, Chinese hamster ovary (CHO) cells (including DHFR - 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 producing antibody constructs are reviewed in Yazaki and Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (edited by B.K.C. Lo, Humana Press, Totowa, N.J., 2003).

[0154] In certain embodiments, the host cell can be a transient or stable higher eukaryotic cell line, such as a mammalian cell line. In some embodiments, the host cell can be a mammalian HEK293T, CHO, HeLa, NS0 or COS cell line, or a cell line derived from any of these cell lines. In some embodiments, the host cell can be a stable cell line that permits mature glycosylation of the antibody construct.

[0155] Host cells containing an expression vector encoding an anti-NaPi2b antibody construct can be cultured using conventional methods to produce the anti-NaPi2b antibody construct. Alternatively, in some embodiments, host cells containing an expression vector encoding an anti-NaPi2b antibody construct can be used therapeutically or prophylactically to deliver the anti-NaPi2b antibody construct to a subject, or a polynucleotide or expression vector can be administered ex vivo to cells from a subject and then the cells returned to the subject's body.

[0156] Typically, anti-NaPi2b antibody constructs are purified after expression. Proteins can be isolated or purified in a variety of ways known to those skilled in the art (see, e.g., Protein Purification: Principles and Practice, 3rd ed., Scopes, Springer-Verlag, NY, 1994). Standard purification methods include chromatographic techniques, including ion exchange chromatography, hydrophobic interaction chromatography, affinity chromatography, size exclusion chromatography or gel filtration, and reverse phase chromatography, using systems such as FPLC and HPLC at atmospheric or high pressure. Additional purification methods include electrophoresis, immunoprecipitation, dialysis, and chromatofocusing techniques. The combination of ultrafiltration and diafiltration techniques with protein concentration is also useful. As is well known in the art, a variety of native proteins bind to Fc and antibodies, and these proteins can be used to purify certain antibody constructs. For example, the bacterial proteins A and G bind to the Fc region. Similarly, the bacterial protein L binds to the Fab region of some antibodies. Purification can also be carried out through specific fusion partners. For example, if a GST fusion is used, the antibody can be purified using glutathione resin, if a His tag is used, the antibody can be purified using Ni +2 affinity chromatography, or if a flag tag is used, the antibody can be purified using immobilized anti-flag antibody. The degree of purification required will vary depending on the use of the anti-NaPi2b antibody construct. In some cases, purification may not be necessary.

[0157] In certain embodiments, the anti-NaPi2b antibody construct is substantially pure. As used herein with respect to the anti-NaPi2b antibody constructs described herein, the term "substantially pure" (or "substantially purified") means that the antibody construct is substantially or essentially free of components that normally accompany or interact with a protein as found in its natural environment (such as a native cell, or in the case of a recombinantly produced construct, the host cell). 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.

[0158] Certain embodiments of the present disclosure relate to a method for preparing an anti-NaPi2b antibody construct, the method comprising culturing a host cell that has been introduced with one or more polynucleotides encoding an anti-NaPi2b antibody construct or one or more expression vectors encoding an anti-NaPi2b antibody construct under conditions suitable for expressing the anti-NaPi2b antibody construct, and optionally recovering the anti-NaPi2b antibody construct from the host cell (or from the host cell culture medium).

[0159] Post-translational modification

[0160] In certain embodiments, the anti-NaPi2b antibody constructs described herein may comprise one or more post-translational modifications. Such post-translational modifications may occur in vivo or may be performed in vitro after the anti-NaPi2b antibody construct has been isolated from the host cell.

[0161] Post-translational modifications include the various modifications known in the art (see, for example, Proteins - Structure and Molecular Properties, 2nd Edition, T.E. Creighton, W.H. Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, edited by B.C. Johnson, 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 those embodiments in which the anti-NaPi2b antibody construct comprises one or more post-translational modifications, the construct may comprise the same type of modification at one or several sites, or it may comprise different modifications at different sites.

[0162] Examples of post-translational modifications include glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, formylation, oxidation, reduction, proteolytic cleavage, or specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease or NaBH 4 4.

[0163] Other examples of post - translational modifications include, for example, the addition or removal of N - linked or O - linked carbohydrate chains, chemical modification of N - linked or O - linked carbohydrate chains, processing of the N - terminus or C - terminus, attachment of chemical moieties to the amino acid backbone, and addition or deletion of the N - terminal methionine residue produced by expression in a prokaryotic host cell. Post - translational modifications can also include modification with a detectable label (such as an enzyme label, a fluorescent label, a luminescent label, an isotope label, or an affinity label) to permit detection and isolation of the protein. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, β - galactosidase, and acetylcholinesterase. Examples of suitable cofactor 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.

[0164] Additional examples of post - translational modifications include acetylation, 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 a covalent cross - linker, formation of cysteine, formation of pyroglutamate, γ - carboxylation, GPI - anchor formation, hydroxylation, iodination, methylation, myristoylation, polyethylene glycolylation, prenylation, racemization, selenoylation, sulfation, transfer - RNA - mediated addition of amino acids to proteins such as arginylation and ubiquitination.

[0165] Polynucleotides, Nucleotides, and Host Cells

[0166] Certain embodiments of the present disclosure relate to an isolated polynucleotide or a set of polynucleotides encoding an anti - NaPi2b antibody construct described herein. In this context, the polynucleotide can encode all or a portion of the anti - NaPi2b antibody construct.

[0167] The terms “nucleic acid”, “nucleic acid molecule”, and “polynucleotide” are used interchangeably herein and refer to a polymeric form of nucleotides (deoxyribonucleotides or ribonucleotides or their analogs) of any length. 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.

[0168] A polynucleotide that “encodes” a given polypeptide is a polynucleotide that, when placed under the control of appropriate regulatory sequences, is transcribed (in the case of DNA) or translated (in the case of mRNA) in vivo into the polypeptide. 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' (carboxyl) terminus. A transcription termination sequence may be located 3' of the coding sequence.

[0169] Certain embodiments of the present disclosure relate to vectors (such as expression vectors) comprising one or more polynucleotides encoding an anti-NaPi2b antibody construct as described herein. The one or more polynucleotides may be contained in a single vector, or contained in more than one vector. In some embodiments, the polynucleotide is contained in a polycistronic vector.

[0170] Certain embodiments of the present disclosure relate to host cells comprising a polynucleotide encoding an anti-NaPi2b antibody construct as described herein or one or more vectors containing the polynucleotide. In some embodiments, the host cell is eukaryotic, such as Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or lymphoid cells (such as Y0, NS0, Sp20 cells).

[0171] Antibody-drug conjugate

[0172] Certain embodiments of the present disclosure relate to antibody-drug conjugates (ADCs) comprising an anti-NaPi2b antibody construct conjugated to one or more drug moieties (such as cytotoxins or immunomodulators).

[0173] Generally, in an ADC, the anti-NaPi2b antibody construct is conjugated to the drug moiety via a linker, which can be a cleavable or non-cleavable linker. The anti-NaPi2b antibody construct can be conjugated to a single drug molecule, or it can 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, in the ADCs of the present disclosure, the DAR is in the range of about 1 to about 12, or about 2 to about 12, or about 2 to about 8.

[0174] In certain embodiments, an ADC comprising an anti-NaPi2b antibody construct has the general formula I:

[0175] A-(L-(D) m ) n (I)

[0176] wherein A is an anti-NaPi2b antibody construct as described herein; L is a linker; D is a drug moiety; m is an integer between 1 and about 8, and n is between 1 and about 12.

[0177] In certain embodiments of Formula I, m is between 1 and 6. In some embodiments, m is 1 or 2. In some embodiments, n is between about 1 and about 8, such as between about 2 and about 8.

[0178] A variety of compounds known to be useful as cytotoxic or immunomodulatory ADC payloads can be used as the drug moiety in an ADC comprising an anti-NaPi2b antibody construct. Examples include, but are not limited to, maytansine alkaloids and maytansine alkaloid analogs, benzodiazepines and pyrrolobenzodiazepines duocarmycins (such as CC-1065) and their analogs, 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 TLR7 and / or TLR8 agonists), or STING agonists.

[0179] In certain embodiments, the drug moiety comprised in the ADCs of the present disclosure is an auristatin or auristatin analog, a hemiasterlin or hemiasterlin analog, a camptothecin or camptothecin analog, or eribulin.

[0180] Generally, in the ADCs of the present disclosure, the drug moiety is linked to the anti-NaPi2b antibody construct via a linker. The linker is a bifunctional or multifunctional moiety capable of linking one or more drug molecules to the antibody construct. In some embodiments, the linker can be bifunctional (or monovalent) such that it links a single drug molecule to a single site on the antibody construct. In some embodiments, the linker can be multifunctional (or multivalent) such that it links more than one drug molecule to a single site on the antibody construct. In some embodiments, a multifunctional linker can also be used to link a single drug molecule to more than one site on the antibody construct.

[0181] The linker can be attached to the anti-NaPi2b antibody construct in a variety of ways, such as through surface lysines, reductive coupling to oxidized carbohydrates, or through cysteine residues released via reduction of interchain disulfide bonds. Alternatively, the linker can be attached to the anti-NaPi2b antibody construct by modifying the antibody construct to include additional cysteine residues (see, e.g., U.S. Patent Nos. 7,521,541, 8,455,622, and 9,000,130) or by providing unnatural amino acids that provide reactive handles such as selenomethionine, p-acetylphenylalanine, formylglycine, or p-azidomethyl-L-phenylalanine to allow site-specific conjugation (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., 2014, Bioconj. Chem., 25:351-361). Another option is to use the GlycoConnect TM technology (Synaffix BV, Nijmegen, Netherlands), which involves enzymatic remodeling of the antibody glycan to allow attachment of the linker by metal-free click chemistry (see, e.g., European Patent No. EP 2 911 699).

[0182] Linkers generally include functional groups capable of reacting with one or more target groups on the antigen-binding construct, and one or more functional groups capable of reacting with target groups on the drug moiety. Suitable functional groups are known in the art and include, for example, those described in Bioconjugate Techniques (G.T. Hermanson, 2013, Academic Press). Non-limiting examples of functional groups that react with free cysteine or thiol include maleimide, haloacetamide, haloacetyl, activated esters (such as succinimidyl esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters), acid anhydrides, acyl chlorides, sulfonyl chlorides, isocyanates, and isothiocyanates. In this case, "self-stabilizing" maleimides such as those described by Lyon et al., 2014, Nat. Biotechnol., 32:1059-1062 can also be used. Non-limiting examples of functional groups for reacting with surface lysine and amine include activated esters such as N-hydroxysuccinimide (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 succinimidyl-1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) and benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP). Non-limiting examples of functional groups capable of reacting with electrophilic groups (such as aldehyde or ketone carbonyls) on the antibody construct or drug moiety include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, carboxylic acid hydrazide, and aromatic hydrazide.

[0183] In certain embodiments, linkers can be used that include functional groups that allow for the bridging of two interchain cysteines on the antibody-binding construct, such as ThioBridge TM Linkers (Badescu et al., 2014, Bioconjug. Chem., 25:1124–1136), dithiomaleimide (DTM) linkers (Behrens et al., 2015, Mol. Pharm., 12:3986–3998), dithioaryl (TCEP) pyridazinone-based linkers (Lee et al., 2016, Chem. Sci., 7:799-802), or dibromopyridazinone-based linkers (Maruani et al., 2015, Nat. Commun., 6:6645).

[0184] A variety of linkers for conjugating drugs to antibodies are known in the art, including hydrazone-, disulfide- and peptide-based linkers. The linker can be cleavable or non-cleavable. Cleavable linkers are generally prone to cleavage under intracellular conditions, for example by lysosomal processes. Examples include protease-sensitive, acid-sensitive or reduction-sensitive linkers. In contrast, non-cleavable linkers rely on the degradation of the antibody in the cell, which typically results in the release of the amino acid-linker-drug moiety.

[0185] Examples of cleavable linkers useful in certain embodiments are peptide-containing linkers that are cleavable by intracellular proteases such as lysosomal proteases or endosomal proteases. Examples include dipeptide-containing linkers, such as those containing 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, Me 3 Lys-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.

[0186] Additional useful cleavable linkers include disulfide-containing linkers and linkers that are hydrolyzable at a specific pH or pH range, such as hydrazone linkers. Examples of disulfide-containing linkers include, but are not limited to, N-succinimidyl 4-(2-pyridyldithio)butyrate (SPDB) and N-succinimidyl 4-(2-pyridyldithio)-2-sulfonate butyrate (sulfonyl-SPDB). Disulfide-containing linkers can optionally include additional groups to provide steric hindrance near the disulfide bond to improve the extracellular stability of the linker, for example, containing gem-dimethyl. Linkers containing combinations of these functionalities may also be useful, for example, linkers containing both hydrazone and disulfide bonds are known in the art.

[0187] Another example of a cleavable linker is a linker that contains β-glucuronide, which 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).

[0188] The cleavable linker may optionally also contain one or more additional functional groups, such as self-immolative / self-eliminating groups, spacers or hydrophilic moieties.

[0189] Self-immolative / self-eliminating groups that can be used for the linker include, for example, p-aminobenzyloxycarbonyl (PABC) and p-aminobenzyl ether (PABE) groups and methylated ethylenediamine (MED). Other examples of self-immolative groups include, but are not limited to, aromatic compounds that are electronically similar to PABC or PABE groups, such as heterocyclic derivatives, e.g., 2-aminoimidazole-5-methanol derivatives, as described in U.S. Patent No. 7,375,078. Other examples include groups that cyclize upon hydrolysis of an amide bond, such as substituted and unsubstituted 4-aminobutyramide (Rodrigues et al., 1995, Chemistry Biology, 2:223-227) and 2-aminophenylpropionamide (Amsberry et al., 1990, J. Org. Chem., 55:5867-5877). Self-immolative / self-eliminating groups, either alone or in combination, are generally included in peptide-based linkers, but can also be included in other types of linkers. In some embodiments, the linker may include one or more self-immolative / self-eliminating groups, such as PABC groups, PABE groups, or a combination of PABC or PABE groups and MED.

[0190] Spacers that can be used in the linker for an ADC include, for example, alkylene and spacers based on aliphatic acids, diacids, amines or diamines, such as diglycolate, malonate, caproate and capramide. Other spacers include, for example, glycine-based spacers and polyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG) spacers. PEG and mPEG spacers also act as hydrophilic moieties and can be particularly useful in combination with hydrophobic drugs, but their use in linkers with other drugs is also contemplated in some embodiments.

[0191] An ADC comprising an anti-NaPi2b antibody construct can be prepared by one of several ways known in the art, using standard organic chemical reactions, conditions, and reagents (see, e.g., 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 divalent linker reagent to form an antibody-linker intermediate Ab-L via a covalent bond, followed by reacting with an activated drug moiety D; or (2) reacting a functional group of the drug moiety with the linker reagent to form a drug-linker intermediate D-L via a covalent bond, followed by reacting with a functional group of the antibody construct. Conjugation methods (1) and (2) can be used with a variety of antibody constructs, drug moieties, and linkers to prepare the ADCs described herein.

[0192] A variety of linkers, linker components, and drugs prepared are commercially available or can be prepared using standard synthetic organic chemistry techniques (see, e.g., March’s Advanced Organic Chemistry (Smith and March, 2006, 6th Edition, 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)). In addition, many preformed drug-linkers suitable for reacting with the selected antibody construct are also commercially available. For example, drug-linkers containing DM1, DM4, MMAE, MMAF, or calicheamicin SA are available from Creative BioLabs (Shirley, NY). A variety of antibody-drug conjugation services are also available commercially from companies such as Lonza Inc. (Allendale, NJ), Abzena PLC (Cambridge, UK), ADC Biotechnology (St. Asaph, UK), Baxter BioPharmaSolutions (Baxter Healthcare Corporation, Deerfield, IL), and Piramal PharmaSolutions (Grangemouth, UK).

[0193] Once prepared, the ADC 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.

[0194] Methods of Use

[0195] Certain aspects of the present disclosure relate to the therapeutic or diagnostic use of anti-NaPi2b antibody constructs and ADCs. NaPi2b is overexpressed in a variety of cancers, and thus 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.

[0196] Examples of cancers that can be treated in certain embodiments are carcinomas, including adenocarcinoma and squamous cell carcinoma; melanoma and sarcoma. Carcinomas and sarcomas are also commonly referred to as "solid tumors". Examples of common solid tumors that can be treated in certain embodiments include, but are not limited to, brain cancer, breast cancer, cervical cancer, colon cancer, head and neck cancer, kidney cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, stomach cancer, uterine cancer, non-small cell lung cancer (NSCLC), and colorectal cancer. Various forms of lymphoma can also result in the formation of solid tumors and can thus be considered solid tumors in certain cases. Generally, the cancer to be treated is a cancer that expresses NaPi2b.

[0197] Certain embodiments relate to methods of inhibiting the growth of NaPi2b-positive tumor cells, the method comprising contacting the cells with an anti-NaPi2b antibody construct or ADC as described herein. The cells can be in vitro or in vivo. In certain embodiments, the anti-NaPi2b antibody constructs and ADCs can be used in methods of treating NaPi2b-positive cancers or tumors in a subject.

[0198] Cancers that overexpress NaPi2b are typically solid tumors. Examples include, but are not limited to, ovarian cancer, endometrial cancer, and lung cancer (such as non-small cell lung cancer (NSCLC)).

[0199] Treatment of NaPi2b-positive cancers can result in one or more of the following: alleviation of symptoms, reduction in tumor size, inhibition of tumor growth, reduction of one or more direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or alleviation of the disease state, increased survival rate, increased progression-free survival rate, remission, and / or improvement of prognosis.

[0200] In certain embodiments, when used to treat cancer, the anti-NaPi2b antibody constructs and ADCs can be administered systemically to the subject to be treated, for example, by bolus injection or continuous infusion into the bloodstream of the subject. In certain embodiments, when used to treat cancer, the anti-NaPi2b antibody constructs and ADCs can be administered locally to the subject at the site to be treated.

[0201] The subject anti-NaPi2b antibody constructs and ADCs can be used alone, or in combination with one or more known chemotherapeutic or immunotherapeutic agents commonly used to treat cancer. The combination of the anti-NaPi2b antibody construct or ADC with a standard chemotherapeutic or immunotherapeutic agent can be used to improve the efficacy of the chemotherapeutic or immunotherapeutic agent and, thus, can improve standard cancer therapies. Such an application may be important in the treatment of drug-resistant cancers that do not respond to standard treatment. 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.

[0202] The dosage of the anti-NaPi2b antibody construct or ADC to be administered is not limited by definition, but it will be a therapeutically effective amount. A "therapeutically effective amount" refers to the amount of the anti-NaPi2b antibody construct or ADC described herein that is sufficient to effect treatment of a specific indication when administered to a subject. A therapeutically effective amount of the anti-NaPi2b antibody construct or ADC for cancer treatment can, for example, have one or more of the following effects: reducing the number of cancer cells, reducing the tumor size, inhibiting the infiltration of cancer cells into peripheral organs, inhibiting tumor metastasis, inhibiting tumor growth; increasing survival time and / or alleviating to some extent one or more symptoms associated with cancer. For cancer therapy, efficacy can alternatively be measured, for example, by assessing time to progression (TTP) and / or determining response rate (RR).

[0203] Certain embodiments relate to methods of using the anti-NaPi2b antibody constructs described herein to detect the presence of NaPi2b in a biological sample, such as a sample comprising cells or tissue. In some embodiments, the biological sample can be taken from a patient, such as a patient known or suspected to have cancer. Some embodiments relate to methods of detecting the presence of NaPi2b in a biological sample, the method comprising contacting the sample with the anti-NaPi2b antibody constructs described herein.

[0204] Certain embodiments relate to methods of using the anti-NaPi2b antibody constructs described herein to diagnose a disorder (such as cancer) associated with increased NaPi2b expression. The diagnostic method can be an in vivo method of administering the anti-NaPi2b antibody construct to a subject, or it can be an in vitro method of contacting a sample taken from a subject with the anti-NaPi2b antibody construct. For in vivo methods, the administration can be systemic or local.

[0205] In methods of detecting the presence of NaPi2b or diagnosing a disorder associated with increased NaPi2b expression, the anti-NaPi2b antibody constructs can be labeled with a detectable label, such as a fluorescent, luminescent, chromogenic, chemiluminescent, radioactive, or enzymatic label known in the art.

[0206] Drug composition

[0207] For therapeutic use, the anti-NaPi2b antibody construct and the ADC can be provided in the form of a drug composition, which comprises the anti-NaPi2b antibody construct or the ADC and a pharmaceutically acceptable carrier or diluent. The composition can be prepared by known procedures using well-known and readily available ingredients.

[0208] The drug composition can be formulated for administration to a subject by, for example, parenteral, oral (including, for example, buccal or sublingual), topical, rectal or vaginal routes, or by inhalation or spraying. As used herein, the term "parenteral" includes subcutaneous injection, as well as intradermal, intra-articular, intravenous, intramuscular, intra-vascular, intrasternal, intrathecal injection or infusion. The drug composition will generally be formulated in a form suitable for administration to the subject, such as syrups, elixirs, tablets, troches, lozenges, hard or soft gelatin capsules, pills, suppositories, oily or aqueous suspensions, dispersible powders or granules, emulsions, injections or solutions. The drug composition can be provided as a unit dosage form.

[0209] In certain embodiments, the drug composition comprising the anti-NaPi2b antibody construct or the ADC can be formulated for parenteral administration by infusion or in unit dose injectable form, such as a lyophilized preparation or an aqueous solution.

[0210] Pharmaceutically acceptable carriers are generally non-toxic to the recipient at the dosages and concentrations employed. Examples of such carriers include, but are not limited to: buffers such as phosphates, citrates and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butanol, benzyl alcohol, alkyl esters of p-hydroxybenzoic acid (such as methyl p-hydroxybenzoate or propyl p-hydroxybenzoate), catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol; polypeptides of low molecular weight (less than about 10 residues); proteins such as serum albumin or 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 ions; metal complexes such as Zn-protein complexes; and non-ionic surfactants such as polyethylene glycol (PEG).

[0211] In certain embodiments, a pharmaceutical composition comprising an anti-NaPi2b antibody construct or an ADC can be in the form of a sterile injectable aqueous or oily solution or suspension. Such suspensions can be formulated using suitable dispersing or wetting agents and / or suspending agents known in the art. The sterile injectable solution or suspension can contain the anti-NaPi2b antibody construct or ADC in a non-toxic parenterally acceptable diluent or solvent. Acceptable diluents and solvents that can be employed include, for example, 1,3-butanediol, water, Ringer's solution, or isotonic sodium chloride solution. Additionally, a sterile, fixed oil can be employed as the solvent or suspending medium. For this purpose, various bland fixed oils can be used including synthetic mono- or diglycerides of fatty acids. Additionally, fatty acids such as oleic acid find use in injectable preparations. Adjuvants such as local anesthetics, preservatives, and / or buffering agents can also be included in the injectable solution or suspension.

[0212] In certain embodiments, a pharmaceutical composition comprising an anti-NaPi2b antibody construct or an ADC can be formulated for intravenous administration to a subject, such as a human. Generally, a composition for intravenous administration is a solution in a sterile, isotonic, aqueous buffer. Optionally, the composition can also contain a solubilizing agent and / or a local anesthetic, such as lidocaine, to alleviate pain at the injection site. Typically, the ingredients are provided separately or mixed together in unit dosage forms, for example, as a dry lyophilized powder or an anhydrous concentrate in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of the active agent. When the composition is to be administered by infusion, it can be dispensed in an infusion bottle containing sterile pharmaceutical grade 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.

[0213] Other pharmaceutical compositions and methods of preparing pharmaceutical compositions are known in the art and are described, for example, in "Remington: The Science and Practice of Pharmacy" (formerly "Remingtons Pharmaceutical Sciences"); Gennaro, A., Lippincott, Williams & Wilkins, Philadelphia, PA (2000).

[0214] Drug Kit

[0215] Certain embodiments relate to a drug kit comprising an anti-NaPi2b antibody construct or an ADC as described herein.

[0216] The kit will typically include a container that houses an anti-NaPi2b antibody construct or ADC, as well as a label and / or package insert on or accompanying the container. The label or package insert contains instructions typically included in the commercial packaging of a therapeutic product, providing information about the indications, usage, dosage, administration, contraindications, and / or warnings for using such a therapeutic product. The label or package insert may also include a notice in a form prescribed by a government agency that regulates the manufacture, use, or sale of a pharmaceutical or biological product, which reflects the approval of the manufacturing agency for use or sale for administration to humans or animals. In some embodiments, the container may have a sterile inlet. For example, the container can be an intravenous solution bag or a vial with a stopper pierceable by a subcutaneous injection needle.

[0217] In addition to the container that houses the anti-NaPi2b antibody construct or ADC, the kit may optionally include one or more additional containers that contain other components of the kit. For example, a pharmaceutically acceptable buffer (such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer’s solution, or glucose solution), other buffers, or diluents.

[0218] Suitable containers include, for example, bottles, vials, syringes, and intravenous solution bags, etc. The containers can be made of various materials such as glass or plastic. In appropriate cases, one or more components of the kit can be lyophilized or provided in a dry form (such as a powder or granule), and the kit can additionally contain a suitable solvent for reconstituting the lyophilized or dry components.

[0219] The kit may also include other materials that are desirable from a commercial or user perspective, such as filters, needles, and syringes.

[0220] The following examples are provided for illustrative purposes and are not intended to limit the scope of the invention in any way.

[0221] Examples

[0222] Example 1: Preparation of Anti-NaPi2b Antibody

[0223] An antibody construct that specifically binds to human NaPi2b was generated by immunizing mice with human cells overexpressing NaPi2b, as outlined below.

[0224] 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) according to the manufacturer's instructions for Lipofectamine 2000 (Thermo Fisher Scientific). Ten B6x129 mice were subcutaneously immunized with the transfected HEK293-6E cells over 63 days, after which blood was drawn and spleens were harvested.

[0225] Anti-human NaPi2b antibody titers were determined by flow cytometry using CHO-S cells expressing human NaPi2b. All ten mice had a significant response to human NaPi2b.

[0226] Subsequently, splenocytes from all mice were pooled and used for hybridoma generation. P3X63Ag8.653 cells (ATCC catalog number CRL-1580) were mixed with IgG+ B cells isolated from the spleens and fused using an ECM 2001 electrofusion apparatus (BTX, Harvard Bioscience) with optimized settings. After overnight recovery, the hybridomas were diluted and seeded in selection medium containing the following final concentrations of supplements: 100 μM hypoxanthine, 0.4 μM aminopterin, and 16 μM thymidine. After 14 days of selection, the hybridomas were diluted to an average of one cell per well and seeded into 96-well plates. The binding of cell supernatants containing secreted antibodies was evaluated on CHO-S cells transfected with the same plasmid (pTT5-huNaPi2b) used to transfect HEK293-6E cells. Hybridoma cells from wells containing supernatants with antibodies that bound NaPi2b were harvested for sequencing.

[0227] A murine - human chimeric IgG1 / κ antibody construct v23855 was prepared using the murine VH and VL sequences of one of the identified anti-NaPi2b antibodies. The coding sequences of the antibody variable regions were cloned in-frame into a human IgG1 expression vector (with the human IgG1 constant region starting from alanine 118 according to Kabat numbering) or a human Cκ expression vector (with the human Cκ constant region starting from arginine 108 according to Kabat numbering), both expression vectors being based on pTT5. The activity of the resulting recombinant chimeric antibody construct was confirmed in a specific binding assay and found to be comparable to the parental antibody (data not shown).

[0228] Example 2: Humanization of the anti-NaPi2b antibody construct

[0229] Humanize the chimeric anti-human NaPi2b antibody construct variant v23855 produced as described in Example 1. 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 is performed as described below.

[0230] Table 2.1: CDR Sequences of Anti-NaPi2b Antibody Construct v23855

[0231]

[0232]

[0233] Table 2.2: VH and VL Sequences of Anti-NaPi2b Antibody Construct v23855

[0234]

[0235] 2.1 Humanization

[0236] When the murine VH and VL sequences of v23855 were aligned with the corresponding human germline sequences, IGHV1-46*03 and IGKV1D-39*01 were identified as the closest and most common human germline sequences (and the IGHJ4*03 and IGKJ2*04 joining region germline sequences were selected respectively). The CDR sequences defined according to AbM (see Table 2.1) were grafted onto the frameworks of these selected human germline sequences, as Figure 1 A Figure 1 shown in B. Back mutations to murine residues were included at positions in the resulting sequences that were judged likely to be important for maintaining binding affinity to the antigen NaPi2b, resulting in several humanized sequences, where the sequences generated were built on the previous sequences, and where the first humanized sequence contained no back mutations. As defined by the AbM method, no variants modified the CDRs of the parental antibody.

[0237] This process yielded four variable heavy chain humanized sequences and three variable light chain humanized sequences. Full heavy chain sequences containing the humanized heavy chain variable domain (VH) and the hIgG1 heavy chain constant domains (CH1, hinge, CH2, CH3), and full light chain sequences containing the humanized light chain variable domain (VL) and the human κ light chain constant domain (κCL) were assembled. Then monoclonal antibody (mAb) variants were assembled such that each humanized heavy chain was paired with each humanized light chain to provide 12 humanized variants to be evaluated by experimental methods.

[0238] 2.2 Generation of Humanized Antibody Constructs

[0239] Each of the 12 humanized antibody constructs, as well as the parental v23855 construct, was generated in a full-size antibody (FSA) format containing two identical full-length heavy chains and two identical κ light chains.

[0240] The full-length heavy chain contains the human CH1-hinge-CH2-CH3 domain sequence of IGHG1*01 [SEQ ID NO:33]; (see Table 2.3). The light chain contains the human κ CL sequence of IGKC*01 [SEQ ID NO:34]; (see Table 2.3).

[0241] Table 2.3: Constant heavy and light chain sequences

[0242]

[0243] Each humanized VH domain sequence, as well as the murine VH domain sequence, was appended to the human CH1-hinge-CH2-CH3 domain sequence of IGHG1*01 to provide four humanized full heavy chain sequences and one parental mouse-human chimeric full heavy chain sequence. Each humanized VL domain sequence or murine VL domain sequence was appended to the human κ 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 gene synthesized.

[0244] The heavy chain vector insert fragment 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 terminating at residue G446 (EU numbering) of the CH3 domain were ligated into the pTT5 vector to generate the heavy chain expression vector. The light chain vector insert fragment 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 in Table 2.4 below.

[0245] Table 2.4: Amino acid sequences of humanized VH and VL sequences

[0246]

[0247]

[0248] The heavy and light chains of each humanized antibody variant and the parental mouse-human chimeric antibody variant were expressed in 300 mL of CHO-3E7 cell cultures. Briefly, CHO-3E7 cells were cultured at 37 °C in FreeStyle F-68 (Gibco / Thermo Fisher Scientific, Waltham, MA) supplemented with 4 mM glutamine (Hyclone SH30034.01) and 0.1% TM F17 medium (Thermo Fisher Scientific, Waltham, MA) at a density of 1.7 - 2 x 10 6 cells / mL with a viability > 95%. Using (Polyscience, Inc., Philadelphia, PA), a total of 300 μg of DNA (150 μg of antibody DNA and 150 μg of GFP / AKT / filler fragment DNA) was transfected into a total volume of 300 mL of CHO-3E7 cells + 1x antibiotic / antimycotic (GE Life Sciences, Marlborough, MA) at a DNA:PEI ratio of 1:4 (w / w). Twenty-four hours after adding 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 an additional 6 days before harvest.

[0249] Protein A purification was performed using a 1 mL HiTrap TM MabSelect TM SuRe TM column (Cytiva, Marlborough, MA). The clarified supernatant sample was loaded onto the column equilibrated in Dulbecco’s PBS (DPBS). The column was washed with DPBS. Proteins were eluted with 100 mM sodium citrate buffer (pH 3.0). The pH of the elution fractions was adjusted to a final pH of 6 - 7 by adding 10% (v / v) 1 M HEPES (pH approximately 10.6 - 10.7). The sample buffer was exchanged into DPBS using a 5 mL Zeba TM spin column (ThermoScientific). Proteins were quantified based on the absorbance at 280 nm (A280nm).

[0250] After purification, the purity of the samples was evaluated by SDS-PAGE under non-reducing and reducing conditions. According to the manufacturer's protocol, the protein samples were mixed with LDS sample buffer and Mix the sample with a reducing agent (for reducing conditions only), and then heat the sample at 70 °C for 15 minutes. The processed protein sample containing 1.5 mg of protein and the molecular weight (MW) Precision Plus Protein TM Dual Color (Bio-Rad) standards for MW estimation was loaded onto a NuPAGE 4-12% Bis-Tris gel (15 wells). Using the XCell Mini-Cell system from Life Technologies (Thermo Fisher Scientific) and MOPS SDS running buffer, perform gel electrophoresis at 200 V for 50 minutes. Stain the gel with Biosafe Coomassie solution and capture the gel image using the ChemiDoc TM MP imaging system (Bio-Rad).

[0251] The yields of each of the twelve humanized antibody variants were similar, ranging from approximately 23 - 30 mg (or approximately 77 - 100 mg / L culture), and were approximately 2-fold that of the parental mouse-human chimeric antibody v23855 (14 mg yield). The SDS-PAGE results of these antibody samples are shown in Figure 2A (non-reducing) and Figure 2B (reducing). As can be seen from these figures, non-reducing (NR) and reducing (R) SDS-PAGE reflect a single species corresponding to the full-size antibody and the intact heavy and light chains.

[0252] 2.3 Quality assessment of humanized antibodies

[0253] After protein A purification, the species homogeneity of the humanized antibody variant and parental mouse-human chimeric antibody variant samples was evaluated by UPLC-SEC.

[0254] Using a Waters Acquity UPLC set at 30 °C and equipped with a photodiode array (PDA) detector TMUPLC-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) on an H-Class Bio system. The mobile phase was Dulbecco's phosphate buffered saline (DPBS) (pH 7.4) containing 0.02% Tween 20, and the flow rate was 0.4 mL / min. The total run time for each injection was 7 minutes, 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. Using Waters 3 software, peak integration was performed using Apex Track TM and shoulder features were detected.

[0255] Figure 2C and Figure 2D Figure 1 shows the UPLC-SEC profiles of the parental mouse-human chimeric antibody v23855 and the representative humanized antibody v29456 samples. The UPLC-SEC profile of the representative humanized antibody sample reflects a high species homogeneity comparable to that of the parental mouse-human chimeric antibody sample. Samples from the remaining humanized antibody variants have features similar to those shown for the representative humanized antibody sample.

[0256] 2.4 Purity assessment of humanized antibodies

[0257] The apparent purity of humanized antibody variants was evaluated using mass spectrometry and non-denaturing deglycosylation.

[0258] 10 μg of each sample was incubated with 1 μg of deglycosylation mixture (NEB, P6044) at room temperature for 1 hour and transferred to an incubator at 37 °C for 16 hours.

[0259] 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 PLRP-S column ( Inject 1 μL of the sample at (2.1 x 50 mm, 8 μm) using an Agilent 1290 Infinity II LC system coupled with an Agilent 6545 QTOF, with a dual-jet electrospray ionization source, a column temperature of 70 °C, and a flow rate of 0.3 mL / min. The mobile phase consists of the following: A: LC-MS grade water containing 0.1% v / v formic acid, 0.025% v / v trifluoroacetic acid, and 10% v / v isopropanol, and B: acetonitrile containing 0.1% v / v formic acid and 10% v / v isopropanol. Before injection, pre-equilibrate the column in 20% mobile phase B. Then, apply a 20-minute gradient of 20% to 40% mobile phase B, followed by a 2-minute gradient of 27% to 90% mobile phase B, and perform a 2-minute column wash at 99% mobile phase B.

[0260] Example 3: Characterization of the humanized anti-NaPi2b antibody construct - Assessment of thermal stability

[0261] The thermal stability of the humanized antibody variants was evaluated by differential scanning calorimetry (DSC) as described below.

[0262] 400 μL of the purified sample mainly 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 start of each DSC run, 5 buffer blank injections were performed to stabilize the baseline, and a buffer injection was arranged before each sample injection for reference. Each sample was scanned from 20 °C to 100 °C at a rate of 60 °C / hr using low feedback, an 8-second filter, a 3-minute pre-scan thermostat, and a 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.

[0263] The Fab Tm values determined for the humanized variants are shown in Table 3.1. Compared to the parental antibody v23855 (Fab Tm of approximately 72.4 °C), all humanized variants showed increased thermal stability, with Fab Tm values in the range of approximately 78 - 83 °C.

[0264] Table 3.1: Thermal stability of humanized variants

[0265] Variant Heavy chain and light chain composition Fab Tm (°C) v23855 (parent) Chimera 72.4 v29449 H4L3 80.0 v29450 H3L3 81.0 v29451 H2L3 83.0 v29452 H1L3 79.5 v29453 H4L2 78.9 v29454 H3L2 79.7 v29455 H2L2 81.5 v29456 H1L2 77.9 v29457 H4L1 80.5 v29458 H3L1 81.6 v29459 H2L1 83.1 v29460 H1L1 80.3

[0266] Example 4: Functional characterization of the anti-NaPi2b antibody construct - Competitive binding (epitope grouping)

[0267] To characterize the binding of parental chimeric anti-NaPi2b antibody v23855 to NaPi2b, competitive binding or epitope binning assays were performed against anti-NaPi2b reference antibodies MX-35 (v18992) and rituximab (v18993). Binding was evaluated by flow cytometry using HEK293-6e cells as described below.

[0268] Each of the anti-NaPi2b detection antibodies v23855, v18992, v18993, and palivizumab (anti-RSV, v16955) was conjugated to 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 (1 μg pTT5-NaPi2b per 1 million cells), or transfected with GFP (ATUM, Menlo Park, CA; pD2610-v23, also 1 μg DNA / 1 million cells). After transfection, HEK296-6e cells expressing human NaPi2b and transfected HEK296-6e cells expressing GFP were mixed at a ratio of 4:1. Each well of a V-bottom 96-well plate was inoculated with a mixture of 100,000 cells and incubated with 100 μg / mL unlabeled competitor anti-NaPi2b antibody on ice for one hour. After incubation, the cells were washed and stained with 1 μg / mL AF647-conjugated anti-NaPi2b detection antibody on ice for one hour. After staining and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa TM Cell Analyzer (BD Biosciences, Franklin Lake, NJ), collecting a minimum of 10,000 events per well. Using FlowJo TM Version 10.8.1 (BD Biosciences, Franklin Lake, NJ), the AF647 / APC-A GeoMean (geometric mean of the fluorescence signal, proportional to binding to anti-human AF647) of the FITC / GFP-negative live cell population was calculated. The percent inhibition was calculated using the following formula:

[0269]

[0270] The competitive binding results of parental chimeric anti-NaPi2b antibody v23855 against v18992 (MX35) and v18993 (rituximab) are shown in Table 4.1.

[0271] Table 4.1: Competitive binding. Percent inhibition (relative to negative control antibody). GFP-population

[0272]

[0273] Each tested anti-NaPi2b antibody competed with itself as expected (>95% inhibition, see data in bold text). The chimeric anti-NaPi2b antibody v23855 competed for binding to v18992 and v18993, as demonstrated by comparable % inhibition to itself (>94%). As expected, no competitive binding was observed against the negative control palivizumab (v16955).

[0274] Example 5: Functional Characterization of Humanized Anti-NaPi2b Antibody Constructs - Cynomolgus Monkey and Mouse NaPi2b Binding

[0275] The binding cross-reactivity of the humanized antibody variant v29456 with human, cynomolgus monkey, and mouse NaPi2b was evaluated by flow cytometry using HEK293-6e transfected cells. The reference anti-NaPi2b antibodies MX-35 (v18992) and rifaximuzumab (v18993) were included as comparators, and the anti-RSV antibody palivizumab (v22277) was included as a negative control.

[0276] Briefly, HEK293-6e cells were transfected for approximately 24 hours to transiently express human NaPi2b, cynomolgus monkey, or mouse NaPi2b, 1 μg DNA per 1 million cells. After transfection, 50,000 cells per well were seeded in a V-bottom 96-well plate and incubated with 200 nM primary antibody at 4°C for 18 - 24 hours to prevent internalization. After incubation, the cells were washed and stained with an anti-human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA, catalog number 109-605-098, lot number 124868) at 4°C for 1 hour. After staining and washing, fluorescence was detected by flow cytometry on a BD LSR Fortessa TM cell analyzer (BD Biosciences, Franklin Lake, NJ), collecting a minimum of 10,000 events per well. The AF647 / APC-A GeoMean (geometric mean of the fluorescence signal, proportional to binding to anti-human AF647) of the live single-cell population for each primary antibody was calculated using FlowJo TM v8 software (BD Biosciences, Franklin Lake, NJ). The Bmax and Kd for each primary antibody were calculated using GraphPad Prism version 9 (GraphPad Software, San Diego, CA)

[0277] Binding results of the humanized antibody variants v29456, MX-35 (v18992) and rituximab (v18993) are shown in Table 5.1 and Figure 3A (human NaPi2b), Figure 3B (cynomolgus monkey NaPi2b) and Figure 3C (mouse NaPi2b).

[0278] Table 5.1: Cross-reactivity of v29456 with cynomolgus monkey and mouse NaPi2b

[0279]

[0280]

[0281] * Apparent Kd values are greater than the highest antibody test concentration (>200 nM).

[0282] v29456 and v18992 show binding to human, cynomolgus monkey and mouse NaPi2b on transfected HEK296-6e cells. Rituximab (v18993) shows binding to human and cynomolgus monkey NaPi2b and shows minimal binding to HEK293-6e cells transfected with mouse NaPi2b.

[0283] v29456 has apparent Kd values comparable to v18992 and v18993 in terms of human NaPi2b binding and shows the greatest binding to cynomolgus monkey NaPi2b, generating apparent Kds that are 1 / 10 and 1 / 2 of v18992 (MX35) and v18993 (rituximab), respectively. As expected, the negative control palivizumab (v22277) does not bind to any of the tested species.

[0284] Example 6: Functional characterization of anti-NaPi2b antibody constructs - cell binding of monovalent antibodies by KINEXA

[0285] The binding affinities of anti-NaPi2b antibody constructs were evaluated by Kinexa in the cell line IGROV-1 expressing endogenous NaPi2b. The affinities of the antibody constructs were evaluated in monovalent format to reduce the effects of avidity and internalization and were directly compared to the parental chimeric antibody variants (also in monovalent format). v29814 (monovalent format of parental chimeric variant 23855), v36123 (monovalent format of humanized antibody variant 29452) and v36124 (monovalent format of humanized antibody variant 29456) were evaluated. The experiments were conducted as described below.

[0286] IGROV-1 cell preparation:

[0287] 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 a T175 culture flask (Corning, Corning, NY) and incubated at 37 °C and 5% CO 2 until 80% confluence was reached. Cells were detached from the culture vessel by incubation with cell dissociation buffer (Invitrogen, Waltham, MA) at 37 °C and 5% CO 2 for 30 - 60 minutes, and the cells were collected by neutralizing the cell dissociation buffer with at least 5 volumes of ATCC-modified RPMI 1640 medium supplemented with 10% FBS and kept on ice until use. Cells were counted using a Vi-Cell TM XR cell viability analyzer (Beckman Coulter, Brea, California).

[0288] KinExA Preparation:

[0289] A solid phase was prepared by coating a 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 with gentle rotation at room temperature for 2 hours. The beads were allowed to settle, the supernatant was removed, and the beads were 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) and 10 mg / mL BSA (Sigma-Aldrich, St. Louis, Missouri) in PBS pH 7.4 and rotated for 1 hour at room temperature. The beads were allowed to settle, the supernatant was removed, and the beads were rinsed five times with PBS pH 7.4. The final step of solid phase preparation was completed by coating with 30 μg / mL biotinylated goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) for 1 hour at room temperature with rotation.

[0290] Cell Binding Assay:

[0291] Cell binding assays were established at two different concentrations of 50 pM and 500 pM using an antibody construct or variant as a constant binding partner. For the titration curve of the antibody variant fixed at 50 pM, at least 10 million IGROV-1 cells were used as the titrant. For the titration curve of the antibody variant fixed at 500 pM, at least 5 million cells were used as the titrant. The antibody variant and cells were mixed in PBS pH 7.4, 1 mg / mL BSA, 0.2% NaN 3 and incubated at 4 °C for 7 days with gentle rotation until equilibrium was reached. After incubation, the mixture of the antibody variant and cells was centrifuged to separate the cells from the unbound free antibody variant. The free antibody variant was loaded onto a KinExA 3200 (Sapidyne, Boise, Idaho) where biotinylated anti-human IgG PMMA was used as the solid phase and 0.5 μg / mL of Alexa 647 goat anti-human IgG (Jackson ImmunoResearch, West Grove, PA) was used as the detection antibody.

[0292] The results of the parental chimeric monovalent antibody variant (v29814) and two humanized monovalent antibody variants are shown in Table 6.1. Affinity and receptor expression levels were calculated using N-curve analysis with the concentration of the antibody variant as the reference point. The affinity and receptor expression levels obtained narrow 95% confidence intervals with a fitting error % of less than 1.5%. The N-curve analysis is shown in Figure 4A (v29814), Figure 4B (v36123), and Figure 4C (v36124). For each graph, the right curve shows the data for the 500 pM constant binding partner and the left curve shows the data for the 50 pM constant binding partner.

[0293] Table 6.1 Binding of anti-Napi2b antibody constructs to IGROV-1 cells

[0294]

[0295] # Parentheses indicate 95% confidence intervals

[0296] Compared to the chimeric parental antibody construct, all humanized anti-NaPi2b antibody variants showed similar binding characteristics to each other and a binding affinity that was approximately 1 / 2 lower. The calculated receptor expression levels were between 0.9 - 1.3 million per cell.

[0297] Example 7: Functional characterization of anti-NaPi2b antibody constructs - Internalization

[0298] The internalization of the chimeric parental anti-NaPi2b antibody v23855 and the representative humanized variant v29456 (H1L2) in cell lines expressing NaPi2b (HCC-78 and NCI-H441) was determined by flow cytometry as described below. The anti-NaPi2b antibodies rituximab (v18993) and MX35 (v18992) were used as positive controls, and the anti-RSV antibody palivizumab (v22277) was used as a negative control.

[0299] Briefly, the antibodies were fluorescently labeled by conjugation with the Fab fragment AF488 conjugate targeting anti-human IgG Fc (Jackson ImmunoResearch Labs, West Grove, PA; catalog number 109-547-008) at a 1:1 molar ratio in PBS pH 7.4 (ThermoFisher Scientific, Waltham, MA; catalog number 10010-023) at 4 °C for 24 hours. The cells were seeded at 50,000 cells / well in ATCC-modified RPMI1640 (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (ThermoFisher Scientific, Waltham, MA) in a 48-well plate and incubated overnight under standard culture conditions (37 °C / 5% CO 2 ) to allow attachment. The conjugated antibodies were added to the cells at 10 nM the next day and incubated for 5 - 24 hours under standard culture conditions to allow internalization. After incubation, the cells were dissociated, washed, and the surface AF488 fluorescence was quenched with anti-AF488 antibody (Life Technologies, Carlsbad, CA; catalog number A-11094) at 100 nM for 30 minutes at 4 °C. The quenched AF488 fluorescence (internalization fluorescence) was detected by flow cytometry on a BD LSRFortessa TM cell analyzer (BD Biosciences, Franklin Lake, NJ), and 1,000 minimum events were collected per well. The AF488 / FITC-AGeoMean (geometric mean of the fluorescence signal, proportional to the anti-human Fab AF488 labeling) of the live single-cell population was calculated using FlowJo TM version 10.8.1 (BD Biosciences, Franklin Lake, NJ) and graphed using GraphPad Prism version 9 (GraphPad Software, San Diego, CA).

[0300] The results are shown in Figure 5A(HCC-78) and Figure 5B (NCI-H441) as well as in Table 7.1 below.

[0301] Table 7.1: Internalization of Antibody Constructs

[0302]

[0303] The chimeric parental antibody v23855 and the humanized antibody variant v29456 showed comparable internalization levels to the humanized antibody MX35 (v18992) and much higher internalization levels compared to the humanized antibody rituximab (v18993) on both HCC-78 and NCI-H441 cells at 10 nM antibody treatment at all time points (5 hours and 24 hours). For example, after 5 hours of incubation in HCC-78, v23855 and v29456 showed 21.9-fold and 25.3-fold increases in internalization fluorescence, respectively, compared to the negative control palivizumab. Similarly, after 24 hours of incubation in HCC-78 cells, v23855 and v29456 showed 50.9-fold and 59.9-fold increases in internalization fluorescence, respectively, compared to the negative control palivizumab.

[0304] Example 8: Evaluating the Developability of Anti-NaPi2b Antibodies

[0305] The isoelectric points, self-aggregation tendencies, and non-specific bindings of the anti-NaPi2b antibodies v23855 (parental chimeric), v29452 (H1L3), and v29456 (H1L2) were determined to evaluate the developability of these antibodies. The isoelectric points were measured by capillary isoelectric focusing (cIEF), the self-aggregation tendencies were measured by affinity capture self-interaction nanoparticle spectroscopy (AC-SINS), and the non-specific bindings were measured by NS-ELISA, as described below.

[0306] Capillary Isoelectric Focusing (cIEF)

[0307] The cIEF was performed using the Maurice C. system, system suitability kit, and method development kit. The system suitability standards, fluorescence calibration standards, cartridges, and samples were prepared according to the supplier's recommendations. The capillary was automatically calibrated with the fluorescence standards pretreated with the Maurice cIEF system suitability kit to ensure normal operation of the capillary. The antibody samples were in Gibco TMDilute to a concentration of 0.5 mg / mL in distilled water to a final volume of 40 μL, and mix the Maurice cIEF method development kit samples. Then vortex, centrifuge the samples, and pipette the supernatant into individual wells of a 96-well plate. Detect all electrophoregrams using UV absorbance at 280 nm. All data analysis was performed using the vendor software Compass for iCE was carried out. The Compass software aligned each electrophoregram using pI markers such that the x-axis showed the normalized pI for each injection.

[0308] AC-SINS assay

[0309] The AC-SINS method was carried out in a 384-well plate format ( #3702). Initially, 20 nm gold nanoparticles washed with 0.22 μm filtered Gibco TM distilled water (Ted Pella, Inc., #15705) were coated with a mixture of capture antibody - 80% AffiniPure goat anti-human IgG (H+L) (Jackson ImmunoResearch #109-005-088) and non-capture antibody - 20% ChromPure goat IgG whole molecule (Jackson ImmunoResearch #005-000-003), which was initially buffer exchanged into 20 mM sodium acetate pH 4.3 and diluted to 0.4 mg / mL. The mixture of gold nanoparticles, capture antibody, and non-capture antibody was incubated at room temperature in the dark for 18 hours. Unoccupied sites on the gold nanoparticles were blocked with 1 μM thiolated polyethylene glycol (2 kD) in 20 mM sodium acetate (pH 4.3) to a final concentration of 0.1 μM and then incubated at room temperature for 1 hour. Then the coated nanoparticles were concentrated by centrifugation at 21,000 xg 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 the concentrated nanoparticles was added to Gibco in a 384-well plate TMIn 45 μL of 0.05 mg / mL antibody in PBS pH 7.4. The coated nanoparticles were incubated with the target antibody at room temperature in the dark for 4 hours. Absorbance was read from 450 - 700 nm in 1 nm increments, and the maximum absorbance was identified using a Microsoft Excel macro, the data was smoothed, and the data was fitted with a second-order polynomial. Δλ (nm) was calculated based on the smoothed maximum absorbance from the antibody sample minus the smoothed maximum absorbance of the average blank (only PBS) to determine the antibody AC-SINS score. Antibody-antibody interactions are directly related to the shift in the maximum absorption wavelength of gold nanoparticles coated with the target antibody. A cutoff value of Δλ 10 nm was set for high self-aggregation tendency of the antibody.

[0310] NS-ELISA

[0311] NS-ELISA was used to measure the tendency of antibodies to bind a range of biomolecules to mimic undesired non-specific interactions with biological matrices in vivo as described below.

[0312] NS-ELISA was performed in 96-well EIA / RIA Easy Wash TM transparent flat-bottomed polystyrene high-binding microtiter plates, which were coated overnight at 4 °C with 50 mL of heparin (Sigma, H3149), which was diluted to a final concentration of 250 μg / mL with 50 mM sodium carbonate pH 9.6. The plates were incubated at room temperature for 2 days, and the wells coated with heparin were left uncovered to allow air drying. Insulin ( I9278) and KLH ( H8283) were each diluted to a final concentration of 5 μg / mL with 50 mM sodium carbonate pH 9.6. The ssDNA ( D8899) and dsDNA ( D4553) were diluted to a final concentration of 10 μg / mL with Gibco TM PBS pH 7.4. 50 μL each of insulin, KLH, dsDNA, and ssDNA were added to the 96-well plates and then incubated at 37 °C for 2 hours. The coating material was removed, and the plates were blocked with 200 μL of Gibco TM PBS pH 7.4, 0.1% 20 and incubated with shaking at 200 rpm at room temperature for 1 hour. The plates were washed 3 times with Gibco TM PBS pH 7.4, 0.1% Tween 20. 50 μL in Gibco TM PBS pH 7.4, 0.1% Each of 100 nM of each mAb (15 mg / mL) in 20 was added in duplicate to the wells and incubated with shaking at 200 rpm for 1 hour at room temperature. The plates were washed three times with Gibco TM PBS pH 7.4, 0.1% Tween 20 and 50 μL of 50 ng / mL anti-human IgG HRP (Thermofisher H10307) was added to each well. The plates were incubated for 1 hour at room temperature with shaking at 200 rpm. The plates were washed three times with Gibco TM PBS pH 7.4, 0.1% Tween 20 and 100 μL of TMB substrate (Cell Signaling 7004P6) was added to each well. The reaction was terminated by adding 100 μL of 1 M HCl to each well after approximately 10 minutes and the absorbance was read at 450 nm. The binding score was calculated as the ratio of the ELISA signal of the antibody (antibody-treated) to the signal of the wells containing buffer instead of the primary antibody (untreated). The cut-off values considered for each binding molecule (ssDNA, KLH, insulin, dsDNA, and heparin) were calculated internally based on the mean of the antibodies produced by Zymeworks Inc. and the antibody benchmark values published in the literature.

[0313] The results of all three assays are shown in Table 8.1. In these assays, scores above the cut-off value were used to indicate potentially less desirable biophysical characteristics.

[0314] Table 8.1: Results of the developability assessment of anti-NaPi2b antibodies

[0315]

[0316] The pI values determined for the major isoforms of variants v23855, v29452, and v29456 were 8.35, 8.53, and 8.53, respectively, and they all fell within the typical range for therapeutic antibodies. Analysis of Δλ showed no potential problems on the AC-SINS for all variants. In addition, no potential problems were found by NS-ELISA.

[0317] Example 9: Stability of humanized antibody variants in mouse plasma or PBS

[0318] The purpose of this experiment was to evaluate whether antibody variant v29456 and the reference antibody MX35 (v18992) fragmented over time after incubation in mouse plasma or in PBS pH 7.4 at 37 °C.

[0319] Briefly, v29456 or v18992 was each diluted to a final concentration of 0.5 mg / ml in PBS or mouse plasma and incubated at 37°C. Samples were taken at 0, 7, and 14 days and stored at -80°C until characterization. For characterization, the samples were thawed at room temperature and 50 μg was taken and incubated with 5 μg of recombinant EndoS endoglycosidase for one hour at room temperature. By incubating 95 μL / sample of magnetic agarose streptavidin-coated beads with 15 μg / sample of biotinylated goat anti-human IgG Fc capture antibody for 45 minutes, and then washing 4 times with PBS pH 7.4 by means of a DynaMag TM -2 magnet (Invitrogen TM ), an immunoprecipitation slurry was obtained.

[0320] After deglycosylation, the plasma-incubated samples were incubated with 95 μL of the immunoprecipitation slurry for 1.5 hours at room temperature. Then, by means of a DynaMag TM -2 magnet (Invitrogen TM ), the slurry was washed 6 times with PBS pH 7.4 and 2 times with LC-MS grade water. A final wash with PBS pH 7.4 was performed before elution. The protein was eluted by incubating the beads with 35 μL of LC-MS grade water (containing 20% acetonitrile and 0.1% formic acid) for one hour at room temperature. PBS samples were not immunoprecipitated.

[0321] 5 μL of the eluted sample was transferred to a glass insert in an LC-MS vial. For LC-MS analysis, a Waters TM Synapt TM G2-Si HDMS coupled Waters TM ACQUITY TM UPLC I-Class HPLC system was used, and 1 μL of the sample was injected into a Waters TM BioSuite Phenyl column, 10 μm, 4.6 mm X 75 mm, where the column temperature was 70°C and the flow rate was 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 isopropanol, and B: acetonitrile containing 0.1% v / v formic acid and 10% v / v isopropanol. Before injection, the column was pre-equilibrated in 10% mobile phase B. Then, a 20-minute gradient of 10% to 27% mobile phase B was applied, followed by a 2-minute gradient of 27% to 90% mobile phase B, and a 2-minute column wash at 99% mobile phase B.

[0322] The column was re-equilibrated to 10% mobile phase B for 2 minutes between runs. ESI was carried out 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 / hr, a desolvation gas flow rate of 800 L / hr, and an atomizer gas flow rate of 6.5 bar. The data format was continuous scan, the analyzer was set to sensitivity mode, and the m / z range was 500 to 7000.

[0323] Peak integration, MS deconvolution, and mass assignment were performed in Protein Metrics v4.0 using a deconvolution window of 60000 - 160000 Da, with an m / z range of 1000 - 4000. For all time points, the highest intensity deconvoluted mass was designated as the reference mass for v29456. The reference mass was defined as the average mass of v29456 or v18992 with two short ends of 2-acetamido-2-deoxy-β-D-glucopyranosyl-(1-4)-[α-L-fucopyranosyl-(1-6)] due to the activity of EndoS on N-glycans, 16 disulfide bonds, and the formation of pyroglutamic acid at the N-terminus (if applicable). The mass tolerance for mass assignment was ±10 Da. Other specified mAb protein forms were: mAb reference mass with a phosphate adduct, mAb reference mass with one fucose unit lost, mAb reference mass with a hexose unit added. A pentasaccharide adduct (possibly pentamannose) relative to the reference mass of v29456 without 2-acetamido-2-deoxy-β-D-glucopyranosyl-(1-4)-[α-L-fucopyranosyl-(1-6)] was also identified. Apparent purity was calculated as the ratio of the deconvoluted peak intensities of all v29456 or v18992 mAb protein forms to the intensities of all observed deconvoluted peaks. Mouse plasma proteins present in the mouse plasma control on day 0 but not observed in the PBS control were not considered in the apparent purity calculation.

[0324] Data are presented in Table 9.1 and show no evidence of fragmentation of v29456 after incubation in mouse plasma or PBS pH 7.4 at 37 °C for 7 and 14 days, as the apparent purity after 7 and 14 days was 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 >10% decrease in apparent purity relative to the day 0 control. No v18992 fragmentation was observed in plasma.

[0325] Table 9.1 Stability of anti-NaPi2b antibody in mouse plasma (% of apparent required protein forms)

[0326]

[0327]

[0328] Example 10: Quantification of NaPi2b on the surface of tumor cells

[0329] Surface NaPi2b protein was measured on tumor cell lines by quantitative flow cytometry using a set of beads with known levels of antibody binding capacity (ABC), as described below. The reference humanized antibody MX35 (v18992) conjugated to Alexa AF647 and the control anti-RSV antibody palivizumab (v21995) conjugated to Alexa AF647 were used for fluorescent labeling of tumor cells and beads. Variant 22277 differs from the anti-RSV antibody v21995 used in the previous example in that it has a heterodimeric Fc. This 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.

[0330] The conjugation of v18992 and v21995 to Alexa 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 carried out in the dark at room temperature and allowed to proceed for 200 minutes and 150 minutes, respectively. After incubation, the reactants were purified by four and two rounds of purification 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).

[0331] Cells were detached from the culture vessel using cell dissociation buffer (Invitrogen, Waltham, MA) and seeded at 50,000 cells / well in a conical-bottom 96-well plate, in triplicate. The cells and anti-human beads (Bangs Laboratories, Inc., Fishers, IN) were stained with v18992-AF647 conjugated antibody or negative control v21995-AF647 at a predetermined excess level at the same concentration and incubated at 4 °C for 30 minutes. After incubation, the cells and beads were washed in FACS buffer and analyzed on a BD TM Fortessa HTS and analyzed using FlowJo TMProcessed with v8 software (BD Biosciences, Franklin Lake, NJ).

[0332] Using the Bangs Laboratories QuickCal v 2.3 calibration curve template for anti-human IgG lot number 14490, the median AF647 fluorescence intensity of all bead populations was plotted against the relevant ABC values.

[0333] The surface protein expression of the cell lines was calculated based on a monovalent binding model and thus equivalent to ABC minus background (SABC). The median fluorescence intensity of v21995-AF647 stained cells from each cell line in the corresponding cell line was used as the background value for determining SABC.

[0334] The results are shown in Table 10.1. The reported NaPi2b protein / cell is the average of at least two biological replicates. Tumor cell lines are described as high, medium, low, or negative expressors of the target; if the average number of NaPi2b proteins detected is greater than 900,000 per cell, the cell line is called a "high" expressor; if the number is between 40,000 and 900,000 per cell, it is called "medium"; if the number is between 500 and 40,000 per cell, it is called "low"; if the quantity is negative (below the limit of quantification of the calibration beads), it is called "negative".

[0335] Table 10.1. Surface NaPi2b Quantification on Tumor Cell Lines

[0336]

[0337] *Percentage coefficient of variation calculated in two to seven biological replicates

[0338] Example 11: Preparation of Antibody-Drug Conjugates

[0339] Prepare the antibody-drug conjugates (ADCs) shown in Table 11.1. An exemplary protocol for preparing these ADCs is provided below, followed by a description of the structures of the drug-linker (DL) and payload in Table 11.2.

[0340] Table 11.1 Antibody-Drug Conjugates and DAR

[0341]

[0342] Exemplary Protocol

[0343] v29456 - MC - GGFG - AM - DXd1DAR8:A solution (2.47 mL) of humanized variant v29456 (54 mg) in PBS (pH 7.4) was diluted in PBS (pH 7.4) (1.00 mL) and reduced by adding 5 mM diethylenetriaminepentaacetic acid (DTPA) (0.90 mL PBS solution, pH adjusted to 7.4) and 25 mM tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (134 μL, 9.0 equivalents). After 4 hours at 37 °C, the reduced antibody was passed through a Zeba TM spin desalting column (40KDa MWCO; Thermo Scientific TM ) for purification. An aliquot (13.5 mg, 1.32 mL) of the reduced antibody solution was diluted with 1 mM DTPA (33.5 μL PBS solution, pH adjusted to 7.4). 38.3 μL of DMSO and an excess of MC-GGFG-AM-DXd1 (111.7 μL; 12 equivalents) from a 10 mM DMSO stock solution were added to the antibody solution. The conjugation reaction was carried out for 120 minutes at room temperature with mixing. At this point, an additional drug-linker (14 μL, 1.5 equivalents) was added. The conjugation reaction was carried out for an additional 60 minutes at room temperature with mixing. An excess of 10 mM N-acetyl-L-cysteine solution (76.8 μL, 8 equivalents) was added to quench the conjugation reaction.

[0344] v29456 - MT - VC - Compound 1DAR4 A solution (43.9 μL) of humanized variant v29456 (0.5 mg) in PBS (pH 7.4) was diluted in PBS (pH 7.4) (44.3 μL) and reduced by adding 5 mM diethylenetriaminepentaacetic acid (DTPA) (24 μL PBS solution, pH adjusted to 7.4) and 1 mM tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (7.76 μL, 2.25 equivalents). After 2 hours at 37 °C, the reduced antibody was cooled to 0 - 4 °C. An excess of MT-VC-compound 1 (1.72 μL, 10 equivalents) from a 20 mM stock solution was added. The conjugation reaction was carried out for 90 minutes at 0 - 4 °C.

[0345] v23855 - MT - VC - Compound 1 and v23855 - MC - VC - PABC - MMAEA solution (2.52 mL) of chimeric variant v23855 (15 mg) in PBS (pH 7.4) was diluted in PBS (pH 7.4) (457 μL) and reduced by adding 5 mM diethylenetriaminepentaacetic acid (DTPA) (750 μL PBS solution, pH adjusted to 7.4) and 10 mM tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (26.0 μL, 2.6 equivalents). After 2 hours at 37 °C, the reduced antibody was cooled to 0 - 4 °C. An excess of MT-VC-compound 1 or MC-VC-PABC-MMAE (50.0 μL, 10 equivalents) from a 20 mM stock solution was added. The conjugation reaction was carried out at 0 - 4 °C for 60 minutes. An excess of 10 mM N-acetyl-L-cysteine solution (26.7 μL, 8 equivalents) was added to quench the conjugation reaction. The quenching reaction was carried out at 0 - 4 °C for 30 minutes.

[0346] v18992 - MC - GGFG - AM - DXd1DAR8 A solution (120.8 μL) of humanized variant v18992 (1 mg) in PBS (pH 7.4) was diluted in PBS (pH 7.4) (29.6 μL) and reduced by adding 5 mM diethylenetriaminepentaacetic acid (DTPA) (40.0 μL PBS solution, pH adjusted to 7.4) and 10 mM tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (9.62 μL, 14.0 equivalents). After 3 hours at 37 °C, the reduced antibody was purified by a Zeba TM spin desalting column (40KDa MWCO; Thermo Scientific TM ). 20.0 μL of DMSO and an excess of MC-GGFG-AM-DXd1 (10.31 μL; 15 equivalents) from a 10 mM DMSO stock solution were added to the reduced antibody solution. The conjugation reaction was carried out at room temperature and with mixing for 120 minutes.

[0347] v18992 - MT - VC - Compound 1DAR3A solution (4.23 mL) of the humanized variant v18992 (35 mg) in PBS (pH 7.4) was diluted in PBS (pH 7.4) (2.2 mL) and reduced by adding 5 mM diethylenetriaminepentaacetic acid (DTPA) (1.75 mL PBS solution, pH adjusted to 6.7) and 10 mM tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (50.8 μL, 2.1 equivalents). After 2 hours at 37 °C, the reduced antibody was cooled to 0 - 4 °C. Excess MT-VC-compound 1 (120.9 μL, 10 equivalents) from a 20 mM stock solution was added. The conjugation reaction was carried out at 0 - 4 °C for 60 minutes. Excess 10 mM N-acetyl-L-cysteine solution (64.5 μL, 8 equivalents) was added to quench the conjugation reaction. The quenching reaction was carried out at 0 - 4 °C for 30 minutes.

[0348] v18993 - MC - GGFG - AM - DXd1DAR8 A solution (156.5 μL) of the humanized variant v18993 (1 mg) in PBS (pH 7.4) was diluted in PBS (pH 7.4) (1.96 μL) and reduced by adding 5 mM diethylenetriaminepentaacetic acid (DTPA) (42.0 μL PBS solution, pH adjusted to 7.4) and 10 mM tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (9.55 μL, 14.0 equivalents). After 3 hours at 37 °C, the reduced antibody was purified by a Zeba TM spin desalting column (40KDa MWCO; Thermo Scientific TM ). 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 to the reduced antibody solution. The conjugation reaction was carried out at room temperature and with mixing for 120 minutes.

[0349] v18993 - MT - VC - Compound 1DAR4 A solution (78.3 μL) of the humanized variant v18993 (0.5 mg) in PBS (pH 7.4) was diluted in PBS (pH 7.4) (10.25 μL) and reduced by adding 5 mM diethylenetriaminepentaacetic acid (DTPA) (24.0 μL PBS solution, pH adjusted to 7.4) and 1 mM tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (7.50 μL, 2.2 equivalents). After 2 hours at 37 °C, the reduced antibody was cooled to 0 - 4 °C. Excess MT-VC-compound 1 (1.71 μL, 10 equivalents) from a 20 mM stock solution was added. The conjugation reaction was carried out at room temperature for 60 minutes.

[0350] v18993 - MC - VC - PABC - MMAEDAR4A solution (5.84 mL) of the humanized variant v18993 (30 mg) in PBS (pH 7.4) was diluted in PBS (pH 7.4) (0.60 mL) and reduced by adding 5 mM diethylenetriaminepentaacetic acid (DTPA) (1.62 mL PBS solution, pH adjusted to 7.4) and 10 mM tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (55.3 μL, 2.7 equivalents). After 2.5 hours at 37 °C, the reduced antibody was cooled to 0 - 4 °C. PBS (pH 7.4) (0.24 mL) was added, and then an excess of MC-VC-PABC-MMAE (225 μL, 11 equivalents) from a 10 mM stock solution was added. The conjugation reaction was carried out at 0 - 4 °C for 16 hours. An excess of 10 mM N-acetyl-L-cysteine solution (0.90 mL, 44 equivalents) was added to quench the conjugation reaction. The quenching reaction was carried out at room temperature for 60 minutes.

[0351] v22277 - MC - GGFG - AM - DXd1DAR8 A solution (2.18 mL) of the control variant v22277 (10 mg) in PBS (pH 7.4) was diluted in PBS (pH 7.4) (4.1 μL) and reduced by adding 5 mM diethylenetriaminepentaacetic acid (DTPA) (563 μL PBS solution, pH adjusted to 6.7) and 10 mM tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (68.9 μL, 10.0 equivalents). After 3 hours at 37 °C, the reduced antibody was purified by a Zeba TM spin desalting column (40KDa MWCO; Thermo Scientific TM )). 230.0 μL of DMSO and an excess of MC-GGFG-AM-DXd1 (51.7 μL; 15 equivalents) from a 20 mM DMSO stock solution were added to the reduced antibody solution. The conjugation reaction was carried out at room temperature and with mixing for 60 minutes. An excess of 20 mM N-acetyl-L-cysteine solution (51.7 μL, 15 equivalents) was added to quench the conjugation reaction. The quenching reaction was carried out at 0 - 4 °C for 30 minutes.

[0352] v22277 - MT - VC - Compound 1DAR4, v22277 - MC - VC - PABC - MMAEDAR4A solution of control variant v22277 (20 mg) in PBS (pH 7.4) (4.36 mL) was reduced by adding 5 mM diethylenetriaminepentaacetic acid (DTPA) (1.10 mL PBS solution, pH adjusted to 7.4) and 10 mM tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (35.1 μL, 2.55 equivalents). After 1.5 hours 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 equivalents) from a 20 mM stock solution was added. The conjugation reaction was carried out for 60 minutes at room temperature or 0 - 4 °C.

[0353] v22277 - AD - VC - Compound 1DAR4 A solution of variant v22277 (20 mg) (4.31 mL) was diluted with 0.69 mL PBS (pH 7.4). Excess TFP-AD-VC-compound 1 (65.4 μL, 9.5 equivalents) from a 20 mM DMSO stock solution was added, and the conjugation was carried out overnight at room temperature.

[0354] v22277 - MC - VC - PABC - MMAE DAR8 A solution of control variant v22277 (5 mg) in PBS (pH 7.4) (1.08 mL) was diluted in PBS (pH 7.4) (81.1 μL) and reduced by adding 5 mM diethylenetriaminepentaacetic acid (DTPA) (300 μL PBS solution, pH adjusted to 6.7) and 10 mM tris(2-carboxyethyl)phosphine (TCEP) aqueous solution (41.3 μL, 10.0 equivalents). After 3 hours at 37 °C, the reduced antibody was purified by passing through a Zeba TM spin desalting column (40KDa MWCO; Thermo Scientific TM )). Excess MC-VC-PABC-MMAE (20.7 μL; 12 equivalents) from a 20 mM DMSO stock solution was added to the reduced antibody solution. The conjugation reaction was carried out for 60 minutes at room temperature with mixing.

[0355] Table 11.2 Drug-linkers for preparing ADC

[0356] <![CDATA[Drug-linker 1 > Linker Payload DL1 MC - GGFG - AM - DXd1 DL2 MT - VC - Compound 1 DL3 MC - VC - PABC - MMAE DL4 AD - VC Compound 1

[0357] 1 The structures of the linkers and payloads provided below

[0358] Structure:

[0359] MC-GGFG-AM-

[0360]

[0361] MT-VC-

[0362]

[0363] MC-VC-PABC-

[0364]

[0365] TFP-AD-VC-

[0366]

[0367] DXd1

[0368]

[0369] Compound 1

[0370]

[0371] MMAE

[0372]

[0373] Example 12: Purification and Characterization of Antibody-Drug Conjugates

[0374] The ADCs prepared by cysteine conjugation chemistry as described in Example 11 were purified one to two times by passing through a Zeba TM spin desalting column (40 kDa MWCO; Thermo Scientific TM ).

[0375] On an AKTA TM Pure chromatography system (Cytiva Life Sciences, Marlborough, MA), the ADCs prepared on a large scale as described in Example 11 were purified using a 53 mL HiPrep 26 / 10 desalting column (Cytiva Life Sciences, Marlborough, MA), a mobile phase consisting of 10 mM NaOAc (pH 4.5) and 150 mM NaCl, and a flow rate of 10 mL / min. Then the purified ADCs were sterile filtered (0.2 μm).

[0376] The ADCs prepared by lysine conjugation chemistry as described in Example 11 were passed once through a Zeba TM spin desalting column (40 kDa MWCO; Thermo Scientific TM) Purification is carried out and the column is pre-equilibrated with PBS (pH 7.4).

[0377] After purification, the ADC prepared for in vivo use is sterile filtered (0.2 mm).

[0378] After purification, the concentration of the ADC is determined by BCA assay with reference to the standard curve generated using the humanized variant v29456. Alternatively, the extinction coefficient taken from the literature (European Patent No. 3 342 785, for DL1) or experimentally determined (for the remaining drug-linker) is used to estimate the concentration by measuring the absorbance at 280 nm. As described below, the ADC is also characterized by hydrophobic interaction chromatography (HIC), reversed-phase ultra-high performance liquid chromatography mass spectrometry (RP-UPLC-MS), and size exclusion chromatography (SEC).

[0379] Hydrophobic interaction chromatography

[0380] Antibodies and ADCs are analyzed by HIC to estimate the drug-antibody ratio (DAR). Chromatography is performed on an Agilent Infinity II 1290 HPLC (Agilent Technologies, Santa Clara, CA) using a butyl-NPR column (2.5 μm, 4.6 x 35 mm; TOSOH Bioscience GmbH, Griesheim, Germany) with a gradient from 95 / 5% MPA / MPB to 5 / 95% MPA / MPB over 12 minutes at a flow rate of 0.5 mL / min (MPA = 1.5 M (NH 4 ) 2 SO 4 , 25 mM Na x PO 4 , pH 7 and MPB = 75% 25 mM Na x PO 4 , pH 7, 25% isopropanol). Detection is carried out by absorbance at 280 nm.

[0381] Reversed-phase ultra-high performance liquid chromatography mass spectrometry:

[0382] Alternatively, the ADC can be analyzed by RP-UPLC-MS to determine the average drug-to-antibody ratio (DAR).

[0383] Determination of the DAR of lysine-conjugated ADC by RP-UPLC-MS

[0384] The ADC sample was deglycosylated with Endo S for 1 hour at room temperature and injected onto an Agilent 1290 Infinity II LC (Agilent Technologies, Santa Clara, CA) coupled with an Agilent 6545 quadrupole time-of-flight (Q-TOF) mass spectrometer. A PLRP-S column ( 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: aqueous solution of 0.1% formic acid (FA), 0.025% trifluoroacetic acid (TFA), and 10% isopropanol (IPA). Mobile phase B: acetonitrile (ACN) solution of 0.1% FA and 10% IPA.

[0385] Table 12.1: RP-HPLC-MS gradient

[0386] Time (min) Buffer A % Buffer B % 0 80 20 20 60 40 22 10 90 22.5 1 99 1 99

[0387] The MS source conditions are shown in Table 12.2 and the acquisition parameters are as follows:

[0388] Mode: MS; mass range: 500 to 7000 m / z; acquisition rate: 1 spectrum / s and 1000 ms / spectrum, 3354 transients / spectrum.

[0389] Table 12.2: MS source conditions

[0390] Gas temperature: 300 °C VCap: 5000V Dry gas: 13L / min Nozzle voltage: 2000V Nebulizer: 45psig Fragmentation voltage: 170V Sheath gas temperature: 400 °C Skimmer cone: 65V Sheath gas flow: 12L / min Oct RF Vpp: 750V

[0391] Qualitative analysis using MassHunter software (Agilent Technologies, Santa Clara, CA) was employed for deconvolution and data analysis. The deconvolution parameters are as follows:

[0392] Deconvolution algorithm: maximum entropy; mass range: 70000 - 160000; mass step: 1.0; finite m / z range used: 1000 - 7000; baseline subtraction: 7.0; adduct: proton; isotope width: automatic; height filter: peak signal-to-noise ratio >= 30.0; maximum number of peaks: limited by height 100.

[0393] The average DAR was calculated from the deconvoluted spectra using the following formula:

[0394]

[0395] Determination of the DAR of cysteine-conjugated ADC by RP-UPLC-MS

[0396] The ADC sample was deglycosylated with Endo S for 1 hour at room temperature, reduced and denatured by incubating with TCEP for 1 hour at 70 °C, and injected onto an Agilent 1290 Infinity II LC (Agilent Technologies, Santa Clara, CA) coupled with an Agilent 6545 quadrupole time-of-flight (Q-TOF) mass spectrometer. A PLRP-S column ( 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: aqueous solution of 0.1% formic acid (FA), 0.025% trifluoroacetic acid (TFA), and 10% isopropanol (IPA). Mobile phase B: acetonitrile (ACN) solution of 0.1% FA and 10% IPA.

[0397] Table 12.3: RP-HPLC-MS gradient

[0398] Time (min) Buffer A % Buffer B % 0 80 20 20 60 40 22 10 90 22.5 1 99 24 1 99

[0399] The MS source conditions are shown in Table 12.4, and the acquisition parameters are as follows: Mode: MS; Mass range: 500 to 7000 m / z; Acquisition rate: 1 spectrum / s and 1000 ms / spectrum, 3354 transients / spectrum.

[0400] Table 12.4: MS source conditions

[0401]

[0402]

[0403] Qualitative analysis using MassHunter software (Agilent Technologies, Santa Clara, CA) was employed for deconvolution and data analysis. For a typical IgG1 ADC, reduction of the sample should separate distinct species, with the light chain (LC) eluting first (DAR 0, followed by DAR 1, 2), and the heavy chain (HC) eluting subsequently (DAR 0, followed by DAR 1, 2, 3). Integrate the TIC in two regions corresponding to the light and heavy chains, and then perform deconvolution. The deconvolution parameters are as follows:

[0404] Deconvolution algorithm: Maximum entropy; Mass range: 20000 - 60000; Mass step: 1.0; Finite m / z range used: 700 - 3000; Baseline subtraction: 7.0; Adduct: Proton; Isotope width: Automatic; Height filter: Peak signal-to-noise ratio >= 30.0; Maximum number of peaks: Limited by height 100.

[0405] The average DAR was calculated from the deconvoluted spectrum using the following formula:

[0406]

[0407] Size-exclusion chromatography

[0408] 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), a mobile phase consisting of 150 mM phosphate (pH 6.95), and a flow rate of 1 mL / min, the aggregation levels of antibodies and ADCs (approx. 15 - 150 mg, 5 mL injection volume) were evaluated by SEC. Detection was performed by absorbance at 280 nm.

[0409] Results

[0410] When determining the DAR by HIC, the individual contributions of the DAR0, DAR2, DAR4, DAR6, and DAR8 species to the average DAR of the purified ADC were evaluated by integration of the HPLC - HIC chromatogram. The average drug - antibody ratio (DAR) for each ADC was determined as the weighted average of each DAR species. Regardless of the method used, 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.

[0411] The aggregation levels and monomer contents were evaluated by integration of the HPLC - SEC chromatogram. The monomer peak for each ADC was identified as the peak having the same retention time as the unconjugated antibody that gave rise to each ADC. All peaks having an earlier retention time relative to the monomer species were determined to be aggregated species. The percentage of monomer species determined for each ADC is shown in Table 12.1.

[0412] Table 12.1 Characterization of ADCs

[0413]

[0414] Example 13: Functional Characterization of Anti - NaPi2b Antibody Constructs - Cell Binding of Bivalent Antibodies by Flow Cytometry

[0415] The ability of the parental chimeric antibody construct v23855 and the humanized antibody variants described in Examples 2 and 3 to bind to NaPi2b expressed on the cell surface was evaluated by flow cytometry on the cell line IGROV - 1, which expresses endogenous NaPi2b at a high level.

[0416] Briefly, cells were seeded at 50,000 cells / well in a conical-bottom 96-well plate and treated with the test antibodies at 4 °C for 24 h to prevent internalization. Palivizumab (anti-RSV antibody, v22277) was included as a negative control. The reference anti-NaPi2b antibodies, Rituximab (v18993) and MX35 (v18992), conjugated to a maleimide-functionalized auristatin drug linker (DL2), were included as comparators; conjugation of the drug linker was shown to have no effect on antibody binding ability (data not shown). After incubation, the cells were washed and stained with an anti-human IgG Fc AF647 conjugate (Jackson ImmunoResearch Labs, West Grove, PA; catalog number 109-605-098) at 4 °C for 30 min. After incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa TM cell analyzer (BD Biosciences, Franklin Lake, NJ), collecting 1,000 minimum events per well. The AF647 / APC-A GeoMean (geometric mean of the fluorescence signal, proportional to binding to anti-human AF647) of the live cell population was calculated using FlowJo TM 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).

[0417] The results for the parental chimeric construct (v23855) and all humanized antibody variants are shown in Table 13.1. Figure 6 The complete dose-response binding curves for the parental chimeric antibody (v23855) and two representative humanized antibody variants (v29452, v29456) are shown in

[0418] Table 13.1: Binding of parental chimeric and humanized antibody variants to IGROV-1 cells

[0419]

[0420]

[0421] All humanized antibody variants similarly bind to IGROV-1 cells, yielding apparent Kd values within 2-fold and comparable Bmax values. The reference antibody MX35-DL2 ADC shows binding comparable to the chimeric v23855 and the humanized antibody. The humanized antibody rituximab-DL2 ADC shows lower binding compared to all other targeted antibodies, with a lower Bmax value and a larger apparent Kd value. As expected, the negative control palivizumab (v22277) shows no cell binding (NB).

[0422] Example 14: Functional Characterization of Anti-NaPi2b ADCs - Cell Binding by Flow Cytometry

[0423] The ability of the ADCs to bind to cells expressing NaPi2b was evaluated. ADCs of the representative humanized variant antibody v29456 (H1L2) were prepared by conjugation with DL2 and DL1 as described in Example 11. Binding to the cell lines IGROV-1 and HCC-78, which endogenously overexpress NaPi2b, was evaluated by flow cytometry according to the method described in Example 5.

[0424] The results are shown in Table 14.1 and plotted for IGROV-1 cells in Figure 7A and for HCC-78 cells in Figure 7B .

[0425] Table 14.1 Cell Binding of Antibodies and Antibody-Drug Conjugates

[0426]

[0427] Both antibody-drug conjugates yield similar apparent Kd and Bmax values on the NaPi2b-overexpressing cell lines IGROV-1 and HCC-78 compared to their unconjugated parental antibody v29456. The negative control palivizumab (v22277) did not bind to either cell line, as expected.

[0428] These results indicate that the ability of the representative antibody variant v29456 to bind to cells expressing NaPi2b is not affected by conjugation to the drug-linker.

[0429] Example 15: Functional Characterization of Anti-NaPi2b ADCs - In Vitro Cytotoxicity in 2D Monolayer Cultures

[0430] Cytotoxicity measured by cell growth inhibition of the humanized variant v29456 (H1L2) conjugated to various drug-linkers was evaluated in a panel of cell lines expressing NaPi2b, as described below. The cell lines used were OVCAR-3 (ovarian cancer), IGROV-1 (ovarian cancer), and HCC-78 (lung cancer). An ADC with the antibody palivizumab (anti-RSV) (v22277) was used as a non-targeting control.

[0431] OVCAR-3 was 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 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 at 50 μL / well in standard medium and treated with 20 μL / well of titrated test article prepared in ATCC-modified RPMI 1640 medium (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS. Cells were incubated for 4 days under standard culture conditions. After incubation, reagent (Promega Corporation, Madison, WI) was added to all wells and luminescence corresponding to ATP present in each well was measured using a Synergy TM H1 microplate reader (BioTek Instruments, Winooski, VT). The percentage cytotoxicity value was calculated as follows: (1 - (luminescence of treated cells / average luminescence of untreated cells)) × 100, and the test article concentration was plotted using GraphPad Prism 9 software (GraphPad Software, San Diego, CA). The EC50 value was calculated by GraphPad Prism 9 based on nonlinear regression log(agonist) vs. response, variable slope (four parameters).

[0432] 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).

[0433] Table 15.1: In Vitro Cytotoxicity - 2D Monolayer

[0434]

[0435] *Incomplete curve

[0436] When conjugates containing the same drug linkers (DL2 and MC-GGFG-DXd) were compared against all three NaPi2b-expressing cell lines, the humanized variant v29456 and the humanized antibody MX35 (v18992) showed comparable potency to each other and greater potency than the humanized antibody rituximab (v18993). The EC50 value for NaPi2b-targeted killing of DXd ADC in OVCAR-3 cells could not be calculated (incomplete curve). The chimeric antibody v23855 conjugated to DL3 showed NaPi2b-targeted killing with sub-nanomolar potency against the three cell lines. As expected, the palivizumab control ADC showed EC50 values more than a log-fold greater than those of the NaPi2b-targeted ADC against all three NaPi2b-expressing cell lines.

[0437] Example 16: In Vivo Activity of Chimeric Antibody-Drug Conjugates

[0438] The in vivo antitumor activity of the chimeric v23855 ADC was evaluated in an OVCAR3 ovarian cancer xenograft model expressing high levels of NaPi2b. The antitumor activities of ADCs based on the reference antibodies rituximab (v18993) and MX35 (v18992) were also evaluated for comparison. The studies were conducted as described below.

[0439] These data indicate that the v23855-based ADC is active against NaPi2b-expressing tumors in vivo.

[0440] For the OVCAR3 ovarian cancer model with high Napi2b expression, tumor fragments (approx. 1 mm 3 ) from stock mice were implanted subcutaneously into female CB.17 SCID mice. When the average tumor volume reached approximately 100 - 150 mm 3 , the animals were grouped with n = 8 per group and a single IV dose of the test article was administered on Day 1 of the study. Tumor volume and body weight were measured twice a week and the study duration was 39 - 49 days. For statistical analysis, a linear mixed-effects model was used to fit the log-transformed tumor volume, followed by an F-test on the null hypothesis of equal mean growth rates and post hoc pairwise comparisons. Two studies were conducted for the treatment groups described in Tables 16.1 and 16.2 below.

[0441] Table 16.1: Treatment Groups in OVCAR3 Study 1

[0442] Group Treatment DAR Dose (mg / kg) 1 Vehicle 0 2 v23855 - DL2 4 6 3 v23855 - DL2 4 18 4 v18992 - DL2 4 6 5 v18992 - DL2 4 18

[0443] Table 16.2: Treatment Groups in OVCAR3 Study 2

[0444] Group Treatment DAR Dose (mg / kg) 1 Vehicle 0 2 v23855 - DL2 4 6 3 v18993 - DL2 4 6 4 v23855 - DL3 4 6 5 v23855 - DL3 4 18 6 v18993 - DL3 4 6

[0445] The results of OVCAR3 Study 1 are shown in Figure 9A and indicate that compared to the vehicle control, the parental chimeric antibody v23855-DL2 at 6 mg / kg and 18 mg / kg doses resulted in significant inhibition of tumor growth (p < 0.05). At the 6 mg / kg or 18 mg / kg dose levels, the inhibition of tumor growth by v23855-DL2 was comparable to that of the reference antibody v18992-DL2.

[0446] The results of OVCAR3 Study 2 are shown in Figure 9B and indicate that compared to the vehicle control, the chimeric v23855-DL2 at 6 mg / kg dose resulted in significant inhibition of tumor growth (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.

[0447] In summary, these studies indicate that when conjugated to DL2 or DL3, the ADCs containing the parental chimeric v23855 demonstrated anti-tumor activity superior to that of the ADCs containing the reference antibody v18993. When conjugated to DL2, v23855 demonstrated activity not inferior to that of the reference antibody v18992.

[0448] Example 17: In Vivo Activity of Humanized Antibody-Drug Conjugates

[0449] The in vivo anti-tumor activity of the ADC of the humanized antibody variant v29456 conjugated to DXd1 was evaluated in OVCAR3 ovarian cancer xenograft model and NCI-H441 lung cancer xenograft model, in which tumors expressed high levels of NaPi2b. The study was conducted as described below.

[0450] For the high NaPi2b-expressing OVCAR3 ovarian cancer model, tumor fragments (approx. 1 mm 3 ) were implanted subcutaneously into female CB.17 SCID mice. When the average tumor volume reached approximately 100 - 150 mm 3At that time, the animals were grouped with n = 8 in each group, and a single IV dose of the test article was administered on Day 1 of the study. Tumor volume and body weight were measured twice a week, and the study duration was 60 days. The treatment groups are described in Table 17.1.

[0451] Table 17.1: Treatment groups for the OVCAR3 study

[0452] Group Treatment DAR Dose (mg / kg) 1 Vehicle 0 2 v29456 - DL1 8 1 3 v29456 - DL1 8 3 4 v29456 - DL1 8 10

[0453] For the NCI-H441 model of lung cancer, 5 x 10^6 cells in 0.1 ml of 1:1 PBS:Matrigel were implanted subcutaneously into male NU-Foxn1nu mice. When the average tumor volume reached approximately 145 mm 3 At that time, the animals were grouped with n = 6 in each group, and a single IV dose of the test article was administered on Day 0 of the study. Tumor volume and body weight were measured twice a week, and the study duration was 28 - 35 days. For statistical analysis, a linear mixed-effects model was used to fit the log-transformed tumor volume, and then an F-test was performed on the null hypothesis of equal mean growth rates, followed by post hoc pairwise comparisons. Two studies were conducted in the NCI-H441 model, and the treatment groups are described in Tables 17.2 and 17.3.

[0454] Table 17.2 Treatment groups for NCI-H441 Study 1

[0455] Group Treatment DAR Dose (mg / kg) 1 Vehicle 0 2 v29456 - DL1 8 1 3 v29456 - DL1 8 3 4 v29456 - DL1 8 10

[0456] Table 17.3: Treatment groups for NCI-H441 Study 2

[0457] Grouping Treatment DAR Dose (mg / kg) 1 Vehicle 0 2 v29456-DL1 8 0.3 3 v29456-DL1 8 1 4 21995 (Palivizumab)-DL1 8 1

[0458] The results of the OVCAR3 model are shown in Figure 10 indicating that v29456-DL1 caused strong tumor growth inhibition at 1, 3, and 10 mg / kg.

[0459] The results of NCI-H441 Study 1 are shown in Figure 11A indicating that v29456-DL1 caused strong tumor growth inhibition at 1, 3, and 10 mg / kg.

[0460] The results of NCI-H441 Study 2 are shown in Figure 11B and indicate that v29456-DL1 caused moderate inhibition of tumor growth at 0.3 mg / kg and strong tumor growth inhibition at 1 mg / kg. At 1 mg / kg, the activity of the non-targeted control v21995 (palivizumab)-DL1 was lower than that of v29456-DL1, indicating target-dependent activity of the V29456 ADC.

[0461] Example 18: Specificity Assessment of Anti-NaPi2b Antibody

[0462] Using Membrane Proteome Array TM (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 an amino acid sequence substantially identical to v29456. The DNA sequence encoding the v38591 heavy chain is the same as the DNA sequence encoding v29456, except that the heavy chain contains a C-terminal lysine. Once this C-terminal lysine is secreted from the cell that produces it, the C-terminal lysine is cleaved from most antibody products.

[0463] Briefly, the study consisted of three phases: phase (1) determination of screening conditions, phase (2) membrane proteome array (library) screening, and phase (3) protein target validation. In phase (1), conditions suitable for detecting v38591 binding by high-throughput flow cytometry were determined, including the optimal antibody concentration and cell type for screening (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 6000 human membrane proteins (expressed individually 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 (potential off-target interaction) from the screening phase was evaluated in a titration experiment using flow cytometry.

[0464] Phase (1) determined that the HEK293T cell type and an antibody concentration of 20 mg / mL were optimal for library screening. As Figure 12A shown, library screening resulted in a primary target of NaPi2b and a validated protein target hit of FcgR1A that binds to the Fc portion of the antibody. Another validated protein target hit was CLDN3. CLDN3 validation data showed that it is a very weak binder of humanized v38591, as indicated by low binding signals (MFI of approximately 60 - 275) to v38591 at a series of concentrations compared to strong binding signals (MFI of approximately 3500 - 7000) in the case of NaPi2b in the validation assay ( Figure 12B ). Generally, this data indicates high specificity of humanized v38591 for the primary target NaPi2b.

[0465] Example 19: Functional Characterization of Anti-NaPi2b Antibodies - Cellular Binding of Anti-NaPi2b Antibodies by Flow Cytometry

[0466] The ability of the humanized antibody variants v38591 and v29456 to bind to NaPi2b expressed on the cell surface was evaluated by flow cytometry on the tumor cell lines IGROV-1 (ovarian adenocarcinoma) and TOV-21G (ovarian cancer) that express endogenous NaPi2b. IGROV-1 and TOV-21G cells express endogenous NaPi2b at high and medium levels, respectively, as described in Example 10.

[0467] Cellular binding was performed according to the method described in Example 13. The anti-NaPi2b antibody rifatuzumab (v18993) and the anti-RSV antibody palivizumab (v22277) were included as positive and negative controls, respectively.

[0468] The results are shown in Table 19.1 and plotted in Figure 13 Both v38591 and v29456 exhibited comparable cellular binding to the IGROV-1 and TOV-21G cell lines, indicating that addition of a C-terminal lysine to the heavy chain of the antibody had little effect on the antibody's cellular binding ability. Compared to v38591, the anti-NaPi2b antibody control v18993 showed slightly poorer binding on IGROV-1 and TOV-21G, with the upper limit of Bmax reduced by approximately 1.2-fold and 1.4-fold, respectively. As expected, the negative control palivizumab (v22277) showed no cellular binding.

[0469] Table 19.1: Cellular Binding of Anti-NaPi2b Antibodies

[0470]

[0471] *NB = No binding (apparent Kd value greater than the highest antibody test concentration > 150 nM)

[0472] Example 20: Functional Characterization of Anti-NaPi2b Antibodies - NaPi2b Specificity

[0473] The binding cross-reactivity of the humanized antibody variant v38591 with human NaPi2b, NaPi2a, and NaPi2c was evaluated by flow cytometry using HEK293-6e transfected cells. The reference anti-NaPi2b antibodies MX35 (v18992) and rituximab (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.

[0474] HEK293-6e cells were maintained under standard culture conditions (37 °C / 5% CO2), suspended with shaking at 110 rpm, and cultured in FreeStyle TM 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). 293Fectin TM Transfection Reagent (Thermo Fisher Scientific, Waltham, MA) and Opti-MEM TMIscove's Modified Dulbecco's Medium (Thermo Fisher Scientific, Waltham, MA) was used to transfect cells with human NaPi2b (pTT5-huNaPi2b) (CL#13432), human NaPi2a (CL#13435), or human NaPi2c (CL#13436) (all obtained from GenScript Biotech, Piscataway, NJ) at 1 μg DNA per 1 million cells 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 a conical-bottom 96-well plate and treated with the test antibody at 4 °C for 24 hours 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 TM cell analyzer (BD Biosciences, Franklin Lake, NJ), and 1,000 minimum events were collected per well. FlowJo TM version 10.8.1 (BD Biosciences, Franklin Lake, NJ) was used to calculate the AF647 / APC-A GeoMean (geometric mean of the fluorescence signal, proportional to the binding of anti-human AF647) of the live cell population, and each test antibody was plotted using GraphPad Prism version 9 (GraphPad Software, San Diego, CA).

[0475] The results are shown in Table 20.1. Antibody variant v38591, as well as reference antibodies v18993 and v18992, showed comparable binding to each other on HEK293-6e cells transfected with NaPi2b, as well as minimal binding to HEK293-6e cells transfected with NaPi2a and NaPi2c. The positive NaPi2a-binding control antibody obtained from Atlas Bio showed binding to NaPi2a-transfected cells, some binding to NaPi2c-transfected cells, and no binding to NaPi2b-transfected cells. As expected, the positive NaPi2c-binding control antibody obtained 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.

[0476] Table 20.1: Cross-reactivity of v38591 ADC, parental antibodies, and controls with cynomolgus monkey and mouse NaPi2b

[0477]

[0478] NB = No binding (apparent Kd value greater than the highest antibody test concentration, >200 nM for human antibodies and >50 nM for rabbit antibodies)

[0479] IC = Incomplete curve

[0480] Example 21: Pharmacokinetic evaluation of anti-NaPi2b antibodies in TG32 mice

[0481] The pharmacokinetics (PK) of antibody v29456 and v18993 (rifatuzumab)-MCvcPABC-MMAE DAR4 were evaluated in humanized FcRn Tg32 mice. This mouse model can predict the pharmacokinetics of drugs in humans (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).

[0482] All test articles were administered intravenously at 5 mg / kg to hFcRn Tg32 mice (The Jackson Laboratory, Sacramento, CA; catalog no. 014565) as shown in Table 26.1. For each test article, blood was collected from n = 4 animals by retro-orbital bleeding at 1, 3, and 6 hours and at 1, 3, 7, 10, 14, and 21 days after dosing. Blood was processed into serum and stored frozen at -80 °C in 96-well storage plates prior to pharmacokinetic analysis.

[0483] Table 26.1 Test articles and doses for PK assessment

[0484]

[0485] Mouse sera containing the v29456 antibody and v18993 (rituximab)-MCvcPABC-MMAE DAR4 were captured onto 384-well plates coated with goat anti-human IgG Fc antibody (Jackson 109-005-098). Total antibody was detected with goat anti-human IgG Fab biotin antibody (Jackson 109-065-097) and then with streptavidin SULFO-TAG conjugate (Mesoscale). After addition of MSD GOLD Read Buffer A, the SULFO-TAG-labeled electrochemiluminescence (ECL) signal was measured using a plate reader (Mesoscale).

[0486] The PK curves obtained are shown in Figure 14 . The anti-NaPi2b antibody v29456 exhibited typical antibody PK characteristics and was largely comparable between the test article and the rituximab-MMAE comparator.

[0487] The disclosures of all patents, patent applications, publications, and database entries mentioned in this specification are hereby expressly incorporated by reference in their entirety to the same extent as if each such individual patent, patent application, publication, and database entry were specifically and individually indicated to be incorporated by reference.

[0488] Modifications of the specific embodiments described herein that will be apparent to those skilled in the art are intended to be within the scope of the following claims.

[0489] Additional Sequence Listing (SEQ ID NO: 36-59)

[0490] Table A: Clone numbers of variants

[0491]

[0492] Table B: Cloned amino acid sequences (full chain)

[0493]

[0494]

[0495]

[0496]

[0497]

[0498]

[0499]

[0500] Table C: Cloned DNA Sequences (VH and VL of v29449 - 29460)

[0501]

[0502]

[0503]

Claims

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

18.

2. The antibody-drug construct according to claim 1, wherein said antigen-binding domain comprises: a) a VH domain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 24 and a VL domain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 29; b) a VH domain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 24 and a VL domain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 30; c) a VH domain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 26 and a VL domain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 30; d) a VH domain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 25 and a VL domain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 30; e) a VH domain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 27 and a VL domain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 30; f) a VH domain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 27 and a VL domain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 29; g) a VH domain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 26 and a VL domain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 29; h) a VH domain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 25 and a VL domain having at least 90% sequence identity with the sequence shown in SEQ ID NO: 29; i) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:27 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:28; j) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:26 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:28; k) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:25 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:28; l) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:24 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:28; or m) a VH domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:31 and a VL domain having at least 90% sequence identity to the sequence shown in SEQ ID NO:

32.

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

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

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

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

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

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

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

10. An expression vector or a set of expression vectors comprising the polynucleotide or set of polynucleotides according to 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 according to any one of claims 1 to 8 conjugated to one or more drug moieties.

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

14. The antibody-drug conjugate according to claim 13, wherein the antibody-drug conjugate has the general formula I: A-(L-(D) m ) n (I) wherein: A is the antibody construct; L is a linker; D is a drug moiety; m is an integer between 1 and about 8, and n is between 1 and about 12.

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

16. The antibody-drug conjugate according to claim 14 or 15, wherein n is between about 2 and about 8.

17. The antibody-drug conjugate according to any one of claims 13 to 16, wherein the drug moiety is maytansine alkaloid, maytansine alkaloid analog, benzodiazepine pyrrolobenzodiazepine calicheamicin, calicheamicin analog, auristatin, auristatin analog, halichondrin, halichondrin analog, tubulysin, tubulysin analog, amanitin, amanitin analog, camptothecin, camptothecin analog, eribulin, TLR agonist or STING agonist.

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

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

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

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

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

23. A method of inhibiting the growth of NaPi2b-positive tumour cells, which comprises contacting the cells with the antibody construct according to any one of claims 1 to 8 or the antibody-drug conjugate according to any one of claims 12 to 18.

24. A method of treating a subject suffering from cancer, which comprises administering to the subject an effective amount of the antibody construct according to any one of claims 1 to 8 or the antibody-drug conjugate according to any one of claims 12 to 18.

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