Antibody constructs binding folate receptor alpha and NAPI2b, conjugates and methods of use

Abstract

Bispecific antibodies that bind to folate receptor alpha (FRα) and NaPi2b, conjugates comprising the bispecific antibodies conjugated to one or more drug moieties and methods of using the antibodies and conjugates in the treatment of cancers expressing FRα, NaPi2b or both FRα and NaPi2b.

Description

ANTIBODY CONSTRUCTS BINDING FOLATE RECEPTOR ALPHA AND NAPI2B, CONJUGATES AND METHODS OF USEFIELD

[0001] The present disclosure relates to the field of antibody therapeutics and, in particular, to bispecific antibodies that bind to folate receptor alpha and NaPi2b, conjugates comprising the bispecific antibodies conjugated to one or more drugs and methods of using the antibodies and conjugates.BACKGROUND

[0002] Both folate receptor alpha (FRa) and sodium-dependent phosphate transporter 2B (NaPi2b) are overexpressed in a variety of cancers including ovarian cancer, endometrial cancer, and lung cancer. Given the overexpression of these two proteins in certain types of cancers, therapeutic agents targeted to one or the other have been studied in clinical trials for the treatment of cancer.

[0003] For example, several clinical studies involving FRa-targeted agents in the treatment of cancer are currently ongoing, including the anti-FRa antibody, farletuzumab, and the FRa-targeted antibody-drug conjugates (ADCs), mirvetuximab soravtansine (ImmunoGen, Inc.), MORAb-202 (Eisai Inc.) and STRO-002 (Sutro Biopharma, Inc.).

[0004] In addition, the ADC upifitamab rilsodotin (Mersana Therapeutics), comprising the NaPi2b-targeting antibody MX-35 with an auristatin-F payload has been studied in patients with platinum-resistant ovarian cancer or non-small cell lung cancer (NSCLC), and lifastuzumab vedotin (Genentech), an ADC of the NaPi2b-targeting antibody lifastuzumab with an MMAE payload has been studied in patients with ovarian cancer or NSCLC.

[0005] This background information is provided for the purpose of making known information believed by the applicant to be of possible relevance to the present disclosure. No admission is necessarily intended, nor should be construed, that any of the preceding information constitutes prior art against the claimed invention(s).SUMMARY

[0006] Described herein are bispecific antibody constructs that bind folate receptor alpha (FRa) and NaPi2b, conjugates comprising the bispecific antibody constructs and methods of using the antibody constructs and conjugates. One aspect of the present disclosure relates to a bispecific antibody construct comprising one or more NaPi2b antigen-binding domains that specifically bind to NaPi2b, one or more folate receptor alpha (FRa) antigen-binding domains that specifically bind to FRa, and an immunoglobulin (IgG) Fc region comprising a first Fc polypeptide and a second Fc polypeptide, wherein at least two of the antigen-binding domains are linked to the IgG Fc region.

[0007] Another aspect of the present disclosure relates to a bispecific antibody construct comprising one or more NaPi2b antigen-binding domains that specifically bind to NaPi2b, one or more folate receptor alpha (FRa) antigen-binding domains that specifically bind to FRa, and an immunoglobulin (IgG) Fc region comprising a first Fc polypeptide and a second Fc polypeptide, wherein at least two of the antigen-binding domains are linked to the IgG Fc region, and wherein the one or more NaPi2b antigen-binding domains comprise the heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) of the VH domain as set forth in SEQ ID NO: 58, and the light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) of the VL domain as set forth in SEQ ID NOs: 59.

[0008] Another aspect of the present disclosure relates to a bispecific antibody construct comprising one or more NaPi2b antigen-binding domains that specifically bind to NaPi2b, one or more folate receptor alpha (FRa) antigen-binding domains that specifically bind to FRa, and an immunoglobulin (IgG) Fc region comprising a first Fc polypeptide and a second Fc polypeptide, wherein at least two of the antigen-binding domains are linked to the IgG Fc region, and wherein the one or more NaPi2b antigen-binding domains comprise heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 52, 53 and 54, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 55, 56 and 57.

[0009] Another aspect of the present disclosure relates to a polynucleotide or set of polynucleotides encoding a bispecific antibody construct as described herein.

[0010] Another aspect of the present disclosure relates to an expression vector or set of expression vectors comprising a polynucleotide or set of polynucleotides encoding a bispecific antibody construct as described herein.

[0011] Another aspect of the present disclosure relates to a host cell comprising the expression vector or set of expression vectors comprising a polynucleotide or set of polynucleotides encoding a bispecific antibody construct as described herein.

[0012] Another aspect of the present disclosure relates to an antibody-drug conjugate comprising a bispecific antibody construct as described herein conjugated to one or more drug moieties.

[0013] Another aspect of the present disclosure relates to an antibody-drug conjugate having general Formula I:A-(L-(D)m)n(I)wherein:A is a bispecific antibody construct as described herein;L is a linker;D is a drug moiety;m is between 1 and about 8, andn is 1 and about 12.

[0014] Another aspect of the present disclosure relates to a pharmaceutical composition comprising a bispecific antibody construct as described herein, and a pharmaceutically acceptable carrier or diluent.

[0015] Another aspect of the present disclosure relates to a pharmaceutical composition comprising an antibody-drug conjugate as described herein, and a pharmaceutically acceptable carrier or diluent.

[0016] Another aspect of the present disclosure relates to a bispecific antibody construct as described herein for use in therapy, for example in the treatment of cancer.

[0017] Another aspect of the present disclosure relates to an antibody-drug conjugate as described herein for use in therapy, for example in the treatment of cancer.

[0018] Another aspect of the present disclosure relates to a use of a bispecific antibody construct as described herein in the manufacture of a medicament for the treatment of cancer.

[0019] Another aspect of the present disclosure relates to a use of an antibody-drug conjugate as described herein in the manufacture of a medicament for the treatment of cancer.

[0020] Another aspect of the present disclosure relates to a method of inhibiting the growth of tumor cells that express FRa, NaPi2b or both FRa and NaPi2b, comprising contacting the cells with a bispecific antibody construct as described herein.

[0021] Another aspect of the present disclosure relates to a method of inhibiting the growth of tumor cells that express FRa, NaPi2b or both FRa and NaPi2b, comprising contacting the cells with an antibody-drug conjugate as described herein.

[0022] Another aspect of the present disclosure relates to a method of treating a subject having a cancer comprising administering to the subject an effective amount of a bispecific antibody construct as described herein.

[0023] Another aspect of the present disclosure relates to a method of treating a subject having a cancer comprising administering to the subject an effective amount of the antibody-drug conjugate as described herein.

[0024] Another aspect of the present disclosure relates to an antibody construct comprising an antigen-binding domain that specifically binds to folate receptor alpha (FRa), the antigen-binding domain comprising the heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) of the VH sequence as set forth in SEQ ID NO: 29, and the light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) of the VL sequence as set forth in SEQ ID NO: 33.

[0025] Another aspect of the present disclosure relates to an antibody construct comprising an antigen-binding domain that specifically binds to folate receptor alpha (FRa), the antigen-binding domain comprising heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 10, 11 and 7, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 18, 19 and 17.

[0026] Another aspect of the present disclosure relates to a polynucleotide or set of polynucleotides encoding an antibody construct comprising an antigen-binding domain that specifically binds to FRa as described herein.

[0027] Another aspect of the present disclosure relates to an expression vector or set of expression vectors comprising the polynucleotide or set of polynucleotides encoding an antibody construct comprising an antigen-binding domain that specifically binds to FRa as described herein.

[0028] Another aspect of the present disclosure relates to a host cell comprising the expression vector or set of expression vectors comprising the polynucleotide or set of polynucleotides encoding an antibody construct comprising an antigen-binding domain that specifically binds to FRa as described herein.BRIEF DESCRIPTION OF THE FIGURES

[0029] Fig. 1 depicts schematically examples of bispecific antibody formats comprising Fab antigen-binding domains in accordance with certain embodiments of the present disclosure, (A) bivalent format comprising two Fab antigen-binding domains, (B) and (C) trivalent formats comprising three Fab antigen-binding domains, and (D) and (E) tetravalent formats comprising four Fab antigen-binding domains.

[0030] Fig. 2 depicts schematically examples of bispecific antibody formats comprising one or more Fab antigen-binding domains and an scFv antigen-binding domain in accordance with certain embodiments of the present disclosure, (A) bivalent format comprising one Fab antigen-binding domain and one scFv antigen-binding domain, (B) and (C) trivalent formats comprising two Fab antigen-binding domains and one scFv antigen-binding domain, and (D), (E) and (F) tetravalent formats comprising three Fab antigen-binding domains and one scFv antigen-binding domain.

[0031] Fig. 3 shows the profiles from electrophoresis under (A) non-reducing (NR) and (B) reducing (R) conditions after preparative SEC purification of parental chimeric anti-FRa antibody v31625 (far left-hand side under NR conditions (A); not present under R conditions (B)) and 12 humanized variants.

[0032] Fig.4 shows the UPLC-SEC profiles of two representative humanized variants of chimeric anti-FRa antibody v31625 after preparative SEC purification, (A) variant v32977, and (B) variant v32983.

[0033] Fig. 5 shows the profiles from electrophoresis under (A) non-reducing (NR) and (B) reducing (R) conditions after preparative SEC purification of selected half-antibody constructs (from left to right under each of (A) and (B): Format 2 12A10, Format2 12A10b, Format 3 10L18, Format 3 76, Format 4b 10L18, Format 4b 76, Format 4 10L18, Format 476, Format 42L16 and Format 5 10L18).

[0034] Fig. 6 shows the UPLC-SEC profiles of three representative half-antibody constructs after preparative SEC purification (A) Format 2 12A10, (B) Format 5b 12A10, and (C) Format 4b 10L18.

[0035] Fig. 7 shows the internalization of various FRa x NaPi2b bispecific antibodies in Fab format in (A) IGROV-1, (B) T-47D, and (C) TOV-21G cells.

[0036] Fig. 8 shows the internalization of various FRa x NaPi2b bispecific antibodies in scFv format in (A) IGROV-1, (B) T-47D, and (C) TOV-21G cells.

[0037] Fig. 9 shows the average number of FRa and NaPi2b proteins per cell for select cancer cell lines determined as described in Example 3 herein.

[0038] Fig. 10 shows the cell-binding activity (pICso) of various FRa x NaPi2b bispecific antibodies in (A) IGROV-1 cells, (B) HCC-78 cells, (C) TOV-21G cells, (D) T-47D cells, and (E) JEG-3 cells.

[0039] Fig. 11 shows the differences in cellular binding for FRa x NaPi2b bispecific antibodies in Fab format observed in (A) HCC-78 cells and (B) TOV-21G cells. Open circles indicate bispecificantibodies in 1+1 format, triangles indicate bispecific antibodies in 2+1 format, and open squares indicate bispecific antibodies in 2+2 format.

[0040] Fig. 12 shows the differences in cellular binding for FRa x NaPi2b bispecific antibodies in scFv format observed in (A) T-47D cells and (B) TOV-21G cells. Open circles indicate bispecific antibodies in 1+1 format, triangles indicate bispecific antibodies in 2+1 format, and open squares indicate bispecific antibodies in 2+2 format.

[0041] Fig. 13 shows the extent of internalization of various FRa x NaPi2b bispecific antibodies into (A) IGROV-1 cells, (B) HCC-78 cells, (C) TOV-21G cells, (D) T-47D cells, and (E) H441 cells.

[0042] Fig. 14 shows the cytotoxicity capabilities (pICso) of select ADCs comprising FRa x NaPi2b bispecific antibodies conjugated to Drug-Linker 001 in (A) IGROV-1 cells, (B) HCC-78 cells, (C) TOV-21G cells, (D) T-47D cells, (E) JEG-3 cells, (F) H441 cells, and (G) H2110 cells.

[0043] Fig. 15 shows anti-FRa paratope-based differences in cytotoxic activity for ADCs comprising FRa x NaPi2b bispecific antibodies in Fab format in (A) JEG-3 cells, and (B) H2110 cells. Black circles / solid lines indicate bispecific ADCs comprising the 10L18 anti-FRa paratope, grey triangles / dashed lines indicate bispecific ADCs comprising the 76 anti-FRa paratope, open diamonds / dotted lines indicate bispecific ADCs comprising the 2L16 anti-FRa paratope, open circles / dotted lines indicate negative control ADC.

[0044] Fig. 16 shows cytotoxicity dose-response curves for ADCs comprising FRa x NaPi2b bispecific antibodies in (A) IGROV-1 cells, (B) JEG-3 cells, (C) H2110 cells, and (D) T-47D cells. Squares / dashed lines indicate bispecific antibodies in scFv format and circles / solid lines indicate bispecific antibodies in Fab format.

[0045] Fig. 17 shows cytotoxicity dose-response curves for ADCs comprising FRa x NaPi2b bispecific antibodies in Fab format in (A) TOV-21G cells, (B) HCC78 cells, (C) H1781 cells, and (D) EBC-1 cells.

[0046] Fig. 18 shows cytotoxicity (pICso) vs. cellular internalization (fold-over control) correlation plots for ADCs comprising FRa x NaPi2b Fab bispecific antibodies in (A) IGROV-1cells, (B) HCC-78 cells, and (C) H441 cells. (D) shows a correlation plot of cytotoxicity (pICso) in TOV-21G cells vs. cellular internalization (fold-over control) in T-47D cells for ADCs comprising FRa x NaPi2b Fab bispecific antibodies.

[0047] Fig. 19 shows (A) cytotoxicity (pICso) vs. cellular internalization (fold-over control) correlation plots for ADCs comprising FRa x NaPi2b scFv bispecific antibodies in IGROV-1 cells, (B) a correlation plot of cytotoxicity (pICso) in H2110 cells vs. cellular internalization (fold-over control) in T-47D cells for ADCs comprising FRa x NaPi2b scFv bispecific antibodies, and (C) a correlation plot of cytotoxicity (pICso) in IGROV-1 cells vs. cellular internalization (fold-over control) in T-47D cells for ADCs comprising FRa x NaPi2b scFv bispecific antibodies.

[0048] Fig. 20 shows the UPLC-SEC profiles for two representative FRa x NaPi2b bispecific antibodies reflecting high species homogeneity, with purity ranging from -97-100% monomeric species, (A) variant v41369 and (B) variant v41328.

[0049] Fig. 21 shows the extent of internalization of certain FRa x NaPi2b bispecific antibodies into (A) IGROV-1 cells, (B) H2110 cells, (C) H441 cells, (D) TOV-21G cells, and (E) JEG-3 cells.

[0050] Fig. 22 shows the complement-dependent cytotoxicity (CDC) mediated by certain FRa x NaPi2b bispecific antibodies against IGROV-1 cells in the presence of (A) 25% (v / v) human serum, (B) 25% (v / v) rabbit serum, and (C) no serum.

[0051] Fig. 23 shows the extent of penetration of certain FRa x NaPi2b bispecific antibodies into spheroids formed by co-culture of JEG-3 and HCC-78 cells after 24h incubation.

[0052] Fig. 24 shows the tumor growth inhibition of an FRa-expressing OV-90 ovarian CDX model in immunocompromised mice by certain FRa x NaPi2b bispecific ADCs compared to the vehicle treated control.

[0053] Fig. 25 shows the total antibody levels in serum from immunocompromised mice harbouring OV-90 ovarian CDX tumors and treated with FRa x NaPi2b bispecific ADCs.

[0054] Fig. 26 depicts schematically the formats of the FRa x NaPi2b bispecific antibodies v41321, v41324, v41328, v41330, v41336, v41337, v41340, v41365, v41369 and v41376.DETAILED DESCRIPTION

[0055] The present disclosure relates to bispecific antibodies that bind to folate receptor alpha (FRa) and NaPi2b (FRa x NaPi2b antibody constructs), antibody-drug conjugates (ADCs) comprising the FRa x NaPi2b antibody constructs and methods of using the FRa x NaPi2b antibody constructs and ADCs to treat cancers that express either FRa or NaPi2b, or both FRa and NaPi2b. Certain embodiments of the present disclosure relate to methods of using the FRa x NaPi2b antibody constructs and ADCs to treat a gynecological cancer, such as ovarian cancer.

[0056] Bispecific antibodies and bispecific ADCs that can target two different tumor associated antigens (TAAs) are a promising approach to overcoming challenges associated with spatial and temporal target antigen heterogeneity. A traditional bispecific design employs a bivalent IgG where one paratope interacts with a first target antigen and the other paratope interacts with a second target antigen. This design ensures that the molecule is maximally active when both target antigens are present. However, this enhanced specificity approach has limitations, as it requires cellular co-expression of both target antigens to be effective. In contrast, a bispecific antibody or ADC capable of targeting two different antigens independently could increase the addressable patient population relative to a monospecific antibody or ADC. For example, a bispecific antibody or ADC with the potential for both independent and dual targeting of folate receptor alpha (FRa) and NaPi2b, as described herein, could significantly expand the number of patients who could benefit relative to an antibody or ADC against either target antigen alone, and may further help to address issues of target antigen heterogeneity.

[0057] Certain embodiments of the present disclosure relate to novel antibody sequences that specifically bind FRa (anti-FRa antibody sequences). These anti-FRa antibody sequences may be used in the preparation of monospecific, bispecific, or multispecific antibody constructs that bind to FRa (FRa antibody constructs), including the FRa x NaPi2b antibody constructs described herein.Definitions

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0059] As used herein, the term “about” refers to an approximately + / -10% variation from a given value. It is to be understood that such a variation is always included in any given value provided herein, whether or not it is specifically referred to.

[0060] The use of the word “a” or “an” when used herein in conjunction with the term “comprising” may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one” and “one or more than one.”

[0061] As used herein, the terms “comprising,” “having,” “including” and “containing,” and grammatical variations thereof, are inclusive or open-ended and do not exclude additional, unrecited elements and / or method steps. The term “consisting essentially of’ when used herein in connection with a composition, use or method, denotes that additional elements and / or method steps may be present, but that these additions do not materially affect the manner in which the recited composition, method or use functions. The term “consisting of’ when used herein in connection with a composition, use or method, excludes the presence of additional elements and / or method steps. A composition, use or method 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 referred to.

[0062] A “complementarity determining region” or “CDR” is an amino acid sequence that contributes to antigen-binding specificity and affinity. “Framework” regions (FR) can aid in maintaining the proper conformation of the CDRs to promote binding between the antigen-binding region and an antigen. From N-terminus to C-terminus, both the light chain variable region (VL) and the heavy chain variable region (VH) of an antibody typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The three heavy chain CDRs are referred to herein as HCDR1, HCDR2, and HCDR3, and the three light chain CDRs are referred to as LCDR1, LCDR2, and LCDR3. CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope. Often, the three heavy chain CDRs and the three light chain CDRs are required to bind antigen. However, in some instances, even a single variable domain can confer binding specificity to the antigen. Furthermore, as is known in the art, in some cases, antigen-binding mayalso occur through a combination of a minimum of one or more CDRs selected from the VH and / or VL domains, for example HCDR3.

[0063] A number of different definitions of the CDR sequences are in common use, including those described by Kabat et al. (1983, Sequences of Proteins of Immunological Interest, NIH Publication No. 369-847, Bethesda, MD), by Chothia et al. (1987, J Mol Biol, 196:901-917), as well as the IMGT, AbM (University of Bath) and Contact (MacCallum, et al., 1996, J Mol Biol, 262(5):732-745) definitions. By way of example, CDR definitions according to Kabat, Chothia, IMGT, AbM and Contact are provided in Table 1 below. Accordingly, as would be readily apparent to one skilled in the art, the exact numbering and placement of CDRs may differ based on the numbering system employed. However, it is to be understood that the disclosure herein of a VH sequence includes the disclosure of the associated (inherent) heavy chain CDRs (HCDRs) as defined by any of the known numbering systems. Similarly, disclosure herein of a VL sequence includes the disclosure of the associated (inherent) light chain CDRs (LCDRs) as defined by any of the known numbering systems.Table 1: Common CDR Definitions1Definition Heavy Chain Light ChainCDR12CDR2 CDR3 CDR1 CDR2 CDR3 Kabat H31-H35B H50-H65 H95-H102 L24-L34 L50-L56 L89-L97 Chothia H26-H32, H52-H56 H95-H102 L24-L34 L50-L56 L89-L97 H33 orH34IMGT H26-H33, H51-H57 H93-H102 L27-L32 L50-L52 L89-L97 H34, H35,H35A orH35BAbM H26-H35B H50-H58 H95-H102 L24-L34 L50-L56 L89-L97 Contact H30-H35B H47-H58 H93-H101 L30-L36 L46-L55 L89-L961Either the Kabat or Chothia numbering system may be used for HCDR2, HCDR3 and the light chain CDRs for all definitions except Contact, which uses Chothia numbering2Using Kabat numbering. The position in the Kabat numbering scheme that demarcates the end of the Chothia and IMGT CDR-H1 loop varies depending on the length of the loop because Kabat placesinsertions outside of those CDR definitions at positions 35A and 35B. However, the IMGT and Chothia CDR-H1 loop can be unambiguously defined using Chothia numbering. CDR-H1 definitions using Chothia numbering: Kabat H31-H35, Chothia H26-H32, AbM H26-H35, IMGT H26-H33, Contact H30-H35.

[0064] The term “identical” in the context of two or more polynucleotide or polypeptide sequences, refers to two or more sequences or subsequences that are the same. Sequences are “substantially identical” if they have a percentage of amino acid residues or nucleotides that are the same (for example, about 80%, about 85%, about 90%, about 95%, or about 98% identity, over a specified region) when compared and aligned for maximum correspondence over a comparison window or over a designated region as measured using one of the commonly used sequence comparison algorithms as known to persons of ordinary skill in the art or by manual alignment and visual inspection. For sequence comparison, typically test sequences are compared to a designated reference sequence. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0065] A “comparison window” refers to a segment of a sequence comprising contiguous amino acid or nucleotide positions which may be, for example, from about 10 to 600 contiguous amino acid or nucleotide positions, or from about 10 to about 200, or from about 10 to about 150 contiguous amino acid or nucleotide positions over which a test sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are known to those of ordinary skill in the art. Optimal alignment of sequences for comparison can be conducted, for example, by the local homology algorithm of Smith & Waterman, 1970, Adv. Appl. Math., 2:482c; by the homology alignment algorithm of Needleman & Wunsch, 1970, J. Mol. Biol., 48:443; by the search for similarity method of Pearson & Lipman, 1988, Proc. Natl. Acad. Sci. USA, 85:2444, or by computerized implementations of these algorithms (for example, GAP, BESTFIT, FASTA or TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, WI), or by manual alignment and visual inspection (see, for example, Ausubel et al., CurrentProtocols in Molecular Biology, (1995 supplement), Cold Spring Harbor Laboratory Press). Examples of available algorithms suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul etal., 1997, Nuc. Acids Res., 25:3389-3402, and Altschul et al., 1990, J. Mol. BioL, 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the website for the National Center for Biotechnology Information (NCBI).

[0066] The term “subject,” as used herein, refers to an animal, in some embodiments a mammal, which is the object of treatment, observation or experiment. The animal may be a human, a nonhuman primate, a companion animal (for example, dog, cat, or the like), farm animal (for example, cow, sheep, pig, horse, or the like) or a laboratory animal (for example, rat, mouse, guinea pig, non-human primate, or the like). In certain embodiments, the subject is a human.

[0067] It is contemplated that any embodiment discussed herein can be implemented with respect to any method, use or composition disclosed herein, and vice versa.

[0068] Particular features, structures and / or characteristics described in connection with an embodiment disclosed herein may be combined with features, structures and / or characteristics described in connection with another embodiment disclosed herein in any suitable manner to provide one or more further embodiments.

[0069] It is also to be understood that the positive recitation of a feature in one embodiment, serves as a basis for excluding the feature in an alternative embodiment. For example, where a list of options is presented for a given embodiment or claim, it is to be understood that one or more option may be deleted from the list and the shortened list may form an alternative embodiment, whether or not such an alternative embodiment is specifically referred to.

[0070] The amino acid residues for the immunoglobulin heavy and light chains may be numbered according to several conventions. Kabat numbering is used herein for the VH, CHI, CL, and VL domains unless otherwise indicated. EU numbering is used herein for the CH3 and CH2 domains, and the hinge region unless otherwise indicated.BISPECIFIC ANTIBODY CONSTRUCTS

[0071] The present disclosure relates to bispecific antibody constructs that specifically bind to folate receptor alpha (FRa) and to NaPi2b (referred to herein as bispecific FRa x NaPi2b antibody constructs or FRa x NaPi2b antibody constructs). In this context, the term “antibody construct” refers to a polypeptide or a set of polypeptides that comprises two or more antigen-binding domains, where each of the two or more antigen-binding domains specifically binds to an epitope or antigen. In accordance with the present disclosure, the bispecific antibody constructs comprise at least one antigen-binding domain that specifically binds to FRa and at least one antigen-binding domain that specifically binds to NaPi2b. The antibody construct may further comprise a scaffold and at least one of the two or more antigen-binding domains can be operably linked to the scaffold, directly or via a linker, as described herein.Antigen Binding Domains

[0072] The bispecific FRa x NaPi2b antibody constructs of the present disclosure comprise two or more antigen-binding domains. At least one of these antigen-binding domains is capable of binding to FRa and at least one antigen-binding domain is capable of binding to NaPi2b. Each of the two or more antigen-binding domains are typically an immunoglobulin-based binding domain, such as an antigen-binding antibody fragment. Examples of an antigen-binding antibody fragment include, but are not limited to, a Fab fragment, a Fab’ fragment, a single chain Fab (scFab), a single chain Fv (scFv) and a single domain antibody (sdAb). The two or more antigen-binding domains comprised by the FRa x NaPi2b antibody construct may be the same type of immunoglobulin-based domain, such as an antigen-binding antibody fragment, or they may be different.

[0073] A “Fab fragment” contains the constant domain of the light chain (CL) and the first constant domain of the heavy chain (CHI) along with the variable domains of the light and heavy chains (VL and VH, respectively). Fab' fragments differ from Fab fragments by the addition of a few amino acid residues at the C-terminus of the heavy chain CHI domain, including one or more cysteines from the antibody hinge region. A Fab fragment may also be a single-chain Fab molecule, i.e. a Fab molecule in which the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. For example, the C-terminus of the Fab light chain may be connected to the N-terminus of the Fab heavy chain in the single-chain Fab molecule.

[0074] An “scFv” includes a heavy chain variable domain (VH) and a light chain variable domain (VL) of an antibody in a single polypeptide chain. The scFv may optionally further comprise a polypeptide linker between the VH and VL domains which enables the scFv to form a desired structure for antigen binding. For example, an scFv may include a VL connected from its C-terminus to the N-terminus of a VH by a polypeptide linker. Alternately, an scFv may comprise a VH connected through its C-terminus to the N-terminus of a VL by a polypeptide linker (see review in Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994)). Examples of polypeptide linkers include linkers comprising the following amino acid residues: Gly, Ser, Ala or Thr, or a combination thereof. Examples of such polypeptide linkers include, but are not limited to, glycineserine linkers, such as (GS)n, (GSGGS)n(SEQ ID NO: 193), (GGGGS)n(SEQ ID NO: 194) and (GGGS)n (SEQ ID NO: 195) (where n is an integer between 1 and 4), as well as glycine-alanine linkers and alanine-serine linkers having similar configurations.

[0075] An “sdAb” format refers to a single immunoglobulin domain. The sdAb may be, for example, of camelid origin. Camelid antibodies lack light chains and their antigen-binding sites consist of a single domain, termed a “VHH.” An sdAb comprises three CDR / hypervariable loops that form the antigen-binding site: CDR1, CDR2 and CDR3. sdAbs are fairly stable and easy to express, for example, as a fusion with the Fc chain of an antibody (see, for example, Harmsen & De Haard, 2007, Appl. Microbiol Biotechnol., 77(1): 13-22).FRa Antigen-Binding Domains

[0076] In accordance with the present disclosure, the bispecific FRa x NaPi2b antibody construct comprises at least one antigen-binding domain that specifically binds to FRa (an FRa antigenbinding domain). When the bispecific FRa x NaPi2b antibody constructs comprise more than one FRa antigen-binding domain, the FRa antigen-binding domains may be the same or they may be different.

[0077] In certain embodiments, the FRa x NaPi2b antibody constructs of the present disclosure comprise at least one FRa antigen-binding domain that specifically binds to human FRa. In certain embodiments, the at least one FRa antigen-binding domain comprised by the FRa x NaPi2b antibody constructs of the present disclosure may be capable of binding to human FRa and to anFRa from one or more non-human species. In certain embodiments, the at least one FRa antigenbinding domain comprised by the FRa x NaPi2b antibody constructs of the present disclosure is capable of binding to human FRa and cynomolgus monkey FRa.

[0078] Human FRa is also known as “human folate receptor 1” or “FOLR1.” The protein sequences of human FRa from various sources are known in the art and are readily available from publicly accessible databases, such as GenBank or UniProtKB. Examples of hFRa sequences include for example those provided under NCBI reference numbers P15328, AAX29268.1, AAX37119.1, NP_057937.1 and NP_057936.1. An exemplary human FRa protein sequence is provided in Table 2 as SEQ ID NO: 1 (NCBI Reference Sequence: NP 057936.1). An exemplary cynomolgus monkey FRa protein sequence is also provided in Table 2 (SEQ ID NO: 2; NCBI Reference Sequence: XP_005579002.2).Table 2: Human and Cynomolgus Monkey FRa Protein SequencesOrganism Sequence SEQ ID NOHuman MAQRMTTQLLLLLVWVAVVGEAQTRIAWARTELLNVCMN 110 AKHHKEKPGPEDKLHEQCRPWRKNACCSTNTSQEAHKDVS YLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQV DQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKG WNWTSGFNKCAVGAACQPFHFYFPTPTVLCNEIWTHSYKVS NYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWA AWPFLLSLALMLLWLLSCynomolgus MAQRMTTQLLLLLVWVAWGEAQTRTTRARTELLNVCMN 111 Monkey AKHHKEKPGPEDKLHEQCRPWKKNACCSTNTSQEAHKDVS YLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNLGPWIQQV DQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKG WNWTSGFNKCPVGAACQPFHFYFPTPTVLCNEIWTYSYKVS NYSRGSGRCIQMWFDPAQGNPNEEVARFYAAAMSGAGPWA AWPLLLSLALTLLWLLS

[0079] In certain embodiments, the FRa antigen-binding domain retains the ability to bind to FRa-expressing cells when the FRa antigen-binding domain is linked to an IgG Fc in a one-arm antibody (monovalent) format. In some embodiments, the FRa antigen-binding domain retains the ability to bind to and internalize into FRa-expressing cells when the FRa antigen-binding domain is linked to an IgGFc in a one-arm antibody (OAA) format. The ability of an OAA comprising anFRa antigen-binding domain to bind to and / or internalize into FRa-expressing cells may be determined by standard methods known in the art. Cell binding assays are typically conducted by incubating the antibody construct with cells, for example cancer cells, that express FRa (such cells are available commercially). The amount of the OAA bound to the cells can be assessed by flow cytometry, for example, and compared to binding observed in the presence of controls. Internalization assays are typically conducted by labelling the OAA with a detectable label, for example a fluorescent label, incubating the labelled OAA with cells that express FRa under conditions allowing for internalization. The amount of internalized OAA is then detected by flow cytometry, for example, and compared to internalization observed in the presence of controls. Non-limiting examples of assays that may be used to determine cell binding and internalization are described in the Examples herein.

[0080] FRa antigen-binding domains may be derived from anti-FRa antibodies known in the art, including, but not limited to, farletuzumab (described in International Publication Nos. WO 2004 / 003388 and WO 2005 / 080431), mirvetuximab (described in International Publication No. WO2011 / 106528), and the anti-FRa antibodies described in US Patent Nos. 8,388,972 and 9,695,237, and in International Publication Nos. WO2018 / 098277, WO2015 / 196167, WO2016 / 079076, and WO2018 / 071597.

[0081] In certain embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigenbinding domain comprising at least one, two, or all three heavy chain CDRs and at least one, two, or all three light chain CDRs of farletuzumab, mirvetuximab, or any one of the anti-FRa antibodies described in US 8,388,972, US 9,695,237, WO2018 / 098277, WO2015 / 196167, W02016 / 079076 or WO2018 / 071597. In some embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigen-binding domain comprising the three heavy chain CDRs and the three light chain CDRs of farletuzumab, mirvetuximab, or any one of the anti-FRa antibodies described in US 8,388,972, US 9,695,237, WO2018 / 098277, WO2015 / 196167, W02016 / 079076 or WO2018 / 071597. In some embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigen-binding domain comprising a VH sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence of farletuzumab, mirvetuximab, or any one of the anti-FRa antibodies described in US 8,388,972, US 9,695,237, WO2018 / 098277, WO2015 / 196167, W02016 / 079076 or WO2018 / 071597, and a VL sequence that is at least about80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence of farletuzumab, mirvetuximab, or any one of the anti-FRa antibodies described in US 8,388,972, US 9,695,237, WO2018 / 098277, WO2015 / 196167, W02016 / 079076 or WO2018 / 071597. The CDR sequences for mirvetuximab and farletuzumab are provided in Table 3. The VH and VL sequences of these antibodies, and the CDR and VH and VL sequences of the other anti-FRa antibodies can readily be determined by one of skill in the art with reference to the disclosures of WO 2004 / 003388, WO 2005 / 080431, WO2011 / 106528, US 8,388,972, US 9,695,237, WO2018 / 098277, WO2015 / 196167, WO2016 / 079076 and WO2018 / 071597.Table 3: CDR Sequences of Exemplary Anti-FRa AntibodiesDefinition CDR1 CDR2 CDR3 MirvetuximabHeavy Kabat GYFMN RIHPYDGDTFYNQKFQG YDGSRAMDY chain (SEQ ID NO: 112) (SEQ ID NO: 113) (SEQ ID NO: 114) Chothia GYTFTGY HPYDGD YDGSRAMDY (SEQ ID NO: 115) (SEQ ID NO: 116) (SEQ ID NO: 114) IMGT GYTFTGYF IHPYDGDT TRYDGSRAMDY(SEQ ID NO: 117) (SEQ ID NO: 118) (SEQ ID NO: 119) AbM GYTFTGYFMN RIHPYDGDTF YDGSRAMDY (SEQ ID NO: 120) (SEQ ID NO: 121) (SEQ ID NO: 114) Light Kabat KASQSVSFAGTSLMH RASNLEA QQSREYPYT chain (SEQ ID NO: 122) (SEQ ID NO: 123) (SEQ ID NO: 124) Chothia KASQSVSFAGTSLMH RASNLEA QQSREYPYT(SEQ ID NO: 122) (SEQ ID NO: 123) (SEQ ID NO: 124) IMGT QSVSFAGTSL RAS QQSREYPYT(SEQ ID NO: 125) (SEQ ID NO: 126) (SEQ ID NO: 124) AbM KASQSVSFAGTSLMH RASNLEA QQSREYPYT(SEQ ID NO: 122) (SEQ ID NO: 123) (SEQ ID NO: 124) FarletuzumabHeavy Kabat GYGLS MISSGGSYTYYADSVKG HGDDPAWFAY chain (SEQ ID NO: 127) (SEQ ID NO: 128) (SEQ ID NO: 129) Chothia GFTFSGY SSGGSY HGDDPAWFAY (SEQ ID NO: 130) (SEQ ID NO: 131) (SEQ ID NO: 129) IMGT GFTFSGYG ISSGGSYT ARHGDDPAWFAY(SEQ ID NO: 132) (SEQ ID NO: 133) (SEQ ID NO: 134)Definition CDR1 CDR2 CDR3 AbM GFTFSGYGLS MISSGGSYTY HGDDPAWFAY (SEQ ID NO: 135) (SEQ ID NO: 136) (SEQ ID NO: 129) Light Kabat SVSSSISSNNLH GTSNLAS QQWSSYPYMYT chain (SEQ ID NO: 137) (SEQ ID NO: 138) (SEQ ID NO: 139) Chothia SVSSSISSNNLH GTSNLAS QQWSSYPYMYT (SEQ ID NO: 137) (SEQ ID NO: 138) (SEQ ID NO: 139) IMGT SSISSNN GTS QQWSSYPYMYT(SEQ ID NO: 140) (SEQ ID NO: 141) (SEQ ID NO: 139) AbM SVSSSISSNNLH GTSNLAS QQWSSYPYMYT (SEQ ID NO: 137) (SEQ ID NO: 138) (SEQ ID NO: 139)

[0082] In certain embodiments, at least one of the one or more FRa antigen-binding domains comprised by the FRa x NaPi2b antibody constructs may be derived from one of the anti-FRa antibodies 2L16 (see International Publication No. WO 2021 / 207827), 10L18 (see International Publication No. WO 2023 / 178451) or 76 (see Example 1 herein). The CDR, VHand VL sequences for these anti-FRa antibodies are provided in Table 2.1 (see Example 2 herein).

[0083] In certain embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigenbinding domain having a VH sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the VH sequence of any one of antibody 2L16 (SEQ ID NO: 50), antibody 10L18 (SEQ ID NO: 42) or antibody 76 (SEQ ID NO: 29), and a VL sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the VL sequence of any one of 2L16 (SEQ ID NO: 51), antibody 10L18 (SEQ ID NO: 43) or antibody 76 (SEQ ID NO: 33). In some embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigen-binding domain having a VH sequence as set forth in any one of SEQ ID NO: 50, SEQ ID NO: 42 or SEQ ID NO: 29, and a VL sequence as set forth in any one of SEQ ID NO: 51, SEQ ID NO: 43 or SEQ ID NO: 33.

[0084] In certain embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigenbinding domain having a VH sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the VH sequence of antibody 2L16 (SEQ ID NO: 50), and a VL sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the VL sequence of antibody 2L16 (SEQ ID NO: 51). In some embodiments, the FRa x NaPi2b antibody construct comprises an FRaantigen-binding domain having a VH sequence as set forth in SEQ ID NO: 50, and a VL sequence as set forth in SEQ ID NO: 51.

[0085] In certain embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigenbinding domain having a VH sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the VH sequence of antibody 10L18 (SEQ ID NO: 42), and a VL sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the VL sequence of antibody 10L18 (SEQ ID NO: 43). In some embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigen-binding domain having a VH sequence as set forth in SEQ ID NO: 42, and a VL sequence as set forth in SEQ ID NO: 43.

[0086] In certain embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigenbinding domain having a VH sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the VH sequence of antibody 76 (SEQ ID NO: 29), and a VL sequence that is at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the VL sequence of antibody 76 (SEQ ID NO: 33). In some embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigenbinding domain having a VH sequence as set forth in SEQ ID NO: 29, and a VL sequence as set forth in SEQ ID NO: 33.

[0087] In certain embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigenbinding domain comprising the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) of the VH sequence as set forth in any one of SEQ ID NO: 50, SEQ ID NO: 42 or SEQ ID NO: 29, and the light chain CDRs (LCDR1, LCDR2 and LCDR3) of the VL sequence as set forth in any one of SEQ ID NO: 51, SEQ ID NO: 43 or SEQ ID NO: 33.

[0088] In certain embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigenbinding domain comprising the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) and the light chain CDRs (LCDR1, LCDR2 and LCDR3) of any one of antibodies 2L16, 10L18 or 76, as described in International Publication Nos. WO 2021 / 207827 and WO 2023 / 178451, and Example 1 herein, respectively.

[0089] In certain embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigenbinding domain comprising the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) and the lightchain CDRs (LCDR1, LCDR2 and LCDR3) of antibody 2L16, as described in International Publication No. WO 2021 / 207827. In some embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigen-binding domain comprising the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) of the VH sequence as set forth in SEQ ID NO: 50, and the light chain CDRs (LCDR1, LCDR2 and LCDR3) of the VL sequence as set forth in SEQ ID NO: 51. In some embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigen-binding domain comprising heavy chain CDRs (HCDR1, HCDR2 and HCDR3) having the sequences as set forth in SEQ ID NOs: 44, 45 and 46, and light chain CDRs (LCDR1, LCDR2 and LCDR3) having the sequences as set forth in SEQ ID NOs: 47, 48 and 49.

[0090] In certain embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigenbinding domain comprising the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) and the light chain CDRs (LCDR1, LCDR2 and LCDR3) of antibody 10L18, as described in International Publication No. WO 2023 / 178451. In some embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigen-binding domain comprising the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) of the VH sequence as set forth in SEQ ID NO: 42, and the light chain CDRs (LCDR1, LCDR2 and LCDR3) of the VL sequence as set forth in SEQ ID NO: 43. In some embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigen-binding domain comprising heavy chain CDRs (HCDR1, HCDR2 and HCDR3) having the sequences as set forth in SEQ ID NOs: 36, 37 and 38, and light chain CDRs (LCDR1, LCDR2 and LCDR3) having the sequences as set forth in SEQ ID NOs: 39, 40 and 41.

[0091] In certain embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigenbinding domain comprising the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) and the light chain CDRs (LCDR1, LCDR2 and LCDR3) of antibody 76, as described in Example 1 herein. In some embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigen-binding domain comprising the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) of the VH sequence as set forth in SEQ ID NO: 29, and the light chain CDRs (LCDR1, LCDR2 and LCDR3) of the VL sequence as set forth in SEQ ID NO: 33. In some embodiments, the FRa x NaPi2b antibody construct comprises an FRa antigen-binding domain comprising heavy chain CDRs (HCDR1, HCDR2 and HCDR3) having the sequences as set forth in SEQ ID NOs: 10, 11 and 7, and lightchain CDRs (LCDR1, LCDR2 and LCDR3) having the sequences as set forth in SEQ ID NOs: 18, 19 and 17.NaPilb Antigen-Binding Domains

[0092] In accordance with the present disclosure, the bispecific FRa x NaPi2b antibody construct comprises at least one antigen-binding domain that specifically binds to NaPi2b (a NaPi2b antigenbinding domain). When the bispecific FRa x NaPi2b antibody constructs comprise more than one NaPi2b antigen-binding domain, the NaPi2b antigen-binding domains may be the same or they may be different.

[0093] In certain embodiments, the FRa x NaPi2b antibody constructs of the present disclosure comprise at least one NaPi2b antigen-binding domain that specifically binds to human NaPi2b. In certain embodiments, the at least one NaPi2b antigen-binding domain comprised by the FRa x NaPi2b antibody constructs of the present disclosure may be capable of binding to human NaPi2b and to NaPi2b from one or more non-human species. In certain embodiments, the at least one NaPi2b antigen-binding domain comprised by the FRa x NaPi2b antibody constructs of the present disclosure is capable of binding to human NaPi2b and cynomolgus monkey NaPi2b.

[0094] NaPi2b (also known as SLC34A2, NAPI-3B, NAPI-IIb and NPTIIb) is a pH-sensitive sodium-dependent phosphate transporter that is expressed in some normal epithelial cells in the lung, gut and mammary gland. NaPi2b is found highly expressed on tumor cells, primarily in lung and ovarian cancers (Lin, etal., 2015, Clin Cancer Res., 21(22):5139-50). NaPi2b is a multispan membrane protein, with extracellular domains of 14, 129, 57 and 6 amino acids. The polypeptide sequence of human NaPi2b is provided under NCBI Reference Sequence: NP_001171470.2 and UniProt 095436, and is shown in Table 4 (SEQ ID NO: 142).Table 4: Human NaPilb Protein SequenceSequence SEQ ID NO MAPWPELGDAQPNPDKYLEGAAGQQPTAPDKSKETNKNNTEAPVTKIEL 142 LPSYSTATLIDEPTEVDDPWNLPTLQDSGIKWSERDTKGKILCFFQGIGRLI LLLGFLYFFVCSLDILSSAFQLVGGKMAGQFFSNSSIMSNPLLGLVIGVLVT VLVQSSSTSTSIWSMVSSSLLTVRAAIPIIMGANIGTSITNTIVALMQVGDRSEFRRAFAGATVHDFFNWLSVLVLLPVEVATHYLEIITQLIVESFHFKNGEDAPDLLKVITKPFTKLIVQLDKKVISQIAMNDEKAKNKSLVKIWCKTFTNK TQINVTVPSTANCTSPSLCWTDGIQNWTMKNVTYKENIAKCQHIFVNFHL PDLAVGTILLILSLLVLCGCLIMIVKILGSVLKGQVATVIKKTINTDFPFPFA WLTGYLAILVGAGMTFIVQSSSVFTSALTPLIGIGVITIERAYPLTLGSNIGT TTTAILAALASPGNALRSSLQIALCHFFFNISGILLWYPIPFTRLPIRMAKGL GNISAKYRWFAVFYLIIFFFLIPLTVFGLSLAGWRVLVGVGVPVVFIIILVLC LRLLQSRCPRVLPKKLQNWNFLPLWMRSLKPWDAVVSKFTGCFQMRCCC CCRVCCRACCLLCDCPKCCRCSKCCEDLEEAQEGQDVPVKAPETFDNITIS REAQGEVPASDSKTECTAL

[0095] In certain embodiments, the NaPi2b antigen-binding domain retains the ability to bind to NaPi2b-expressing cells when the NaPi2b antigen-binding domain is linked to an IgG Fc in a one-arm antibody (monovalent) format. In some embodiments, the NaPi2b antigen-binding domain retains the ability to bind to and internalize into NaPi2b-expressing cells when the NaPi2b antigenbinding domain is linked to an IgG Fc in a one-arm antibody (OAA) format. The ability of an OAA comprising a NaPi2b antigen-binding domain to bind to and / or internalize into NaPi2b-expressing cells may be determined by standard methods known in the art as described above for the FRa antigen-binding domain(s).

[0096] NaPi2b antigen-binding domains may be derived from antibodies known in the art including, but not limited to, lifastuzumab (International Publication No. WO2011 / 066503), MX-35 (International Publication No. W02009 / 097128), and the antibodies described in U. S. Patent Application Publication No. 2017 / 0266311.

[0097] In certain embodiments, the FRa x NaPi2b antibody construct comprises a NaPi2b antigenbinding domain comprising at least one, two, or all three heavy chain CDRs and at least one, two, or all three light chain CDRs of lifastuzumab, MX-35, or any one of the anti-NaPi2b antibodies described in International Publication No. WO2011 / 066503 or W02009 / 097128, or in U. S. Patent Application Publication No. 2017 / 0266311. In some embodiments, the FRa x NaPi2b antibody construct comprises a NaPi2b antigen-binding domain comprising the three heavy chain CDRs and the three light chain CDRs of lifastuzumab, MX-35, or any one of the anti-NaPi2b antibodies described in International Publication No. WO2011 / 066503 or W02009 / 097128, or in U. S. Patent Application Publication No. 2017 / 0266311. In some embodiments, the FRa x NaPi2b antibody construct comprises a NaPi2b antigen-binding domain comprising a VH sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VH sequence oflifastuzumab, MX-35, or any one of the anti-NaPi2b antibodies described in International Publication No. WO2011 / 066503 or W02009 / 097128, or in U. S. Patent Application Publication No. 2017 / 0266311, and a VL sequence that is at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the VL sequence of lifastuzumab, MX-35, or any one of the anti-NaPi2b antibodies described in International Publication No. WO2011 / 066503 or W02009 / 097128, or in U. S. Patent Application Publication No. 2017 / 0266311. The CDR sequences of exemplary anti-NaPi2b antibodies are described in Table 5. The VH and VL sequences of these antibodies, and the CDR and VH and VL sequences of other anti-NaPi2b antibodies can readily be determined by one of skill in the art with reference to the disclosures of International Publication No. WO2011 / 066503 or W02009 / 097128, or U. S. Patent Application Publication No. 2017 / 0266311.Table 5: CDR Sequences of Exemplary Anti-NaPi2b AntibodiesDefinition CDR1 CDR2 CDR3 LifastuzumabHeavy Kabat DFAMS TIGRVAFHTYYPDSMKG HRGFDVGHFDF chain (SEQ ID NO: 143) (SEQ ID NO: 144) (SEQ ID NO: 145) Chothia GFSFSDF GRVAFH HRGFDVGHFDF (SEQ ID NO: 146) (SEQ ID NO: 147) (SEQ ID NO: 145) IMGT GFSFSDFA IGRVAFHT ARHRGFDVGHFDF(SEQ ID NO: 148) (SEQ ID NO: 149) (SEQ ID NO: 150) AbM GFSFSDFAMS TIGRVAFHTY HRGFDVGHFDF (SEQ ID NO: 151) (SEQ ID NO: 152) (SEQ ID NO: 145) Light Kabat RSSETLVHSSGNTYLE RVSNRFS FQGSFNPLT chain (SEQ ID NO: 153) (SEQ ID NO: 154) (SEQ ID NO: 155) Chothia RSSETLVHSSGNTYLE RVSNRFS FQGSFNPLT(SEQ ID NO: 153) (SEQ ID NO: 154) (SEQ ID NO: 155) IMGT ETLVHSSGNTY RVS FQGSFNPLT(SEQ ID NO: 156) (SEQ ID NO: 157) (SEQ ID NO: 155) AbM RSSETLVHSSGNTYLE RVSNRFS FQGSFNPLT(SEQ ID NO: 153) (SEQ ID NO: 154) (SEQ ID NO: 155) MX-35Heavy Kabat GYNIH AIYPGNGDTSYKQKFRG GETARATFAY chain (SEQ ID NO: 158) (SEQ ID NO: 159) (SEQ ID NO: 160)Chothia GYTFTGY YPGNGD GETARATFAY (SEQ ID NO: 161) (SEQ ID NO: 162) (SEQ ID NO: 160) IMGT GYTFTGYN IYPGNGDT ARGETARATFAY(SEQ ID NO: 163) (SEQ ID NO: 164) (SEQ ID NO: 165) AbM GYTFTGYNIH AIYPGNGDTS GETARATFAY (SEQ ID NO: 166) (SEQ ID NO: 167) (SEQ ID NO: 160) Light Kabat SASQDIGNFLN YTSSLYS QQYSKLPLT chain (SEQ ID NO: 168) (SEQ ID NO: 169) (SEQ ID NO: 170) Chothia SASQDIGNFLN YTSSLYS QQYSKLPLT (SEQ ID NO: 168) (SEQ ID NO: 169) (SEQ ID NO: 170) IMGT QDIGNF YTS QQYSKLPLT(SEQ ID NO: 171) (SEQ ID NO: 172) (SEQ ID NO: 170) AbM SASQDIGNFLN YTSSLYS QQYSKLPLT (SEQ ID NO: 168) (SEQ ID NO: 169) (SEQ ID NO: 170)

[0098] In certain embodiments, at least one of the one or more NaPi2b antigen-binding domains comprised by the FRa x NaPi2b antibody constructs may be derived from the anti-NaPi2b antibody 12A10 (see Example 2 herein). The CDR, VH and VL sequences for this antibody are provided in Tables 6 and 7 (below) and Table 2.1 (see Example 2 herein).Table 6: CDR Sequences of Anti-NaPi2b Antibody 12A10Numbering Heavy Chain CDR1 SEQ Heavy Chain CDR2 SEQ Heavy Chain CDR3 SEQ System ID NO ID NO ID NO IMGT GFTFTSYN 173 ISPGNGDL 174 ARGRTGMGAVDY 175 Kabat SYNVH 176 AISPGNGDLSYAQKFQG 177 GRTGMGAVDY 54 Chothia GFTFTSY 178 SPGNGD 179 GRTGMGAVDY 54 AbM GFTFTSYNVH 52 AISPGNGDLS 53 GRTGMGAVDY 54 Contact TSYNVH 180 WMGAISPGNGDLS 181 ARGRTGMGAVD 182 Numbering Light Chain CDR1 SEQ Light Chain CDR2 SEQ Light Chain CDR3 SEQ System ID NO ID NO ID NO IMGT QDVYSY 183 YTS 184 QQYNIFPWT 57 Kabat RASQDVYSYLN 55 YTSRLQS 56 QQYNIFPWT 57 Chothia RASQDVYSYLN 55 YTSRLQS 56 QQYNIFPWT 57 AbM RASQDVYSYLN 55 YTSRLQS 56 QQYNIFPWT 57 Contact YSYLNWY 185 LLIYYTSRLQ 186 QQYNIFPW 187Table 7: VH and VL Sequences of Anti-NaPi2b Antibody 12A10Sequence SEQ ID NO VH QVQLVQSGAEVKKPGASVKVSCKASGFTFTSYNVHWVRQAPGQGL 58 EWMGAISPGNGDLSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDT AVYYCARGRTGMGAVDYWGQGTLVTVSS VL DVQMTQSPSSLSASVGDRVTITCRASQDVYSYLNWYQQKPGKAVK 59 LLIYYTSRLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNIF PWTFGQGTKLEIK

[0099] In certain embodiments, the FRa x NaPi2b antibody construct comprises a NaPi2b antigenbinding domain having a VH sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the VH sequence of antibody 12A10 (SEQ ID NO: 58), and a VL sequence that is at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the VL sequence of antibody 12A10 (SEQ ID NO: 59). In some embodiments, the FRa x NaPi2b antibody construct comprises a NaPi2b antigen-binding domain having a VH sequence as set forth in SEQ ID NO: 58, and a VL sequence as set forth in SEQ ID NO: 59.

[0100] In certain embodiments, the FRa x NaPi2b antibody construct comprises a NaPi2b antigen-binding domain comprising the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) of the VH sequence as set forth in SEQ ID NO: 58, and the light chain CDRs (LCDR1, LCDR2 and LCDR3) of the VL sequence as set forth in SEQ ID NO: 59.

[0101] In certain embodiments, the FRa x NaPi2b antibody construct comprises a NaPi2b antigen-binding domain comprising the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) and the light chain CDRs (LCDR1, LCDR2 and LCDR3) of antibody 12A10, as shown in Table 6 herein. In some embodiments, the NaPi2b antigen-binding domain comprised by the FRa x NaPi2b antibody construct comprises:(a) a heavy chain CDR1 (HCDR1) having the sequence as set forth in any one of SEQ ID NOs: 173, 176, 178, 52 or 180, a heavy chain CDR2 (HCDR2) having the sequence as set forth in any one of SEQIDNOs: 174, 177, 179, 53 or 181, and a heavy chain CDR3(HCDR3) having the sequence as set forth in any one of SEQ ID NOs: 175, 54 or 182, and(b) a light chain CDR1 (LCDR1) having the sequence as set forth in any one of SEQ ID NOs: 183, 55 or 185, a light chain CDR2 (LCDR2) having the sequence as set forth in any one of SEQ ID NOs: 184, 56 or 186, and a light chain CDR3 (LCDR3) having the sequence as set forth in any one of SEQ ID NOs: 57 or 187.

[0102] In some embodiments, the FRa x NaPi2b antibody construct comprises a NaPi2b antigen-binding domain comprising heavy chain CDRs (HCDR1, HCDR2 and HCDR3) having the sequences as set forth in SEQ ID NOs: 52, 53 and 54, and light chain CDRs (LCDR1, LCDR2 and LCDR3) having the sequences as set forth in SEQ ID NOs: 55, 56 and 57.Scaffolds

[0103] In certain embodiments, the bispecific FRa x NaPi2b antibody constructs of the present disclosure comprise two or more antigen-binding domains operably linked to a scaffold. The antigen-binding domain(s) may be in one or a combination of formats, as described below (for example, scFvs, Fabs and / or sdAbs). Examples of suitable scaffolds are described in more detail below and include, but are not limited to, immunoglobulin Fc regions, albumin, albumin analogues and derivatives, heterodimerizing peptides (such as leucine zippers, heterodimer-forming “zipper” peptides derived from Jun and Fos, IgG CHI and CL domains or bamase-barstar toxins), cytokines, chemokines or growth factors. Other examples include antibodies based on the DOCK-AND-LOCK™ (DNL™) technology developed by IBC Pharmaceuticals, Inc. and Immunomedics, Inc. (see, for example, Chang, et al., 2007, Clin. Cancer Res., 13:5586s-5591s).

[0104] A scaffold may be a peptide, polypeptide, polymer, nanoparticle or other chemical entity. Where the scaffold is a polypeptide, an antigen-binding domain of the FRa x NaPi2b antibody construct that is operably linked to the polypeptide scaffold may be linked to either the N- or C-terminus of the polypeptide scaffold. FRa x NaPi2b antibody constructs comprising a polypeptide scaffold in which one or more of the antigen-binding domains are linked to a region other than the N- or C-terminus, for example, via the side chain of an amino acid with or without a linker, are also contemplated in certain embodiments.

[0105] In embodiments where the FRa x NaPi2b antibody construct comprises a scaffold that is a peptide or polypeptide, the antigen-binding domain(s) that are operably linked to the scaffold may be linked by genetic fusion or chemical conjugation. Typically, when the scaffold is a peptide or polypeptide, the antigen-binding domain(s) are linked to the scaffold by genetic fusion. In some embodiments, where the scaffold is a polymer or nanoparticle, the antigen-binding domain(s) may be linked to the scaffold by chemical conjugation.

[0106] A number of protein domains are known in the art that comprise selective pairs of two different polypeptides and may be used to form a scaffold. An example is leucine zipper domains such as Fos and Jun that selectively pair together (Kostelny, et al., J Immunol, 148:1547-53 (1992); Wranik, et al., J. Biol. Chem., 287: 43331-43339 (2012)). Other selectively pairing molecular pairs include, for example, the bamase-barstar pair (Deyev, et al., Nat Biotechnol, 21: 1486-1492 (2003)) and split fluorescent protein pairs (International Patent Publication No. WO 2011 / 135040).

[0107] Other examples of protein scaffolds include immunoglobulin Fc regions, albumin, albumin analogues 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, Muller 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).

[0108] For example, fusing antigen-binding moieties such as scFvs, diabodies or single chain diabodies to albumin has been shown to improve the serum half-life of the antigen-binding moieties (Muller et al., ibid.). Antigen-binding moieties may be fused at the N- and / or C-termini of albumin, optionally via a linker.

[0109] Derivatives of albumin in the form of heteromultimers that comprise two transporter polypeptides obtained by segmentation of an albumin protein such that the transporter polypeptides self-assemble to form quasi-native albumin have been described (see International Patent Publication Nos. WO 2012 / 116453 and WO 2014 / 012082). As a result of the segmentationof albumin, the heteromultimer includes four termini and thus can be fused to up to four different antigen-binding moieties, optionally via linkers.

[0110] In certain embodiments, the FRa x NaPi2b antibody construct may comprise a protein scaffold. In some embodiments, the FRa x NaPi2b antibody construct may comprise a protein scaffold that is based on an immunoglobulin Fc region, an albumin or an albumin analogue or derivative. In some embodiments, the FRa x NaPi2b antibody construct may comprise a protein scaffold that is based on an immunoglobulin Fc region, for example, an IgG Fc region.Fc Regions

[0111] The terms “Fc region,” “Fc” or “Fc domain” as used herein refer to a 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, numbering of amino acid residues in the Fc region and constant regions is according to the EU numbering system, also called 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).

[0112] In certain embodiments, the FRa x NaPi2b antibody construct of the present disclosure may comprise a scaffold that is 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.

[0113] An “Fc polypeptide” in the context of a dimeric Fc refers to one of the two polypeptides forming the dimeric Fc domain, i.e. a polypeptide comprising one or more C-terminal constant regions of an immunoglobulin heavy chain that is capable of stable self-association. When referring to the polypeptides forming a dimeric Fc region, the terms “first Fc polypeptide” and “second Fc polypeptide” may be used interchangeably provided that the Fc region comprises one first Fc polypeptide and one second Fc polypeptide.

[0114] An Fc region may comprise a CH3 domain or it may comprise both a CH3 and a CH2 domain. For example, in certain embodiments, an Fc polypeptide of a dimeric IgG Fc region may comprise an IgG CH2 domain sequence and an IgG CH3 domain sequence. In suchembodiments, the CH3 domain comprises two CH3 sequences, one from each of the two Fc polypeptides of the dimeric Fc region, and the CH2 domain comprises two CH2 sequences, one from each of the two Fc polypeptides of the dimeric Fc region.

[0115] In some embodiments, the FRa x NaPi2b antibody construct may comprise a scaffold that is based on an IgG Fc region. In some embodiments, the FRa x NaPi2b antibody construct may comprise a scaffold that is based on a human IgG Fc region. In some embodiments, the FRa x NaPi2b antibody construct may comprise a scaffold based on an IgGl Fc region. In some embodiments, the FRa x NaPi2b antibody construct may comprise a scaffold based on a human IgGl Fc region.

[0116] In certain embodiments, the FRa x NaPi2b antibody construct may comprise a scaffold based on an IgG Fc region, which is a homodimeric Fc region, comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence, and optionally a CH2 sequence and in which the amino acid sequences of the first and second Fc polypeptides are the same.

[0117] In certain embodiments, the FRa x NaPi2b antibody construct may comprise a scaffold based on an IgG Fc region, which is a heterodimeric Fc region, comprising a first Fc polypeptide and a second Fc polypeptide, each comprising a CH3 sequence, and optionally a CH2 sequence and in which the amino acid sequences of the first and second Fc polypeptides are different. In some embodiments, the FRa x NaPi2b antibody construct may comprise a scaffold based on an Fc region which comprises two CH3 sequences, at least one of which comprises one or more amino acid modifications. In some embodiments, the FRa x NaPi2b antibody construct may comprise a scaffold based on an Fc region which comprises two CH3 sequences and two CH2 sequences, at least one of the CH2 sequences comprising one or more amino acid modifications.

[0118] In some embodiments, the FRa x NaPi2b antibody construct may comprise a heterodimeric Fc region comprising a modified CH3 domain, where the modified CH3 domain is an asymmetrically modified CH3 domain comprising one or more asymmetric amino acid modifications. As used herein, an “asymmetric amino acid modification” refers to a modification, such as a substitution or an insertion, in which an amino acid at a specific position on a first CH3 or CH2 sequence is different to the amino acid on a second CH3 or CH2 sequence at the sameposition. These asymmetric amino acid modifications can be a result of modification of only one of the two amino acids at the same respective amino acid position on each sequence, or different modifications of both amino acids at the same respective position on each of the first and second CH3 or CH2 sequences. Each of the first and second CH3 and / or CH2 sequences of a heterodimeric Fc may comprise one or more than one asymmetric amino acid modification.

[0119] In some embodiments, the FRa x NaPi2b antibody construct may comprise a heterodimeric Fc comprising a modified CH3 domain, where the modified CH3 domain comprises one or more amino acid modifications that promote formation of the heterodimeric Fc over formation of a homodimeric Fc. In some embodiments, one or more of the amino acid modifications are asymmetric amino acid modifications.

[0120] Amino acid modifications that may be made to the CH3 domain of an Fc in order to promote formation of a heterodimeric Fc are known in the art and include, for example, those described in International Publication No. WO 96 / 027011 (“knobs into holes”), Gunasekaran et al., 2010, J Biol Chem, 285, 19637-46 (“electrostatic steering”), Davis et al., 2010, Prot Eng Des Sei, 23(4): 195-202 (strand exchange engineered domain (SEED) technology) and Labrijn et al., 2013, Proc Natl Acad Sci USA, 110(13): 5145-50 (Fab-arm exchange). Other examples include approaches combining positive and negative design strategies to produce stable asymmetrically modified Fc regions as described in International Publication Nos. WO 2012 / 058768 and WO 2013 / 063702. In certain embodiments, the FRa x NaPi2b antibody construct may comprise a scaffold based on a modified Fc region as described in International Publication No. WO 2012 / 058768 or WO 2013 / 063702.

[0121] Table 8 provides the amino acid sequence of the human IgGl Fc sequence (SEQ ID NO: 188), corresponding to amino acids 231 to 447 of the full-length human IgGl heavy chain. The CH3 sequence comprises amino acids 341-447 of the full-length human IgGl heavy chain. Also shown in Table 8 are CH3 domain amino acid modifications that promote formation of a heterodimeric Fc as described in International Publication Nos. WO 2012 / 058768 and WO 2013 / 063702.

[0122] In certain embodiments, the FRa x NaPi2b antibody construct may comprise a heterodimeric Fc scaffold having a modified CH3 domain comprising the modifications of any one of Variant 1, Variant 2, Variant 3, Variant 4 or Variant 5, as shown in Table 5.Table 8: Human IgGl Fc Sequence1and CH3 Domain Amino Acid Modifications Promoting Heterodimer Formation APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 188)Variant No Chain Mutations1 A L351Y_F405A_Y407VB T366L K392M T394W2 A L351Y_F405A_Y407VB T366L K392L T394W3 A T350V_L351 Y_F405A_Y407VB T350V_T366L_K392L_T394W4 A T350V_L351 Y_F405A_Y407VB T350V_T366L_K392M_T394W5 A T350V_L351 Y_S400E_F405A_Y407VB T350 V_T366L_N390R_K392M_T394W1Sequence from positions 231-447 (EU numbering)

[0123] In some embodiments, the FRa x NaPi2b antibody construct may comprise a scaffold based on an Fc region comprising two CH3 sequences and two CH2 sequences, 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 the Fc, such as receptors of the FcyRI, FcyRII and FcyRIII subclasses.

[0124] In some embodiments, the FRa x NaPi2b antibody construct may comprise a scaffold based on an IgG Fc having a modified CH2 domain, where the modification of the CH2 domain results in altered binding to one or more of the FcyRI, FcyRII and FcyRIII receptors.

[0125] A number of amino acid modifications to the CH2 domain that selectively alter the affinity of the Fc for different Fey receptors are known in the art. Amino acid modifications that result in increased binding and amino acid modifications that result in decreased binding can each be useful in certain indications. For example, increasing binding affinity of an Fc for FcyRIIIa (an activating receptor) may result in increased antibody dependent cell-mediated cytotoxicity (ADCC), which in turn results in increased lysis of the target cell. Decreased binding to FcyRIIb (an inhibitory receptor) likewise may be beneficial in some circumstances. In certain indications, a decrease in, or elimination of, ADCC and complement-mediated cytotoxicity (CDC) may be desirable. In such cases, modified CH2 domains comprising amino acid modifications that result in increased binding to FcyRIIb or amino acid modifications that decrease or eliminate binding of the Fc region to all of the Fey receptors (“knock-out” variants) may be useful.

[0126] Examples of amino acid modifications to the CH2 domain that alter binding of the Fc by Fey receptors include, but are not limited to, the following: S298A / E333A / K334A and S298A / E333A / K334A / K326A (increased affinity for FcyRIIIa) (Lu, et al., 2011, J Immunol Methods, 365(1-2): 132-41); F243L / R292P / Y300L / V305I / P396L (increased affinity for FcyRIIIa) (Stavenhagen, et al. 2007, Cancer Res 67(18): 8882-90);F243L / R292P / Y300L / L235V / P396L (increased affinity for FcyRIIIa) (Nordstrom JL, eta!., 2011, Breast Cancer Res, 13(6): R123); F243L (increased affinity for FcyRIIIa) (Stewart, et al., 2011, Protein Eng Des Sei., 24(9):671-8); S298A / E333A / K334A (increased affinity for FcyRIIIa) (Shields, et al., 2001, J Biol Chem, 276(9):6591-604); S239D / I332E / A330L and S239D / I332E (increased affinity for FcyRIIIa) (Lazar, et al., 2006, Proc Natl Acad Sci USA, 103(11):4005-10), and S239D / S267E and S267E / L328F (increased affinity for FcyRIIb) (Chu, et al., 2008, Mol Immunol, 45(15):3926-33). Various amino acid modifications to the CH2 domain that alter binding of the Fc by FcyRIIb are described in International Publication No. WO 2021 / 232162. Additional modifications that affect Fc binding to Fey receptors are described in Therapeutic Antibody Engineering (Strohl & Strohl, Woodhead Publishing series in Biomedicine No 11, ISBN 1 907568 379, Oct 2012, page 283).

[0127] In certain embodiments, the FRa x NaPi2b antibody construct may comprise a scaffold based on an IgG Fc having a modified CH2 domain, in which the modified CH2 domaincomprises one or more amino acid modifications that result in decreased or eliminated binding of the Fc region to all of the Fey receptors (i.e. a “knock-out” variant).

[0128] Various publications describe strategies that have been used to engineer antibodies to produce “knock-out” variants (see, for example, 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 reduction of effector function through modification of glycosylation, use of IgG2 / IgG4 scaffolds, or the introduction of mutations in the hinge or CH2 domain of the Fc (see also, U. S. Patent Application Publication No. 2011 / 0212087, International Publication No. WO 2006 / 105338, U. S. Patent Application Publication No. 2012 / 0225058, U. S. Patent Application Publication No. 2012 / 0251531 and Strop eta!., 2012, J. Mol. Biol., 420: 204-219).

[0129] Examples of mutations that may be introduced into the hinge or CH2 domain to produce a “knock-out” variant include the amino acid modifications L234A / L235A and L234A / L235A / D265S.

[0130] In certain embodiments, the FRa x NaPi2b antibody constructs described herein may comprise a scaffold based on an IgG Fc in which native glycosylation has been modified. As is known in the art, glycosylation of an Fc may 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 an agly coslated Fc that lacks all effector function (Bolt et al., 1993, Eur. J. Immunol., 23:403-411; Tao & Morrison, 1989, J. Immunol., 143:2595-2601).

[0131] Conversely, removal of fucose from heavy chain N297-linked oligosaccharides has been shown to enhance ADCC, based on improved binding to FcyRIIIa (see, for example, Shields et al., 2002, J Biol Chem., 277:26733-26740, and Niwa et al., 2005, J. Immunol. Methods, 306:151-160). Such low fucose antibodies may 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, Lee 13, that has a reduced ability to attach fucose to N297-linked carbohydrates (International Publication No. WO 03 / 035835), or in other cells that generate afucosylated antibodies (see, for example, Li et al., 2006, NatBiotechnol, 24:210-215; Shields et al., 2002, ibid, and Shinkawa et al., 2003, J. Biol. Chem., 278:3466-3473). In addition, International Publication No. WO 2009 / 135181 describes the addition of fucose analogues to culture medium during antibody production to inhibit incorporation of fucose into the carbohydrate on the antibody.

[0132] Other methods of producing antibodies with little or no fucose on the Fc glycosylation site (N297) are well known in the art. For example, the GlymaX® technology (ProBioGen AG) (see von Horsten et al., 2010, Glycobiology, 20(12):1607-1618 and U. S. Patent No. 8,409,572).

[0133] Other glycosylation variants include those with bisected oligosaccharides, for example, variants in which a biantennary oligosaccharide attached to the Fc region of the antibody is bisected by N-acetylglucosamine (GlcNAc). Such glycosylation variants may have reduced fucosylation and / or improved ADCC function (see, for example, International Publication No. WO 2003 / 011878, U. S. Patent No. 6,602,684 and US Patent Application Publication No.2005 / 0123546). Useful glycosylation variants also include those having at least one galactose residue in the oligosaccharide attached to the Fc region, which may have improved CDC function (see, for example, International Publication Nos. WO 1997 / 030087, WO 1998 / 58964 and WO 1999 / 22764).Formats of Bispecific FRa x NaPilb Antibody Constructs

[0134] The bispecific FRa x NaPi2b antibody constructs described herein comprise at least one FRa antigen-binding domain and at least one NaPi2b antigen-binding domain. In certain embodiments, the FRa x NaPi2b antibody constructs may further comprise a scaffold and the antigen-binding domains are linked directly or indirectly (for example, via a linker or via one of the antigen-binding domains) to the scaffold. In certain embodiments, the FRa x NaPi2b antibody constructs are bivalent (i.e. comprise two antigen-binding domains), trivalent (i.e. comprise three antigen-binding domains) or tetravalent (i.e. comprise four antigen-binding domains).

[0135] In certain embodiments, the FRa x NaPi2b antibody constructs are bivalent and comprise one FRa antigen-binding domain and one NaPi2b antigen-binding domain. In some embodiments, the FRa x NaPi2b antibody constructs are trivalent and comprise one FRa antigen-binding domain and two NaPi2b antigen-binding domains, or two FRa antigen-binding domains and one NaPi2b antigen-binding domain. In some embodiments, the FRa x NaPi2b antibody constructs are tetravalent and comprise two FRa antigen-binding domains and two NaPi2b antigen-binding domains, or one FRa antigen-binding domain and three NaPi2b antigen-binding domains, or three FRa antigen-binding domains and one NaPi2b antigen-binding domain.

[0136] In certain embodiments, the FRa x NaPi2b antibody constructs may lack a scaffold and thus comprise two or more antigen-binding domains optionally operably linked by one or more linkers. In such antibody constructs, the antigen-binding domains may be in the form of scFvs, Fabs, sdAbs, or a combination thereof. For example, using scFvs as the antigen-binding domains, formats such as a tandem scFv ((scFv)2 or taFv) may be constructed, in which the scFvs are connected together by a flexible linker. scFvs may also be used to construct diabody formats, which comprise two scFvs connected by a short linker (usually about 5 amino acids in length). The restricted length of the linker results in dimerization of the scFvs in a head-to-tail manner. In any of the preceding formats, the scFvs may be further stabilized by inclusion of an interdomain disulfide bond. For example, a disulfide bond may be introduced between VL and VH through introduction of an additional cysteine residue in each chain (for example, at position 44 in VH and position 100 in VL) (see, for example, Fitzgerald etal., 1997, Protein Engineering, 10: 1221-1225), or a disulfide bond may be introduced between two VHs to provide a construct having a DART format (see, for example, Johnson et al., 2010, J Mol. BioL, 399:436-449).

[0137] Similarly, formats comprising two sdAbs, such as VHs or VHHs, connected together through a suitable linker may be employed in some embodiments. Other examples of antibody construct formats that lack a scaffold include those based on Fab fragments, for example, Fab? and F(ab’)2 formats, in which the Fab fragments are connected through a linker or an IgG hinge region.

[0138] Combinations of antigen-binding domains in different forms may also be employed to generate alternative scaffold-less formats. For example, an scFv or a sdAb may be fused to the C-terminus of either or both of the light and heavy chain of a Fab fragment resulting in a bivalent (Fab-scFv / sdAb) construct.

[0139] In certain embodiments, the FRa x NaPi2b antibody constructs may be in a format that is based on an immunoglobulin (Ig). In certain embodiments, the FRa x NaPi2b antibody constructs may be based on an IgG class immunoglobulin, for example, an IgGl, IgG2, IgG3 or IgG4 immunoglobulin. In some embodiments, the FRa x NaPi2b antibody constructs may be based on an IgGl immunoglobulin. In the context of the present disclosure, when an antibody construct is based on a specified immunoglobulin isotype, it is meant that antibody construct comprises all or a portion of the constant region of the specified immunoglobulin isotype. For example, an FRa x NaPi2b antibody construct based on a given Ig isotype may comprise an Ig scaffold to which at least one antigen-binding domain is operably linked, where the scaffold comprises an Fc region from the given isotype and optionally an Ig hinge region from the same or a different isotype. It is to be understood that the FRa x NaPi2b antibody constructs may also comprise hybrids of isotypes and / or subclasses in some embodiments. It is also to be understood that the Fc region and / or hinge region may optionally be modified to impart one or more desirable functional properties as is known in the art.

[0140] In some embodiments, the FRa x NaPi2b antibody constructs may be derived from two or more immunoglobulins that are from different species, for example, the FRa x NaPi2b antibody construct may be a chimeric antibody or a humanized antibody. The terms “chimeric antibody” and “humanized antibody” both refer generally to antibodies that combine immunoglobulin regions or domains from more than one species.

[0141] A “chimeric antibody” typically comprises at least one variable domain from a nonhuman antibody, such as a rabbit or rodent (for example, murine) antibody, and at least one constant domain from a human antibody. The human constant domain of a chimeric antibody need not be of the same isotype as the non-human constant domain it replaces. Chimeric antibodies are discussed, for example, in Morrison etal., 1984, roc. Natl. Acad. Sci. USA, 81:6851-55, and U. S. Patent No. 4,816,567.

[0142] A “humanized antibody” is a type of chimeric antibody that contains minimal sequence derived from a non-human antibody. Generally, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDR) of the recipient are replaced by residues from a hypervariable region (CDR) of a non-human species(donor antibody), such as mouse, rat, rabbit or non-human primate, having the desired specificity and affinity for a target antigen. This technique for creating humanized antibodies is often referred to as “CDR grafting.”

[0143] In some instances, additional modifications may be made to a humanized antibody to further refine antibody performance. For example, framework region (FR) residues of the human immunoglobulin may be replaced by corresponding non-human residues, or the humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. In general, a variable domain in a humanized antibody will comprise 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 Jones, etal., 1986, Nature, 321:522-525; Riechmann, et al., 1988, Nature, 332:323-329, and Presta, 1992, Curr. Op. Struct. Biol., 2:593-596, for example.

[0144] A number of approaches are known in the art for selecting the most appropriate human frameworks into which to graft the non-human CDRs. Early approaches used a limited subset of well-characterised human antibodies, irrespective of the sequence identity to the non-human antibody providing the CDRs (the “fixed frameworks” approach). More recent approaches 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” approach). An alternative approach is to select fragments of the framework sequences within each light or heavy chain variable region from several different human antibodies. CDR-grafting may in some cases result in a partial or complete loss of affinity of the grafted molecule for its target antigen. In such cases, affinity can be restored by back-mutating some of the residues of human origin to the corresponding non-human ones. Methods for preparing humanized antibodies by these approaches are well-known in the art (see, for example, Tsurushita & Vasquez, 2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science (USA); Jones et al., 1986, Nature, 321:522-525; Riechmann et al., 1988, Nature, 332:323-329; Presta et al., 1997, Cancer Res, 57(20):4593-4599).

[0145] Alternatively, or in addition to, these traditional approaches, more recent technologies may be employed to further reduce the immunogenicity of a CDR-grafted humanizedantibody. For example, frameworks based on human germline sequences or consensus sequences may be employed as acceptor human frameworks rather than human frameworks with somatic mutation(s). Another technique that aims to reduce the potential immunogenicity of non-human CDRs is to graft only specificity-determining residues (SDRs). In this approach, only the minimum CDR residues required for antigen-binding activity (the “SDRs”) are grafted into a human germline framework. This method improves the “humanness” (i.e. the similarity to human germline sequence) of the humanized antibody and thus may help reduce the risk of immunogenicity of the variable region. These techniques have been described in various publications (see, for example, Almagro & Fransson, 2008, Front Biosci, 13:1619-1633; Tan, et al., 2002, J Immunol, 169: 1119-1125; Hwang, et al., 2005, Methods, 36:35-42; Pelat, et al., 2008, J Mol Biol, 384:1400-1407; Tamura, et al., 2000, J Immunol, 164:1432-1441; Gonzales, et al., 2004, Mol Immunol, 1:863-872, and Kashmiri, etal., 2005, Methods, 36:25-34).

[0146] In certain embodiments, the FRa x NaPi2b antibody constructs are bivalent, trivalent or tetravalent and comprise a scaffold based on an immunoglobulin (IgG) Fc region, where the Fc region comprises a first Fc polypeptide and a second Fc polypeptide. In such embodiments in which the FRa x NaPi2b antibody constructs comprise a scaffold based on an immunoglobulin (IgG) Fc region, such as an IgGl or IgG4 Fc region, the Fc region will typically comprise a heterodimeric Fc comprising amino acid substitutions in the CH3 domain that promote formation of a heterodimeric Fc over a homodimeric Fc, as described above. In certain embodiments in which the FRa x NaPi2b antibody constructs comprise a scaffold based on an IgGl or IgG4 Fc region, the Fc region comprises a modified CH3 domain comprising the amino acid substitutions of any one of Variant 1, Variant 2, Variant 3, Variant 4 or Variant 5, as shown in Table 8.

[0147] In certain embodiments, the FRa x NaPi2b antibody constructs comprise at least one FRa antigen-binding domain in Fab format and at least one NaPi2b antigen-binding domain in Fab format. In some embodiments in which the FRa x NaPi2b antibody constructs comprise at least one FRa antigen-binding domain in Fab format and at least one NaPi2b antigen-binding domain in Fab format, the CHI and CL domains of the Fab FRa antigen-binding domain(s) and the CHI and CL domains of the Fab NaPi2b antigen-binding domain(s) may comprise sets of mutations to drive the correct pairing between the heavy and light chains of the FRa antigen-binding domain and between the heavy and light chains of the NaPi2b antigen-binding domain. Examples of sets of mutations that may be used in this context include those described in International Patent Publication Nos. WO 2014 / 082179, WO 2015 / 181805 and WO 2017 / 059551. In certain embodiments, the FRa x NaPi2b antibody constructs comprise sets of amino acid mutations to drive the correct pairing between the heavy and light chains of a Fab FRa antigenbinding domain and between the heavy and light chains of a Fab NaPi2b antigen-binding domain. In some embodiments, the FRa x NaPi2b antibody constructs comprise sets of amino acid mutations to drive the correct pairing between the heavy and light chains of a Fab FRa antigenbinding domain and between the heavy and light chains of a NaPi2b antigen-binding domain, where the sets of amino acid mutations are Design 4478 or Design 2739 as shown in Table 9.Table 9: Sets of CH1 / CL Amino Acid Mutations to Promote Correct PairingDesign Chain1Mutations24478 CH1 HCA A139W / L143E / K145T / Q179ECL LCA F 116A / Q 124R / L 135 V / T 178RCH1 HCB Q179RCL LCB Q 124E / L 135 W / T 178E / T 180E2739 CH1 HCA L143RCL LCA Q124E / V133ECH1 HCB L143E / K145T / Q179ECL LCB Q124R / T178R1HCA=heavy chain A; LCA=light chain A; HCB=heavy chain B, and LCB=heavy chain B, where HCA pairs with LCA, and HCB pairs with LCB.2Numbering of amino acid positions according to Kabat

[0148] As would be appreciated by one of skill in the art, when the FRa x NaPi2b antibody constructs are bivalent, trivalent or tetravalent and comprise a scaffold based on an immunoglobulin (IgG) Fc region, the FRa x NaPi2b antibody constructs may be constructed in various formats. Exemplary, non-limiting formats include those described below and shown in Fig. 1 and Fig. 2.

[0149] In some embodiments, the FRa x NaPi2b antibody constructs are bivalent and comprise two antigen-binding domains, where (a) both antigen-binding domains are in Fab format,or (b) both antigen-binding domains are in scFv format, or (c) one antigen-binding domain is in Fab format and the other is in scFv format. In some embodiments, in the bivalent FRa x NaPi2b antibody constructs, one antigen-binding domain is linked to the N-terminus of one Fc polypeptide and the other antigen-binding domain is linked to the N-terminus of the other Fc polypeptide.

[0150] In some embodiments, the FRa x NaPi2b antibody constructs are trivalent and comprise three antigen-binding domains, where (a) all three antigen-binding domains are in Fab format, or (b) all three antigen-binding domains are in scFv format, or (c) two antigen-binding domains are in Fab format and one antigen-binding domain is in scFv format, or (d) one antigenbinding domain is in Fab format and two antigen-binding domains are in scFv format. In some embodiments, in the trivalent FRa x NaPi2b antibody constructs, a first antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a second antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, and a third antigen-binding domain is linked to the C-terminus of one of the Fc polypeptides. In some embodiments, in the trivalent FRa x NaPi2b antibody constructs, a first antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a second antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, and a third antigen-binding domain is linked to the N-terminus of either the first or the second antigen-binding domain. In some embodiments, in the trivalent FRa x NaPi2b antibody constructs, a first antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a second antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, and a third antigen-binding domain is linked to the C-terminus of either the first or the second antigen-binding domain.

[0151] In some embodiments, the FRa x NaPi2b antibody constructs are tetravalent and comprise four antigen-binding domains, where (a) all four antigen-binding domains are in Fab format, or (b) all four antigen-binding domains are in scFv format, or (c) two antigen-binding domains are in Fab format and two antigen-binding domains are in scFv format, or (d) one antigenbinding domain is in Fab format and three antigen-binding domains are in scFv format, or (e) three antigen-binding domains are in Fab format and one antigen-binding domain is in scFv format. In some embodiments, in the tetravalent FRa x NaPi2b antibody constructs, a first antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a second antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, a third antigen-binding domain is linked tothe C-terminus of one of the Fc polypeptides and a fourth antigen-binding domain is linked to the C-terminus of the other Fc polypeptide. In some embodiments, in the tetravalent FRa x NaPi2b antibody constructs, a first antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a second antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, a third antigen-binding domain is linked to the C-terminus of one of the Fc polypeptides and a fourth antigen-binding domain is linked to the N-terminus of either the first or the second antigen-binding domain. In some embodiments, in the tetravalent FRa x NaPi2b antibody constructs, a first antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a second antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, a third antigen-binding domain is linked to the C-terminus of one of the Fc polypeptides and a fourth antigen-binding domain is linked to the C-terminus of either the first or the second antigen-binding domain. In some embodiments, in the tetravalent FRa x NaPi2b antibody constructs, a first antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a second antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, a third antigen-binding domain is linked to the N-terminus of either the first or the second antigen-binding domain and a fourth antigen-binding domain is linked to the N-terminus of the other of the first or second antigen-binding domain. In some embodiments, in the tetravalent FRa x NaPi2b antibody constructs, a first antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a second antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, a third antigen-binding domain is linked to the C-terminus of either the first or the second antigen-binding domain and a fourth antigen-binding domain is linked to the N-terminus of the other of the first or second antigen-binding domain. In some embodiments, in the tetravalent FRa x NaPi2b antibody constructs, a first antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a second antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, a third antigen-binding domain is linked to the C-terminus of either the first or the second antigen-binding domain and a fourth antigen-binding domain is linked to the C-terminus of the other of the first or second antigen-binding domain.Fab Formats

[0152] In certain embodiments, the FRa x NaPi2b antibody constructs are bivalent, trivalent or tetravalent and comprise a scaffold based on an immunoglobulin (IgG) Fc region,where the Fc region comprises a first Fc polypeptide and a second Fc polypeptide, and in which the two or more antigen-binding domains comprised by the FRa x NaPi2b antibody constructs are in Fab format. Non-limiting examples are shown in Fig. 1A-1E, where Target A is one of FRa or NaPi2b, and Target B is the other of FRa or NaPi2b.

[0153] In certain embodiments, the FRa x NaPi2b antibody constructs are bivalent and comprise one FRa antigen-binding domain in Fab format and one NaPi2b antigen-binding domain in Fab format, in which the FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide and the NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide (see Fig. 1 A).

[0154] In certain embodiments, the FRa x NaPi2b antibody constructs are trivalent and comprise two FRa antigen-binding domains in Fab format and one NaPi2b antigen-binding domain in Fab format, in which a first FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide, the NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide and a second FRa antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain (see Fig. IB). In certain embodiments, the FRa x NaPi2b antibody constructs are trivalent and comprise one FRa antigen-binding domain in Fab format and two NaPi2b antigen-binding domains in Fab format, in which the FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide and a second NaPi2b antigen-binding domain is linked to the N-terminus of the first NaPi2b antigen-binding domain (see Fig. IB).

[0155] In certain embodiments, the FRa x NaPi2b antibody constructs are trivalent and comprise two FRa antigen-binding domains in Fab format and one NaPi2b antigen-binding domain in Fab format, in which a first FRa antigen-binding domain is linked to the N-terminus of a first Fc polypeptide, a second FRa antigen-binding domain is linked to the C-terminus of the first Fc polypeptide, and the NaPi2b antigen-binding domain is linked to the N-terminus of the other (second) Fc polypeptide (see Fig. 1C). In certain embodiments, the FRa x NaPi2b antibody constructs are trivalent and comprise one FRa antigen-binding domain in Fab format and two NaPi2b antigen-binding domains in Fab format, in which the FRa antigen-binding domain is linked to the N-terminus of a first Fc polypeptide, a first NaPi2b antigen-binding domain is linkedto the N-terminus of the other (second) Fc polypeptide, and a second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide (see Fig. 1C).

[0156] In certain embodiments, the FRa x NaPi2b antibody constructs are tetravalent and comprise two FRa antigen-binding domains in Fab format and two NaPi2b antigen-binding domains in Fab format, in which a first FRa antigen-binding domain is linked to the N-terminus of one (first) Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other (second) Fc polypeptide, a second FRa antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain and a second NaPi2b antigen-binding domain is linked to the N-terminus of the first NaPi2b antigen-binding domain (see Fig. ID).

[0157] In certain embodiments, the FRa x NaPi2b antibody constructs are tetravalent and comprise two FRa antigen-binding domains in Fab format and two NaPi2b antigen-binding domains in Fab format, in which a first FRa antigen-binding domain is linked to the N-terminus of a first Fc polypeptide, a second FRa antigen-binding domain is linked to the C-terminus of the first Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other (second) Fc polypeptide, and a second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide (see Fig. IE).

[0158] In certain embodiments, the FRa x NaPi2b antibody constructs are tetravalent and comprise two FRa antigen-binding domains in Fab format and two NaPi2b antigen-binding domains in Fab format, in which a first FRa antigen-binding domain is linked to the N-terminus of one (first) Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other (second) Fc polypeptide, a second FRa antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain and a second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide. In certain embodiments, the FRa x NaPi2b antibody constructs are tetravalent and comprise two FRa antigen-binding domains in Fab format and two NaPi2b antigen-binding domains in Fab format, in which a first FRa antigen-binding domain is linked to the N-terminus of one (first) Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other (second) Fc polypeptide, a second FRa antigenbinding domain is linked to the C-terminus of the first Fc polypeptide and a second NaPi2b antigen-binding domain is linked to the N-terminus of the first NaPi2b antigen-binding domain.

[0159] In certain embodiments in FRa x NaPi2b antibody constructs having the format as shown in any one of Fig. 1 A, IB, 1C, ID or IE, the one or more NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10, and the one or more FRa antigen-binding domains comprise the CDR sequences of antibody 76.

[0160] In certain embodiments in FRa x NaPi2b antibody constructs having the format as shown in any one of Fig. 1 A, IB, 1C, ID or IE, the one or more NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10, and the one or more FRa antigen-binding domains comprise the CDR sequences of antibody 2L16.

[0161] In certain embodiments in FRa x NaPi2b antibody constructs having the format as shown in any one of Fig. 1 A, IB, 1C, ID or IE, the one or more NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10, and the one or more FRa antigen-binding domains comprise the CDR sequences of antibody 10L18.

[0162] In certain embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. 1 A comprising a NaPi2b antigen-binding domain in Fab format and a FRa antigenbinding domain in Fab format, in which the NaPi2b antigen-binding domain comprises the CDR sequences of antibody 12A10. In some embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. 1 A, in which the NaPi2b antigen-binding domain comprises the CDR sequences of antibody 12A10 and the FRa antigen-binding domains comprises the CDR sequences of any one of antibody 76, antibody 2L16 or antibody 10L18.

[0163] In certain embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. IB comprising two FRa antigen-binding domains and one NaPi2b antigen-binding domain, in which the NaPi2b antigen-binding domain comprises the CDR sequences of antibody 12A10. In some embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. IB comprising two FRa antigen-binding domains and one NaPi2b antigen-binding domain, in which the NaPi2b antigen-binding domain comprises the CDR sequences of antibody 12A10 and the two FRa antigen-binding domains comprise the CDR sequences of any one of antibody 76, antibody 2L16 or antibody 10L18. In some embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. IB comprising two FRa antigen-binding domains and one NaPi2b antigen-binding domain, in which the NaPi2b antigen-binding domain comprises the CDRsequences of antibody 12A10 and the two FRa antigen-binding domains comprise the CDR sequences of antibody 76 or antibody 10L18.

[0164] In certain embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. IB comprising one FRa antigen-binding domain and two NaPi2b antigen-binding domains, in which the NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10. In some embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. IB comprising one FRa antigen-binding domain and two NaPi2b antigen-binding domains, in which the NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10 and the FRa antigen-binding domain comprises the CDR sequences of any one of antibody 76, antibody 2L16 or antibody 10L18. In some embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. IB comprising one FRa antigen-binding domain and two NaPi2b antigen-binding domains, in which the NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10 and the FRa antigen-binding domain comprises the CDR sequences of antibody 76.

[0165] In certain embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. 1C comprising two FRa antigen-binding domains and one NaPi2b antigen-binding domain, in which the NaPi2b antigen-binding domain comprises the CDR sequences of antibody 12A10. In some embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. 1C comprising two FRa antigen-binding domains and one NaPi2b antigen-binding domain, in which the NaPi2b antigen-binding domain comprises the CDR sequences of antibody 12A10 and the two FRa antigen-binding domains comprise the CDR sequences of any one of antibody 76, antibody 2L16 or antibody 10L18. In some embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. 1C comprising two FRa antigen-binding domains and one NaPi2b antigen-binding domain, in which the NaPi2b antigen-binding domain comprises the CDR sequences of antibody 12A10 and the two FRa antigen-binding domains comprise the CDR sequences of antibody 10L18.

[0166] In certain embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. ID comprising two FRa antigen-binding domains and two NaPi2b antigen-binding domains, in which the two NaPi2b antigen-binding domains comprise the CDR sequences ofantibody 12A10. In some embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. ID comprising two FRa antigen-binding domains and two NaPi2b antigen-binding domains, in which the two NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10 and the two FRa antigen-binding domains comprise the CDR sequences of any one of antibody 76, antibody 2L16 or antibody 10L18. In some embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. ID comprising two FRa antigen-binding domains and two NaPi2b antigen-binding domains, in which the two NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10 and the two FRa antigen-binding domains comprise the CDR sequences of antibody 76 or antibody 10L18.

[0167] In certain embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. IE comprising two FRa antigen-binding domains and two NaPi2b antigen-binding domains, in which the two NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10. In some embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. IE comprising two FRa antigen-binding domains and two NaPi2b antigen-binding domains, in which the two NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10 and the two FRa antigen-binding domains comprise the CDR sequences of any one of antibody 76, antibody 2L16 or antibody 10L18. In some embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. IE comprising two FRa antigen-binding domains and two NaPi2b antigen-binding domains, in which the two NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10 and the two FRa antigen-binding domains comprise the CDR sequences of antibody 10L18.

[0168] In certain embodiments, the FRa x NaPi2b antibody constructs are bivalent and comprise one FRa antigen-binding domain in Fab format and one NaPi2b antigen-binding domain in Fab format, in which the FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide and the NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide (see Fig. 1A), and in which the FRa antigen-binding domain comprises the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 10, 11 and 7, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 18, 19 and 17; and the NaPi2b antigen-binding domain comprises the heavy chain CDR sequences(HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 52, 53 and 54, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 55, 56 and 57.

[0169] In certain embodiments, the FRa x NaPi2b antibody constructs are trivalent and comprise two FRa antigen-binding domains in Fab format and one NaPi2b antigen-binding domain in Fab format, in which a first FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide, the NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide and a second FRa antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain (see Fig. IB), and in which the NaPi2b antigen-binding domain comprises the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 52, 53 and 54, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 55, 56 and 57; and both FRa antigen-binding domains comprise (a) the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 10, 11 and 7, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 18, 19 and 17, or (b) the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 36, 37 and 38, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 39, 40 and 41.

[0170] In certain embodiments, the FRa x NaPi2b antibody constructs are trivalent and comprise one FRa antigen-binding domain in Fab format and two NaPi2b antigen-binding domains in Fab format, in which the FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide and a second NaPi2b antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain (see Fig. IB), and in which the FRa antigen-binding domain comprises the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 10, 11 and 7, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 18, 19 and 17; and both NaPi2b antigen-binding domains comprise the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 52, 53 and 54, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 55, 56 and 57.

[0171] In certain embodiments, the FRa x NaPi2b antibody constructs are trivalent and comprise two FRa antigen-binding domains in Fab format and one NaPi2b antigen-binding domain in Fab format, in which a first FRa antigen-binding domain is linked to the N-terminus of a first Fc polypeptide, a second FRa antigen-binding domain is linked to the C-terminus of the first Fc polypeptide, and the NaPi2b antigen-binding domain is linked to the N-terminus of the other (second) Fc polypeptide (see Fig. 1C), and in which both FRa antigen-binding domains comprise the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 36, 37 and 38, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 39, 40 and 41; and the NaPi2b antigen-binding domain comprises the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 52, 53 and 54, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 55, 56 and 57.

[0172] In certain embodiments, the FRa x NaPi2b antibody constructs are tetravalent and comprise two FRa antigen-binding domains in Fab format and two NaPi2b antigen-binding domains in Fab format, in which a first FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, a second FRa antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain and a second NaPi2b antigen-binding domain is linked to the N-terminus of the first NaPi2b antigen-binding domain (see Fig. ID), and in which both NaPi2b antigen-binding domains comprise the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 52, 53 and 54, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 55, 56 and 57; and both FRa antigen-binding domains comprise (a) the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 10, 11 and 7, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 18, 19 and 17, or (b) the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 36, 37 and 38, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 39, 40 and 41.

[0173] In certain embodiments, the FRa x NaPi2b antibody constructs are tetravalent and comprise two FRa antigen-binding domains in Fab format and two NaPi2b antigen-binding domains in Fab format, in which a first FRa antigen-binding domain is linked to the N-terminusof a first Fc polypeptide, a second FRa antigen-binding domain is linked to the C-terminus of the first Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other (second) Fc polypeptide, and a second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide (see Fig. IE), and in which both NaPi2b antigen-binding domains comprise the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 52, 53 and 54, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 55, 56 and 57; and both FRa antigen-binding domains comprise the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 36, 37 and 38, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 39, 40 and 41.Chain Composition of Fab Formats

[0174] In those embodiments in which the FRa x NaPi2b antibody constructs are in Fab Format, the Fc region of the antibody construct will typically include a hinge sequence, for example an IgG hinge sequence or a modified version thereof. Non-limiting examples of hinge sequences and modified hinge sequences are described herein. The Fab Format antibody construct may also comprise one or more additional hinge sequences and / or linkers. Linkers are typically employed to join different domains of the antibody construct together, for example, to join an Fc polypeptide to a Fab domain, or to join two Fab domains. Non-limiting examples of linkers are described herein. Non-limiting examples of heavy and light chain compositions for various Fab Format antibody constructs are described below. Heavy and light chains are designated “Hl,” “H2,” “LI” and “L2” solely for the purpose of indicating which heavy chain and light chain(s) form heavy-light chain pairs, i.e. Hl pairs with LI, and H2 pairs with L2. The numbering “1” and “2” is therefore interchangeable, thus in some instances chains designated Hl, LI, H2 and L2 in the following embodiments may be designated H2, L2, Hl and LI, respectively.

[0175] In certain embodiments, in antibody constructs having the format shown in Fig. 1 A, the two heavy chains (Hl and H2) and the two light chains (LI and L2) that make up the antibody construct have the following structures:Hl: VHa-CHl-hinge-CH2-CH3LI: VLa-CLH2: VHb-CHl-hinge-CH2-CH3L2: VLb-CL

[0176] Where VHaand VLaare the VH and VL domains of one antigen-binding domain (either FRa or NaPi2b), VHband VLbare the VH and VL domains of the other antigen-binding domain, and “hinge” is an IgG hinge sequence or modified version thereof.

[0177] In certain embodiments, in antibody constructs having the format shown in Fig. IB, the two heavy chains (Hl and H2) and the two light chains (LI and L2) that make up the antibody construct have the following structures:HL VHa-CHl-hinge-CH2-CH3LI: VLa-CLH2: VHb-CHl -hinge-linker- VHb-CHl-hinge-CH2-CH3L2 x2: VLb-CL

[0178] Where VHaand VLaare the VH and VL domains of one antigen-binding domain (either FRa or NaPi2b), VHband VLbare the VH and VL domains of the other antigen-binding domain, each “hinge” is independently an IgG hinge sequence or modified version thereof, and “linker” is a linker sequence.

[0179] In some embodiments, for antibody constructs having the format shown in Fig. IB, the H2 chain may lack one of the hinge or the linker and have the structure H2a or H2b:H2a: VHb-CHl -hinge- VHb-CHl-hinge-CH2-CH3H2b: VHb-CHl -linker- VHb-CHl -hinge-CH2-CH3

[0180] In certain embodiments, in antibody constructs having the format shown in Fig. 1C, the two heavy chains (Hl and H2) and the two light chains (LI and L2) that make up the antibody construct have the following structures:Hl: VHa-CHl-hinge-CH2-CH3LI: VLa-CLH2: VHb-CH 1 -hinge-CH2-CH3 -linker- VHb-CH 1 -hingeL2 x2: VLb-CL

[0181] Where VHaand VLaare the VH and VL domains of one antigen-binding domain (either FRa or NaPi2b), VHband VLbare the VH and VL domains of the other antigen-binding domain, each “hinge” is independently an IgG hinge sequence or modified version thereof, and “linker” is a linker sequence.

[0182] In some embodiments, for antibody constructs having the format shown in Fig. 1C, the H2 chain may lack the C-terminal hinge and have the structure H2c:H2c: VHb-CHl-hinge-CH2-CH3 -linker- VHb-CHl

[0183] In certain embodiments, in antibody constructs having the format shown in Fig. ID, the two heavy chains (Hl and H2) and the two light chains (LI and L2) that make up the antibody construct have the following structures:Hl: VHa-CHl -hinge-linker- VHa-CHl-hinge-CH2-CH3LI x2: VLa-CLH2: VHb-CHl -hinge-linker- VHb-CHl-hinge-CH2-CH3L2 x2: VLb-CL

[0184] Where VHaand VLaare the VH and VL domains of one antigen-binding domain (either FRa or NaPi2b), VHband VLbare the VH and VL domains of the other antigen-binding domain, each “hinge” is independently an IgG hinge sequence or modified version thereof, and “linker” is a linker sequence.

[0185] In some embodiments, for antibody constructs having the format shown in Fig. ID, the Hl chain may lack one of the hinge or the linker and have the structure Hl a or Hlb:Hla: VHa-CHl -hinge- VHa-CHl-hinge-CH2-CH3Hlb: VHa-CHl -linker- VHa-CHl-hinge-CH2-CH3

[0186] In some embodiments, for antibody constructs having the format shown in Fig. ID, the H2 chain may lack one of the hinge or the linker and have the structure H2d or H2e:H2d: VHb-CHl -hinge- VHb-CHl-hinge-CH2-CH3H2e: VHb-CHl -linker- VHb-CHl-hinge-CH2-CH3

[0187] In certain embodiments, in antibody constructs having the format shown in Fig. IE, the two heavy chains (Hl and H2) and the two light chains (LI and L2) that make up the antibody construct have the following structures:Hl: VHa-CHl-hinge-CH2-CH3 -linker- VHa-CHl -hingeLI x2: VLa-CLH2: VHb-CH 1 -hinge-CH2-CH3 -linker- VHb-CH 1 -hingeL2 x2: VLb-CL

[0188] Where VHaand VLaare the VH and VL domains of one antigen-binding domain (either FRa or NaPi2b), VHband VLbare the VH and VL domains of the other antigen-binding domain, each “hinge” is independently an IgG hinge sequence or modified version thereof, and “linker” is a linker sequence.

[0189] In some embodiments, for antibody constructs having the format shown in Fig. IE, the H2 chain may lack the C-terminal hinge and have the structure Hlc:Hlc: VHa-CHl-hinge-CH2-CH3 -linker- VHa-CHl

[0190] In some embodiments, for antibody constructs having the format shown in Fig. IE, the H2 chain may lack the C-terminal hinge and have the structure H2f:H2f: VHb-CHl-hinge-CH2-CH3 -linker- VHb-CHl

[0191] In certain embodiments, the FRa x NaPi2b antibody constructs are in Fab Format and comprise a first heavy chain (Hl), a first light chain (LI), a second heavy chain (H2) and a second light chain (L2), where:

[0192] (i) Hl comprises a sequence as set forth in SEQ ID NO: 250, LI comprises a sequence as set forth in SEQ ID NO: 252, H2 comprises a sequence as set forth in SEQ ID NO: 251, and L2 comprises a sequence as set forth in SEQ ID NO: 253; or

[0193] (ii) Hl comprises a sequence as set forth in SEQ ID NO: 254, LI comprises a sequence as set forth in SEQ ID NO: 256, H2 comprises a sequence as set forth in SEQ ID NO: 255, and L2 comprises a sequence as set forth in SEQ ID NO: 257; or

[0194] (iii) Hl comprises a sequence as set forth in SEQ ID NO: 258, LI comprises a sequence as set forth in SEQ ID NO: 260, H2 comprises a sequence as set forth in SEQ ID NO: 259, and L2 comprises a sequence as set forth in SEQ ID NO: 261; or

[0195] (iv) Hl comprises a sequence as set forth in SEQ ID NO: 262, LI comprises a sequence as set forth in SEQ ID NO: 264, H2 comprises a sequence as set forth in SEQ ID NO: 263, and L2 comprises a sequence as set forth in SEQ ID NO: 265; or

[0196] (v) Hl comprises a sequence as set forth in SEQ ID NO: 266, LI comprises a sequence as set forth in SEQ ID NO: 268, H2 comprises a sequence as set forth in SEQ ID NO: 267, and L2 comprises a sequence as set forth in SEQ ID NO: 269; or

[0197] (vi) Hl comprises a sequence as set forth in SEQ ID NO: 270, LI comprises a sequence as set forth in SEQ ID NO: 272, H2 comprises a sequence as set forth in SEQ ID NO: 271, and L2 comprises a sequence as set forth in SEQ ID NO: 273; or

[0198] (vii) Hl comprises a sequence as set forth in SEQ ID NO: 278, LI comprises a sequence as set forth in SEQ ID NO: 280, H2 comprises a sequence as set forth in SEQ ID NO: 279, and L2 comprises a sequence as set forth in SEQ ID NO: 281; or

[0199] (viii) Hl comprises a sequence as set forth in SEQ ID NO: 282, LI comprises a sequence as set forth in SEQ ID NO: 284, H2 comprises a sequence as set forth in SEQ ID NO: 283, and L2 comprises a sequence as set forth in SEQ ID NO: 285.scFv Formats

[0200] In certain embodiments, the FRa x NaPi2b antibody constructs are bivalent, trivalent or tetravalent and comprise a scaffold based on an immunoglobulin (IgG) Fc region, where the Fc region comprises a first Fc polypeptide and a second Fc polypeptide, and in which at least one of the two or more antigen-binding domains comprised by the FRa x NaPi2b antibody constructs is in scFv format. In some embodiments, the FRa x NaPi2b antibody constructs are bivalent, trivalent or tetravalent and comprise a scaffold based on an immunoglobulin (IgG) Fc region, where the Fc region comprises a first Fc polypeptide and a second Fc polypeptide, and in which one of the two or more antigen-binding domains comprised by the FRa x NaPi2b antibody constructs is in scFv format and the other antigen-binding domains are in Fab format. Non-limiting examples are shown in Fig. 2A-2F.

[0201] In certain embodiments, the FRa x NaPi2b antibody constructs are bivalent and comprise (a) one FRa antigen-binding domain in Fab format and one NaPi2b antigen-binding domain in scFv format, or (b) one FRa antigen-binding domain in scFv format and one NaPi2b antigen-binding domain in Fab format, and in which the FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide and the NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide (see Fig. 2A).

[0202] In certain embodiments, the FRa x NaPi2b antibody constructs are trivalent and comprise one FRa antigen-binding domain in Fab format, a first NaPi2b antigen-binding domain in Fab format and a second NaPi2b antigen-binding domain in scFv format, in which the FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide, the first NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, and the second NaPi2b antigen-binding domain is linked to the N-terminus of the first NaPi2b antigen-binding domain (see Fig. 2B). In certain embodiments, the FRa x NaPi2b antibody constructs are trivalent and comprise a first FRa antigen-binding domain in Fab format, a second FRa antigen-binding domain in scFv format and one NaPi2b antigen-binding domain in Fab format, in which the first FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide, the second FRa antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain, and the NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide (see Fig. 2B).

[0203] In certain embodiments, the FRa x NaPi2b antibody constructs are trivalent and comprise one FRa antigen-binding domain in Fab format, a first NaPi2b antigen-binding domain in Fab format and a second NaPi2b antigen-binding domain in scFv format, in which the FRa antigen-binding domain is linked to the N-terminus of one (first) Fc polypeptide, the first NaPi2b antigen-binding domain is linked to the N-terminus of the other (second) Fc polypeptide, and the second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide (see Fig. 2C). In certain embodiments, the FRa x NaPi2b antibody constructs are trivalent and comprise a first FRa antigen-binding domain in Fab format, a second FRa antigen-binding domain in scFv format and one NaPi2b antigen-binding domain in Fab format, in which the first FRa antigen-binding domain is linked to the N-terminus of one (first) Fc polypeptide, the second FRa antigen-binding domain is linked to the C-terminus of the first Fc polypeptide, and the NaPi2b antigen-binding domain is linked to the N-terminus of the other (second) Fc polypeptide (see Fig.2C).

[0204] In certain embodiments, the FRa x NaPi2b antibody constructs are tetravalent and comprise two FRa antigen-binding domains in Fab format, a first NaPi2b antigen-binding domain in Fab format and a second NaPi2b antigen-binding domain in scFv format, in which a first FRa antigen-binding domain is linked to the N-terminus of one (first) Fc polypeptide, a second FRa antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain, the first NaPi2b antigen-binding domain is linked to the N-terminus of the other (second) Fc polypeptide, and the second NaPi2b antigen-binding domain is linked to the N-terminus of the first NaPi2b antigen-binding domain (see Fig. 2D). In certain embodiments, the FRa x NaPi2b antibody constructs are tetravalent and comprise a first FRa antigen-binding domain in Fab format, a second FRa antigen-binding domain in scFv format and two NaPi2b antigen-binding domains in Fab format, in which the first FRa antigen-binding domain is linked to the N-terminus of one (first) Fc polypeptide, the second FRa antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other (second) Fc polypeptide, and a second NaPi2b antigen-binding domain is linked to the N-terminus of the first NaPi2b antigen-binding domain (see Fig. 2D).

[0205] In certain embodiments, the FRa x NaPi2b antibody constructs are tetravalent and comprise two FRa antigen-binding domains in Fab format, a first NaPi2b antigen-binding domainin Fab format and a second NaPi2b antigen-binding domain in scFv format, in which a first FRa antigen-binding domain is linked to the N-terminus of one (first) Fc polypeptide, a second FRa antigen-binding domain is linked to the C-terminus of the first Fc polypeptide, the first NaPi2b antigen-binding domain is linked to the N-terminus of the other (second) Fc polypeptide, and the second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide (see Fig. 2E). In certain embodiments, the FRa x NaPi2b antibody constructs are tetravalent and comprise a first FRa antigen-binding domain in Fab format, a second FRa antigen-binding domain in scFv format and two NaPi2b antigen-binding domains in Fab format, in which the first FRa antigen-binding domain is linked to the N-terminus of one (first) Fc polypeptide, the second FRa antigen-binding domain is linked to the C-terminus of the first Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other (second) Fc polypeptide, and a second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide (see Fig. 2E).

[0206] In certain embodiments, the FRa x NaPi2b antibody constructs are tetravalent and comprise two FRa antigen-binding domains in Fab format, a first NaPi2b antigen-binding domain in Fab format and a second NaPi2b antigen-binding domain in scFv format, in which a first FRa antigen-binding domain is linked to the N-terminus of one (first) Fc polypeptide, a second FRa antigen-binding domain is linked to the C-terminus of the first Fc polypeptide, the first NaPi2b antigen-binding domain is linked to the N-terminus of the other (second) Fc polypeptide, and the second NaPi2b antigen-binding domain is linked to the N-terminus of the first FRa antigenbinding domain (see Fig. 2F). In certain embodiments, the FRa x NaPi2b antibody constructs are tetravalent and comprise a first FRa antigen-binding domain in Fab format, a second FRa antigenbinding domain in scFv format and two NaPi2b antigen-binding domains in Fab format, in which the first FRa antigen-binding domain is linked to the N-terminus of one (first) Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other (second) Fc polypeptide, a second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide, and the second FRa antigen-binding domain is linked to the N-terminus of the first NaPi2b antigen-binding domain (see Fig. 2F).

[0207] In certain embodiments in FRa x NaPi2b antibody constructs having the format as shown in any one of Fig. 2A, 2B, 2C, 2D, 2E or 2F, the one or more NaPi2b antigen-bindingdomains comprise the CDR sequences of antibody 12A10, and the one or more FRa antigenbinding domains comprise the CDR sequences of antibody 76.

[0208] In certain embodiments in FRa x NaPi2b antibody constructs having the format as shown in any one of Fig. 2A, 2B, 2C, 2D, 2E or 2F, the one or more NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10, and the one or more FRa antigenbinding domains comprise the CDR sequences of antibody 2L16.

[0209] In certain embodiments in FRa x NaPi2b antibody constructs having the format as shown in any one of Fig. 2A, 2B, 2C, 2D, 2E or 2F, the one or more NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10, and the one or more FRa antigenbinding domains comprise the CDR sequences of antibody 10L18.

[0210] In certain embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. 2D comprising two FRa antigen-binding domains and two NaPi2b antigen-binding domains, in which the two NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10. In some embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. 2D comprising two FRa antigen-binding domains and two NaPi2b antigen-binding domains, in which the two NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10 and the two FRa antigen-binding domains comprise the CDR sequences of any one of antibody 76, antibody 2L16 or antibody 10L18. In some embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. 2D comprising two FRa antigen-binding domains and two NaPi2b antigen-binding domains, in which the two NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10 and the two FRa antigen-binding domains comprise the CDR sequences of antibody 10L18.

[0211] In certain embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. 2F comprising two FRa antigen-binding domains and two NaPi2b antigen-binding domains, in which the two NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10. In some embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. 2F comprising two FRa antigen-binding domains and two NaPi2b antigen-binding domains, in which the two NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10 and the two FRa antigen-binding domains comprise the CDR sequences of anyone of antibody 76, antibody 2L16 or antibody 10L18. In some embodiments, the FRa x NaPi2b antibody construct has the format as shown in Fig. 2F comprising two FRa antigen-binding domains and two NaPi2b antigen-binding domains, in which the two NaPi2b antigen-binding domains comprise the CDR sequences of antibody 12A10 and the two FRa antigen-binding domains comprise the CDR sequences of antibody 10L18.

[0212] In certain embodiments, the FRa x NaPi2b antibody constructs are tetravalent and comprise two FRa antigen-binding domains in Fab format, a first NaPi2b antigen-binding domain in Fab format and a second NaPi2b antigen-binding domain in scFv format, in which a first FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a second FRa antigenbinding domain is linked to the N-terminus of the first FRa antigen-binding domain, the first NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, and the second NaPi2b antigen-binding domain is linked to the N-terminus of the first NaPi2b antigenbinding domain (see Fig. 2D), and in which both NaPi2b antigen-binding domains comprise the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 52, 53 and 54, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 55, 56 and 57; and both FRa antigen-binding domains comprise the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 36, 37 and 38, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 39, 40 and 41.

[0213] In certain embodiments, the FRa x NaPi2b antibody constructs are tetravalent and comprise a first FRa antigen-binding domain in Fab format, a second FRa antigen-binding domain in scFv format and two NaPi2b antigen-binding domains in Fab format, in which the first FRa antigen-binding domain is linked to the N-terminus of one (first) Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other (second) Fc polypeptide, a second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide, and the second FRa antigen-binding domain is linked to the N-terminus of the first NaPi2b antigenbinding domain (see Fig. 2F), and in which both NaPi2b antigen-binding domains comprise the heavy chain CDR sequences (HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 52, 53 and 54, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 55, 56 and 57; and both FRa antigen-binding domains comprise the heavy chain CDR sequences(HCDR1, HCDR2, HCDR3) as set forth in SEQ ID NOs: 36, 37 and 38, and the light chain CDR sequences (LCDR1, LCDR2, LCDR3) as set forth in SEQ ID NOs: 39, 40 and 41.Chain Composition of scFv Formats

[0214] In those embodiments in which the FRa x NaPi2b antibody constructs are in scFv Format, the Fc region of the antibody construct will typically include a hinge sequence, for example an IgG hinge sequence or a modified version thereof. Non-limiting examples of hinge sequences and modified hinge sequences are described herein. The scFv Format antibody construct may also comprise one or more additional hinge sequences and / or linkers. Linkers are typically employed to join different domains of the antibody construct together, for example, to join an Fc polypeptide to a Fab domain or an scFv domain, or to join a Fab domain and an scFv, or to join the VH and VL chains within an scFv. Non-limiting examples of linkers are described herein. Nonlimiting examples of heavy and light chain compositions for various scFv Format antibody constructs are described below. Heavy and light chains are designated “Hl,” “H2,” “LI” and “L2” solely for the purpose of indicating which heavy chain and light chain(s) form heavy-light chain pairs, i.e. Hl pairs with LI, and H2 pairs with L2. The numbering “1” and “2” is therefore interchangeable, thus in some instances chains designated Hl, LI, H2 and L2 in the following embodiments may be designated H2, L2, Hl and LI, respectively.

[0215] In certain embodiments, in antibody constructs having the format shown in Fig. 2A, the two heavy chains (Hl and H2) and the light chain (L2) that make up the antibody construct have the structures shown in (A) or (B) below:(A) Hl: VHa-linker-VLa-hinge-CH2-CH3H2: VHb-CHl-hinge-CH2-CH3L2: VLb-CL(B) Hl: VLa-linker-VHa-hinge-CH2-CH3H2: VHb-CHl-hinge-CH2-CH3L2: VLb-CL

[0216] Where VHaand VLaare the VH and VL domains of one antigen-binding domain (either FRa or NaPi2b), VHband VLbare the VH and VL domains of the other antigen-binding domain, “hinge” is an IgG hinge sequence or modified version thereof, and “linker” is a linker sequence.

[0217] In certain embodiments, in antibody constructs having the format shown in Fig. 2B, the two heavy chains (Hl and H2) and the two light chains (LI and L2) that make up the antibody construct have the structures shown in (A) or (B) below:(A) HL VHa-linker-VLa-linker-VHa-CHl -hinge-CH2-CH3LI: VLa-CLH2: VHb-CHl-hinge-CH2-CH3L2: VLb-CL(B) Hl: VLa-linker-VHa-linker-VHa-CHl -hinge-CH2-CH3LI: VLa-CLH2: VHb-CHl-hinge-CH2-CH3L2: VLb-CL

[0218] Where VHaand VLaare the VH and VL domains of one antigen-binding domain (either FRa or NaPi2b), VHband VLbare the VH and VL domains of the other antigen-binding domain, “hinge” is an IgG hinge sequence or modified version thereof, and each “linker” is indpendently a linker sequence.

[0219] In certain embodiments, in antibody constructs having the format shown in Fig. 2C, the two heavy chains (Hl and H2) and the two light chains (LI and L2) that make up the antibody construct have the structures shown in (A) or (B) below:(A) Hl: VHa-CHl-hinge-CH2-CH3 -linker- VHa-linker-VLaLI: VLa-CLH2: VHb-CHl-hinge-CH2-CH3L2: VLb-CL(B) Hl: VHa-CHl-hinge-CH2-CH3 -linker- VLa-linker-VHaLI: VLa-CLH2: VHb-CHl-hinge-CH2-CH3L2: VLb-CL

[0220] Where VHaand VLaare the VH and VL domains of one antigen-binding domain (either FRa or NaPi2b), VHband VLbare the VH and VL domains of the other antigen-binding domain, “hinge” is an IgG hinge sequence or modified version thereof, and each “linker” is independently a linker sequence.

[0221] In certain embodiments, in antibody constructs having the format shown in Fig. 2D, the two heavy chains (Hl and H2) and the two light chains (LI and L2) that make up the antibody construct have the structures shown in (A) or (B) below:(A) Hl: VHa-linker-VLa-linker-VHa-CHl -hinge-CH2-CH3LI: VLa-CLH2: VHb-CHl -hinge-linker- VHb-CHl-hinge-CH2-CH3L2 x2: VLb-CL(B) Hl: VLa-linker-VHa-linker-VHa-CHl -hinge-CH2-CH3LI: VLa-CLH2: VHb-CHl -hinge-linker- VHb-CHl-hinge-CH2-CH3L2 x2: VLb-CL

[0222] Where VHaand VLaare the VH and VL domains of one antigen-binding domain (either FRa or NaPi2b), VHband VLbare the VH and VL domains of the other antigen-binding domain, each “hinge” is independently an IgG hinge sequence or modified version thereof, and each “linker” is independently a linker sequence.

[0223] In some embodiments, for antibody constructs having the format shown in Fig. 2D, the H2 chain in either option (A) or (B) may lack one of the hinge or the linker and have the structure H2a or H2b:H2a: VHb-CHl -hinge- VHb-CHl-hinge-CH2-CH3H2b: VHb-CHl -linker- VHb-CHl -hinge-CH2-CH3

[0224] In certain embodiments, in antibody constructs having the format shown in Fig. 2E, the two heavy chains (Hl and H2) and the two light chains (LI and L2) that make up the antibody construct have the structures shown in (A) or (B) below:(A) HL VHa-CHl-hinge-CH2-CH3 -linker- VHa-linker-VLaLI: VLa-CLH2: VHb-CH 1 -hinge-CH2-CH3 -linker- VHb-CH 1 -hingeL2 x2: VLb-CL(B) Hl: VHa-CHl-hinge-CH2-CH3 -linker- VLa-linker-VHaLI: VLa-CLH2: VHb-CH 1 -hinge-CH2-CH3 -linker- VHb-CH 1 -hingeL2 x2: VLb-CL

[0225] Where VHaand VLaare the VH and VL domains of one antigen-binding domain (either FRa or NaPi2b), VHband VLbare the VH and VL domains of the other antigen-binding domain, each “hinge” is independently an IgG hinge sequence or modified version thereof, and each “linker” is independently a linker sequence.

[0226] In some embodiments, for antibody constructs having the format shown in Fig. 2E, the H2 chain of either option (A) or (B) may lack the C-terminal hinge and have the structure H2c:H2c: VHb-CHl-hinge-CH2-CH3 -linker- VHb-CHl

[0227] In certain embodiments, in antibody constructs having the format shown in Fig. 2F, the two heavy chains (Hl and H2) and the two light chains (LI and L2) that make up the antibody construct have the structures shown in (A) or (B) below:(A) Hl: VHb-linker- VLb-linker- VHa-CHl-hinge-CH2-CH3-linker-VHa-CHl-hingeLI x2: VLa-CLH2: VHb-CHl-hinge-CH2-CH3L2: VLb-CL(B) Hl: VLb-linker-VHb-linker- VHa-CHl-hinge-CH2-CH3-linker-VHa-CHl-hingeLI x2: VLa-CLH2: VHb-CHl-hinge-CH2-CH3L2: VLb-CL

[0228] Where VHaand VLaare the VH and VL domains of one antigen-binding domain (either FRa or NaPi2b), VHband VLbare the VH and VL domains of the other antigen-binding domain, each “hinge” is independently an IgG hinge sequence or modified version thereof, and each “linker” is independently a linker sequence.

[0229] In some embodiments, for antibody constructs having the format shown in Fig. 2F, the Hl chain of either option (A) or (B) may lack the C-terminal hinge and have the structure Hl a:Hla: VLb-linker-VHb-linker-VHa-CHl-hinge-CH2-CH3 -linker- VHa-CHl

[0230] In certain embodiments, the FRa x NaPi2b antibody constructs are in scFv Format and comprise a first heavy chain (Hl), a first light chain (LI), a second heavy chain (H2) and a second light chain (L2), where:

[0231] (i) Hl comprises a sequence as set forth in SEQ ID NO: 274, LI comprises a sequence as set forth in SEQ ID NO: 276, H2 comprises a sequence as set forth in SEQ ID NO: 275, and L2 comprises a sequence as set forth in SEQ ID NO: 277; or

[0232] (ii) Hl comprises a sequence as set forth in SEQ ID NO: 286, LI comprises a sequence as set forth in SEQ ID NO: 288, H2 comprises a sequence as set forth in SEQ ID NO: 287, and L2 comprises a sequence as set forth in SEQ ID NO: 289.Hinge and Linker Sequences

[0233] In certain embodiments, the FRa x NaPi2b antibody constructs may comprise one or more hinge sequences and / or one or more linker sequences. For example, in those embodiments in which the antibody construct comprises an IgG Fc region or an antigen-binding domain in a Fab format, the Fc region may comprise an IgG hinge sequence or modified version thereof. Similarly, in those embodiments in which the antibody construct comprises an antigen-binding domain in an scFv format, or two antigen binding domains joined together, the antibody construct may comprise one or more linker sequences.

[0234] Non-limiting examples of wild-type IgG hinge region sequences, of which all or a portion (for example, at least 5, 6, 7, 8, 9, 10 or more contiguous amino acids) may be comprised by the FRa xNaPi2b antibody constructs, include EPKSCDKTHTCPPCP (SEQ ID NO: 61) (IgGl); ERKCCVECPPCP (SEQ ID NO: 189) (IgG2); ELKTPLGDTTHTCPRCP (SEQ ID NO: 190) (IgG3-Hl); EPKSCDTPPPCPRCP (SEQ ID NO: 191) (IgG3-H2, IgG3-H3 and IgG3-H4), and ESKYGPPCPSCP (SEQ ID NO: 192) (IgG4). In certain embodiments, the FRa xNaPi2b antibody constructs comprise one or more wild-type hinge sequences. In some embodiments, the FRa xNaPi2b antibody constructs comprise one or more hinge sequences that comprise all or a portion of the wild-type IgGl hinge sequence. In some embodiments, the FRa x NaPi2b antibody constructs comprise one or more hinge sequences having the sequence EPKSCDKTHTCPPCP (SEQ ID NO: 61). In some embodiments, the FRa x NaPi2b antibody constructs comprise one or more hinge sequences that are modified versions of a wild-type hinge sequence. In someembodiments, the FRa x NaPi2b antibody constructs comprise one or more hinge sequences that are modified versions of a wild-type IgGl hinge sequence. In some embodiments, the FRa x NaPi2b antibody constructs comprise one or more hinge sequences having the sequence AAEPKSSDKTHTCPPCP (SEQ ID NO: 62).

[0235] Various linker sequences that are suitable for use in antibody constructs are known in the art. Examples include linker sequences comprising the amino acid residues Gly, Ser, Ala or Thr, or a combination thereof. Examples of such polypeptide linkers include, but are not limited to, glycine-serine linkers, such as (GS)n, (GSGGS)n(SEQ ID NO: 193), (GGGGS)n(SEQ ID NO: 194) and (GGGS)n(SEQ ID NO: 195) (where n is an integer between 1 and 4), as well as glycine-alanine linkers and alanine-serine linkers having similar configurations. In certain embodiments, the FRa x NaPi2b antibody constructs comprise one or more linker sequences. In some embodiments, the FRa x NaPi2b antibody constructs comprise one or more glycine-serine linker sequences, for example, (G4S)2 (SEQ ID NO: 67), (G4S)s (SEQ ID NO: 68) or (G4S)4 (SEQ ID NO: 69).ANTI-FRa ANTIBODY CONSTRUCTS

[0236] An aspect of the present disclosure relates to antibody constructs or antigen-binding fragments thereof that specifically bind to FRa (anti-FRa antibody constructs) that are based on antibody 76 (see Example 1 herein). These anti-FRa antibody constructs and antigen-binding fragments comprise the CDR sequences of the VH and VL sequences as set forth in Table 1.2. In certain embodiments, the anti-FRa antibody constructs and antigen-binding fragments comprise heavy chain CDRs (HCDR1, HCDR2 and HCDR3) and light chain CDRs (LCDR1, LCDR2 and LCDR3) having the sequences as set forth in Table 1.1. In some embodiments, the anti-FRa antibody constructs and antigen-binding fragments comprise heavy chain CDRs (HCDR1, HCDR2 and HCDR3) having the sequences as set forth in SEQ ID NOs: 10, 11 and 7, and light chain CDRs (LCDR1, LCDR2 and LCDR3) having the sequences as set forth in SEQ ID NOs: 18, 19 and 17.

[0237] In certain embodiments, the anti-FRa antibody constructs comprise VH and VL sequences that are humanized versions of the VH and VL sequences of v31625 (see Table 1.2). In some embodiments, the anti-FRa antibody construct comprises a VH sequence having at least85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the VH sequence of any one of variants v32977, v32978, v32979, v32983, v32984 or v32985, derived from the antibody 76, and a VL sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the VL sequence of any one of variants v32977, v32978, v32979, v32983, v32984 or v32985, derived from the antibody 76. In some embodiments, the anti-FRa antibody construct comprises a VH sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the VH sequence of variant v32977, and a VL sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to the VL sequence of variant v32977.

[0238] In certain embodiments, the anti-FRa antibody construct comprises a VH sequence having a sequence as set forth in SEQ ID NO: 29 and a VL sequence having a sequence as set forth in any one of SEQ ID NOs: 33, 34 or 35. In some embodiments, the anti-FRa antibody construct comprises a VH sequence having a sequence as set forth in SEQ ID NO: 29 and a VL sequence having a sequence as set forth in SEQ ID NOs: 33.

[0239] In certain embodiments, the anti-FRa antibody construct comprises a VH sequence having a sequence as set forth in SEQ ID NO: 32 and a VL sequence having a sequence as set forth in any one of SEQ ID NOs: 33, 34 or 35.

[0240] The anti-FRa CDR, VH and VL sequences described above may be used to construct various formats of antibody constructs as is known in the art. For example, these sequences may be used to construct Fab fragments or scFvs, which may be linked to a scaffold such as an Fc or other scaffold as described herein. The antibody constructs may be monovalent, bivalent or multivalent. In some embodiments, the antibody constructs are monospecific. In some embodiments, the antibody constructs are monospecific and in the naturally occurring format (FSA).

[0241] The anti-FRa VH and VL sequences described above may further be used in the construction of bispecific or multispecific antibodies, such as the FRa x NaPi2b antibody constructs described herein, or other antibody constructs comprising at least one antigen-binding domain that binds to FRa.METHODS OF PREPARING ANTIBODY CONSTRUCTS

[0242] The FRa x NaPi2b antibody constructs and anti-FRa antibody constructs described herein may 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” 2ndEdition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014).

[0243] Typically, for recombinant production of an antibody construct, a polynucleotide or set of polynucleotides encoding the antibody construct is generated and inserted into one or more vectors for further cloning and / or expression in a host cell. Polynucleotide(s) encoding the antibody construct may be produced by standard methods known in the art (see, for example, Ausubel el al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1994 & update, and “ Antibodies: A Laboratory Manual,” 2ndEdition, Ed. Greenfield, Cold Spring Harbor Laboratory Press, New York, 2014). As would be appreciated by one of skill in the art, the number of polynucleotides required for expression of the antibody construct will be dependent on the format of the construct, including whether or not the antibody construct comprises a scaffold. For example, when a bispecific antibody construct is in a mAb format with two Fab antigen-binding domains, two polynucleotides each encoding one heavy chain and two polynucleotides each encoding a light chain will be required. When multiple polynucleotides are required, they may be incorporated into one vector (for example, a multi ci str onic vector) or into more than one vector.

[0244] Generally, for expression, the polynucleotide or set of polynucleotides is incorporated into an expression vector or 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. One skilled in the art will appreciate that the choice of regulatory elements is dependent on the host cell selected for expression of the antibody construct and that such regulatory elements may be derived from a variety of sources, including bacterial, fungal, viral, mammalian or insect genes. The expression vector may 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 (“His-tags”), avidin / streptavidin encoding sequences,glutathione-S-transferase (GST) encoding sequences and biotin encoding sequences. The expression vector may be an extrachromosomal vector or an integrating vector.

[0245] Suitable host cells for cloning or expression of the antibody constructs include various prokaryotic or eukaryotic cells as 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, E. coli, A. salmonicida or B. subtilis cells.

[0246] In certain embodiments, the antibody construct may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, as described for example in U. S. Patent Nos. 5,648,237; 5,789,199, and 5,840,523, and in Charlton, Methods in Molecular Biology, Vol. 248, pp. 245-254, B. K. C. Lo, ed., Humana Press, Totowa, N. J., 2003.

[0247] Eukaryotic microbes such as filamentous fungi or yeast may be suitable expression host cells in certain embodiments, in particular fungi and yeast strains whose glycosylation pathways have been “humanized” resulting in the production of an antibody construct with a partially or fully human glycosylation pattern (see, for example, Gemgross, 2004, Nat. Biotech. 22:1409-1414, and Li etal., 2006, Nat. Biotech. 24:210-215).

[0248] Suitable host cells for the expression of glycosylated antibody constructs are usually eukaryotic cells. For example, U. S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 describe PLANTIBODIES™ technology for producing antigen-binding constructs in transgenic plants. Mammalian cell lines adapted to grow in suspension may be particularly useful for expression of 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, for example, Graham et al., 1977, J. Gen Virol., 36:59), baby hamster kidney cells (BHK), mouse sertoli TM4 cells (see, for example, Mather, 1980, Biol Reprod, 23:243-251), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma (HeLa) cells, canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumour (MMT 060562), TRI cells (see, for example, 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 Set USA, 77:4216), and myeloma cell lines (such as YO, NSO and Sp2 / 0). Exemplary mammalian host cell lines suitable for production of antibody constructs are reviewed in Yazaki & Wu, Methods in Molecular Biology, Vol. 248, pp. 255-268 (B. K. C. Lo, ed., Humana Press, Totowa, N. J., 2003).

[0249] In certain embodiments, the host cell used to produce the antibody constructs may be a transient or stable higher eukaryotic cell line, such as a mammalian cell line. In some embodiments, the host cell may be a mammalian HEK293T, CHO, HeLa, NSO or COS cell line, or a cell line derived from any one of these cell lines. In some embodiments, the host cell may be a stable cell line that allows for mature glycosylation of the antibody construct.

[0250] The host cells comprising the expression vector(s) encoding the antibody construct may be cultured using routine methods to produce the antibody construct. Alternatively, in some embodiments, host cells comprising the expression vector(s) encoding the antibody construct may be used therapeutically or prophylactically to deliver the antibody construct to a subject, or polynucleotides or expression vectors may be administered to a cell from a subject ex vivo and the cell then returned to the body of the subject.

[0251] Typically, the antibody constructs are purified after expression. Proteins may be isolated or purified in a variety of ways known to those skilled in the art (see, for example, Protein Purification: Principles and Practice, 3rdEd., Scopes, Springer-Verlag, NY, 1994). Standard purification methods include chromatographic techniques, including ion exchange, hydrophobic interaction, affinity, sizing or gel filtration, and reverse-phase, carried out at atmospheric pressure or at high pressure using systems such as FPLC and HPLC. Additional purification methods include electrophoretic, immunological, precipitation, dialysis and chromatofocusing techniques. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. As is well known in the art, a variety of natural proteins bind Fc and antibodies, and these proteins may be used for purification of certain antibody constructs. For example, the bacterial proteins A and G bind to the Fc region. Likewise, the bacterial protein L binds to the Fab region of some antibodies. Purification may also be enabled by a particular fusion partner. For example, antibodies may be purified using glutathione resin if a GST fusion is employed, Ni+2affinity chromatography if a His-tag is employed or immobilized anti-flag antibody if a flag-tag is used.The degree of purification necessary will vary depending on the use of the antibody constructs. In some instances, no purification may be necessary.

[0252] In certain embodiments, the antibody constructs are substantially pure. The term “substantially pure” (or “substantially purified”) when used in reference to an antibody construct described herein, means that the antibody construct is substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, such as a native cell, or a host cell in the case of recombinantly produced construct. In certain embodiments, an antibody construct that is substantially pure 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.

[0253] Certain embodiments of the present disclosure relate to a method of making a FRa x NaPi2b antibody construct or an anti-FRa antibody construct comprising culturing a host cell into which one or more polynucleotides encoding the antibody construct, or one or more expression vectors encoding the antibody construct, have been introduced, under conditions suitable for expression of the antibody construct, and optionally recovering the antibody construct from the host cell (or from host cell culture medium).Post-Translational Modifications

[0254] In certain embodiments, the antibody constructs described herein may comprise one or more post-translational modifications. Such post-translational modifications may occur in vivo, or they be conducted in vitro after isolation of the antibody construct from the host cell.

[0255] Post-translational modifications include various modifications as are known in the art (see, for example, Proteins - Structure and Molecular Properties, 2nd Ed., T. E. Creighton, W. H. Freeman and Company, New York, 1993; Post-Translational Covalent Modification of Proteins, B. C. Johnson, Ed., Academic Press, New York, pgs. 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 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.

[0256] 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 NaBH4.

[0257] Other examples of post-translational modifications include, for example, addition or removal of N-linked or O-linked carbohydrate chains, chemical modifications of N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends, attachment of chemical moieties to the amino acid backbone, and addition or deletion of an N-terminal methionine residue resulting from prokaryotic host cell expression. Post-translational modifications may also include modification with a detectable label, such as an enzymatic, fluorescent, luminescent, isotopic or affinity label to allow for detection and isolation of the protein. Examples of suitable enzyme labels include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase and acetylcholinesterase. Examples of suitable prosthetic group complexes include, but are not limited to, streptavidin / biotin and avidin / biotin. Examples of suitable fluorescent materials include, but are not limited to, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride and phycoerythrin. Examples of luminescent materials include luminol, and bioluminescent materials such as luciferase, luciferin and aequorin. Examples of suitable radioactive materials include iodine, carbon, sulfur, tritium, indium, technetium, thallium, gallium, palladium, molybdenum, xenon and fluorine.

[0258] Additional examples of post-translational modifications include acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, gamma-carboxylation, GPI anchor formation, hydroxylation, iodination, methylation, myristylation, pegylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.POLYNUCLEOTIDES, VECTORS AND HOST CELLS

[0259] Certain embodiments of the present disclosure relate to an isolated polynucleotide or a set of polynucleotides encoding a bispecific FRa x Napi2b antibody construct described herein. Some embodiments of the present disclosure relate to an isolated polynucleotide or a set of polynucleotides encoding an anti-FRa antibody construct described herein. A polynucleotide in this context may encode all or part of a given antibody construct.

[0260] The terms “nucleic acid,” “nucleic acid molecule” and “polynucleotide” are used interchangeably herein and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogues thereof. Non-limiting examples of polynucleotides include a gene, a gene fragment, messenger RNA (mRNA), cDNA, recombinant polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.

[0261] A polynucleotide that “encodes” a given polypeptide is a polynucleotide that is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A transcription termination sequence may be located 3' to the coding sequence.

[0262] Certain embodiments of the present disclosure relate to vectors (such as expression vectors) comprising one or more polynucleotides encoding an FRa x NaPi2b antibody construct described herein or an anti-FRa antibody construct described herein. The polynucleotide(s) may be comprised by a single vector or by more than one vector. In some embodiments, the polynucleotides are comprised by a multi ci stronic vector.

[0263] Certain embodiments of the present disclosure relate to host cells comprising polynucleotide(s) encoding an FRa x NaPi2b antibody construct described herein or an anti-FRa antibody construct described herein or one or more vectors comprising the polynucleotide(s). In some embodiments, the host cell is eukaryotic, for example, a Chinese Hamster Ovary (CHO) cell, a human embryonic kidney (HEK) cell or a lymphoid cell (e.g. Y0, NS0, Sp20 cell).ANTIBODY-DRUG CONJUGATES

[0264] Certain embodiments of the present disclosure relate to antibody-drug conjugates (ADCs) comprising a bispecific FRa x NaPi2b antibody construct or an anti-FRa antibody construct as described herein conjugated to one or more drug moieties, such as cytotoxins or immune modulators.

[0265] Typically, in an ADC, the FRa x NaPi2b antibody construct or anti-FRa antibody construct is conjugated to a drug moiety via a linker, which may be a cleavable or non-cleavable linker. The antibody construct may be conjugated to a single drug molecule, or it may be conjugated to multiple drug molecules. The number of drug molecules conjugated to a single 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 from about 1 to about 12, or from about 2 to about 12, or from about 2 to about 8.

[0266] In certain embodiments, the ADCs comprising an FRa x NaPi2b antibody construct or anti-FRa antibody construct have the general Formula I:A-(L-(D)m)n(I)

[0267] where A is an FRa x NaPi2b antibody construct or anti-FRa antibody construct as described herein; L is a linker; D is a drug moiety; m is between 1 and about 8, and n is between 1 and about 12.

[0268] In certain embodiments in 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, for example, between about 2 and about 8, or between about 4 and about 8. In some embodiments, m is 1 or 2 and n is between about 4 and about 8.

[0269] Various compounds known to be useful as cytotoxic or immunomodulatory ADC payloads may be employed as the drug moiety in the ADCs comprising the FRa x NaPi2b antibody constructs or anti-FRa antibody constructs. Examples include, but are not limited to, maytansinoids and maytansinoid analogues, benzodiazepines and pyrrolobenzodiazepines (PBDs, including PBD dimers), duocarmycins such as CC-1065 and analogues thereof, calicheamicinsand calicheamicin analogues, auristatins and auristatin analogues, hemiasterlins and hemiasterlin analogues, tubulysins and tubulysin analogues, amatoxins and amatoxin analogues, topoisomerase 1 inhibitors including camptothecins and camptothecin analogues, eribulin, TLR agonists (such as agonists of TLR7 and / or TLR8) and STING agonists.

[0270] In certain embodiments, the drug moiety comprised by the ADCs of the present disclosure is an auristatin or auristatin analogue, a hemiasterlin or a hemiasterlin analogue, a camptothecin or camptothecin analogue, or eribulin.

[0271] Typically, in the ADCs of the present disclosure, the drug moiety is linked to the antibody construct by a linker. Linkers are bifunctional or multifunctional moieties capable of linking one or more drug molecules to the antibody construct. In some embodiments, the linker may 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 may be multifunctional (or polyvalent) such that it links more than one drug molecule to a single site on the antibody construct. Multifunctional linkers may also be used to link one drug molecule to more than one site on the antibody construct in some embodiments.

[0272] Attachment of a linker to an antibody construct can be accomplished in a variety of ways, such as through surface lysines, reductive-coupling to oxidized carbohydrates, or through cysteine residues liberated by reducing interchain disulfide linkages. Alternatively, attachment of a linker to an antibody construct may be achieved by modification of the antibody construct to include additional cysteine residues (see, for example, U. S. Patent Nos. 7,521,541; 8,455,622 and 9,000, 130) or non-natural amino acids that provide reactive handles, such as selenomethionine, / ?-acetylphenylalanine, formylglycine or / ?-azidomethyl-L-phenylalanine to allow for site-specific conjugation (see, for example, 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 etal., 2014, Bioconj. Chem., 25:351-361). A further option is the use of GlycoConnect™ technology (Synaffix BV, Nijmegen, Netherlands), which involves enzymatic remodelling of the antibody glycans to allow for attachment of a linker by metal-free click chemistry (see, for example, European Patent No. EP 2911 699).

[0273] Linkers typically include a functional group capable of reacting with the target group or groups on the antigen binding construct and one or more functional groups capable of reacting with a target group on the drug moiety. Suitable functional groups are known in the art and include those described, for example, in Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press). Non-limiting examples of functional groups for reacting with free cysteines or thiols include maleimide, haloacetamide, haloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. Also useful in this context are “selfstabilizing” maleimides such as those described in Lyon et al., 2014, Nat. Biotechnol., 32:1059-1062. Non-limiting examples of functional groups for reacting with surface lysines and amines include activated esters (such as N-hydroxy succinamide (NHS) esters and sulfo-NHS esters), imido esters (such as Traut’s reagent), isothiocyanates, aldehydes and acid anhydrides (such as diethylenetriaminepentaacetic anhydride (DTP A)). Other examples include the use of succinimido-l,l,3,3-tetra-methyluronium tetrafluoroborate (TSTU) or benzotriazol- 1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBOP) to convert a carboxylic acid to an activated ester, which may then be reacted with an amine. Non-limiting examples of functional groups capable of reacting with an electrophilic group on the antibody construct or drug moiety (such as an aldehyde or ketone carbonyl group) include hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide.

[0274] In certain embodiments, a linker that includes a functional group that allows for bridging of two interchain cysteines on the antibody binding construct may be used, such as a ThioBridge™ linker (Badescu et al., 2014, Bioconjug. Chem., 25:1124-1136), a dithiomaleimide (DTM) linker (Behrens et al., 2015, Mol. Pharm., 12:3986-3998), a dithioaryl(TCEP)pyridazinedione-based linker (Lee et al., 2016, Chem. Sci., 7:799-802) or a dibromopyridazinedione-based linker (Maruani etal., 2015, Nat. Commun., 6:6645).

[0275] A variety of linkers for linking drugs to antibodies are known in the art, including hydrazone-, disulfide- and peptide-based linkers. Linkers may be cleavable or non-cleavable. A cleavable linker is typically susceptible to cleavage under intracellular conditions, for example, through lysosomal processes. Examples include linkers that are protease-sensitive, acid-sensitiveor reduction-sensitive. Non-cleavable linkers by contrast, rely on the degradation of the antibody in the cell, which typically results in the release of an amino acid-linker-drug moiety.

[0276] An example of a cleavable linker that may be useful in certain embodiments is a peptide-containing linker cleavable by an intracellular protease, such as lysosomal protease or an endosomal protease. Examples include dipeptide-containing linkers, such as those comprising 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, Vai-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, MesLys-Pro, PhenylGly-(D)Lys, Met-(D)Lys, Asn-(D)Lys, Pro-(D)Lys or Met-(D)Lys; tripeptide-containing linkers such as those comprising the tripeptides Met-Cit-Val, Gly-Cit-Val, (D)Phe-Phe-Lys or (D)Ala-Phe-Lys, and tetrapeptide-containing linkers such as those comprising the tetrapeptides Gly-Phe-Leu-Gly, Gly-Gly-Phe-Gly or Ala-Leu- Ala-Leu.

[0277] Additional useful cleavable linkers include disulfide-containing linkers and linkers hydrolyzable at a specific pH or within a pH range, such as hydrazone linkers. Examples of disulfide-containing linkers include, but are not limited to, N-succinimydyl-4-(2-pyridyldithio) butanoate (SPDB) and N-succinimydyl-4-(2-pyridyldithio)-2-sulfo butanoate (sulfo-SPDB). Disulfide-containing linkers may optionally include additional groups to provide steric hindrance adjacent to the disulfide bond in order to improve the extracellular stability of the linker, for example, inclusion of a geminal dimethyl group. Linkers comprising combinations of these functionalities may also be useful, for example, linkers comprising both a hydrazone and a disulfide are known in the art.

[0278] A further example of a cleavable linker is a linker comprising a P-glucuronide, which is cleavable by P-glucuronidase, an enzyme present in lysosomes and tumor interstitium (see, for example, De Graaf etal., 2002, Curr. Pharm. Des., 8:1391-1403).

[0279] Cleavable linkers may optionally further comprise one or more additional functionalities such as self-immolative / self-elimination groups, stretchers or hydrophilic moieties.

[0280] Self-immolative and self-elimination groups that find use in linkers include, for example, / ?-aminobenzyl (PAB) and / ?-aminobenzyloxycarbonyl (PABC) groups, methylatedethylene diamine (MED) and hemi-aminal groups. Other examples of self-immolative groups include, but are not limited to, aromatic compounds that are electronically similar to the PABC or PABE group such as heterocyclic derivatives, for example 2-aminoimidazol-5-methanol derivatives as described in U. S. Patent No. 7,375,078. Other examples include groups that undergo cyclization upon amide bond hydrolysis, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al., 1995, Chemistry Biology, 2'223-221') and 2-aminophenylpropionic acid amides (Amsberry, et al., 1990, J. Org. Chem., 55:5867-5877). Self-immolative / self-elimination groups, alone or in combination, are often included in peptide-based linkers but may also be included in other types of linkers. In some embodiments, the linker may include one or more self-immolative / self-elimination groups, for example, a PABC group, a PABE group, or a combination of a PABC or PABE group and an MED.

[0281] Various stretchers that find use in linkers for ADCs are known in the art and include, for example, alkylene groups and stretchers based on aliphatic acids, diacids, amines or diamines, such as diglycolate, malonate, caproate and caproamide. Other stretchers include, for example, glycine-based stretchers and polyethylene glycol (PEG) or monomethoxy polyethylene glycol (mPEG) stretchers. PEG and mPEG stretchers also function as hydrophilic moieties and may be particularly useful with hydrophobic drugs, although their use in linkers with other drugs is also contemplated in some embodiments.

[0282] In certain embodiments, a stretcher may have one of the following structures:0 0 o — (CH2)t— c —. — (CH2CH2O)U— c—. — (CH2)t— (CH2CH2O)U— c—.o o— (CH2CH2O)U— (CH2)t— C —. — (CH2)t— (CH2CH2O)U— (CH2)t— C —O R O O R O— (CH2)t— C— N— (CH2)t— C —or— (CH2)t— C— N— (CH2CH2O)U— C—wherein:R is H or Ci-Ce alkyl;t is an integer between 2 and 10, andu is an integer between 1 and 10.

[0283] In some embodiments, in ADCs of Formula I, linker, L, is a cleavable linker having Formula II:«-z-[S,rJ-qAA1-[AA2]^x]r%(II)wherein:Z is a linking group that joins the linker to a target group on the antibody construct, A; Str is a stretcher;AAi and AA2 are each independently an amino acid, wherein AAi-[AA2]rforms a protease cleavage site;X is a self-immolative group;q is 0 or 1;r is 1, 2 or 3;s is 0, 1 or 2;# is the point of attachment to the antibody construct, A, and% is the point of attachment to the drug moiety, D.

[0284] In some embodiments, in linkers of Formula (II):[2 N— *Zis O where # is the point of attachment to A, and * is the point of attachment to the remainder of the linker.

[0285] In some embodiments, in linkers of Formula (II), Str is selected from:O O O— (CH2)t— C —. — (CH2CH2O)U— C—. — (CH2)t— (CH2CH2O)U— C—.o o— (CH2CH2O)U— (CH2)t— C —. — (CH2)t— (CH2CH2O)U— (CH2)t— C —O R O O R O— (CH2)t— C-N— (CH2)t— c—and— (CH2)t— C-N— (CH2CH2O)U-C—wherein:R is H or Ci-Ce alkyl;t is an integer between 2 and 10, andu is an integer between 1 and 10.

[0286] In some embodiments, ADCs of Formula I may comprise a disulfide-containing linker. In some embodiments, in ADCs of Formula I, linker, L, is a cleavable linker having Formula III:R R(HI)wherein:Z is a linking group that joins the linker to a target group on the antibody construct, A; Q is -(CEhjp- or -(CEECEEOjq-, wherein p and q are each independently an integer between 1 and 10;each R is independently H or Ci-Ce alkyl;n is 1, 2 or 3;# is the point of attachment to the antibody construct, A, and% is the point of attachment to the drug moiety, D.

[0287] In some embodiments, ADCs of Formula I may comprise a P-glucuroni decontaining linker.

[0288] Various non-cleavable linkers are known in the art for linking drugs to antibodies and may be useful in the ADCs of the present disclosure in certain embodiments. Examples of non-cleavable linkers include linkers having an N-succinimidyl ester or N-sulfosuccinimidyl ester moiety for reaction with the antibody, as well as a maleimido- or haloacetyl-based moiety for reaction with the drug, or vice versa. An example of such a non-cleavable linker is based on sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-l -carboxylate (sulfo-SMCC). Sulfo-SMCC conjugation typically occurs via a maleimide group which reacts with sulfhydryls (thiols, — SH), while the sulfo-NHS ester is reactive toward primary amines. Other non-limiting examples of such linkers include those based on N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), N-succinimidyl-4-(N-maleimidomethyl)-cyclohexane-l-carboxy-(6-amidocaproate) (“long chain” SMCC or LC-SMCC), K-maleimidoundecanoic acid N-succinimidyl ester (KMUA), y-maleimidobutyric acid N-succinimidyl ester (GMBS), 8-maleimidocaproic acid N-hydroxysuccinimide ester (EMCS), m-maleimidobenzoyl-N-hydroxy succinimide ester (MBS), N-(a-maleimidoacetoxy)-succinimide ester (AMAS), succinimidyl-6-(P-maleimidopropionamido)hexanoate (SMPH), N-succinimidyl 4-(p-maleimidophenyl)-butyrate (SMPB) and N-(p-maleimidophenyl)isocyanate (PMPI). Other examples include those comprising a haloacetyl-based functional group such as N-succinimidyl-4-(iodoacetyl)-aminobenzoate (SIAB), N-succinimidyl iodoacetate (SIA), N-succinimidyl bromoacetate (SBA) and N-succinimidyl 3-(bromoacetamido)propionate (SBAP).

[0289] ADCs comprising an FRa x NaPi2b antibody construct or anti-FRa antibody construct as described herein may be prepared by one of several routes known in the art, employing standard organic chemistry reactions, conditions, and reagents (see, for example, Bioconjugate Techniques (G. T. Hermanson, 2013, Academic Press). For example, conjugation may be achieved by (1) reaction of a functional group of an antibody construct with a bivalent linker reagent, to form antibody -linker intermediate A-L, via a covalent bond, followed by reaction of A-L with an activated drug moiety D; or (2) reaction of a functional group of a drug moiety with a linker reagent, to form drug-linker intermediate D-L, via a covalent bond, followed by reaction of D-L with a functional group of an antibody construct. Conjugation methods (1) and (2) may be employed with a variety of antibody constructs, drug moieties, and linkers to prepare the ADCs described herein.

[0290] Various prepared linkers, linker components and drugs are commercially available or may be prepared using standard synthetic organic chemistry techniques (see, for example, March’s Advanced Organic Chemistry (Smith & March, 2006, Sixth Ed., Wiley); Toki etal., 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, a number of pre-formed drug-linkers suitable for reaction with a selected antibody construct are also available commercially, for example, drug-linkers comprising DM1, DM4, MMAE, MMAF or Duocarmycin SA are available from Creative BioLabs (Shirley, NY). Various 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 BioPharma Solutions (Baxter Healthcare Corporation, Deerfield, IL) and Piramal Pharma Solutions (Grangemouth, UK).

[0291] The ADCs, once prepared, may be purified by standard techniques such as chromatography (for example, HPLC, size-exclusion, adsorption, ion exchange and / or affinity capture), dialysis and / or tangential flow filtration.PHARMACEUTICAL COMPOSITIONS

[0292] For therapeutic use, the bispecific FRa x NaPi2b antibody constructs, anti-FRa antibody constructs and ADCs may be provided in the form of pharmaceutical compositions comprising the antibody construct or ADC and a pharmaceutically acceptable carrier or diluent. The compositions may be prepared by known procedures using well-known and readily available ingredients.

[0293] Pharmaceutical compositions may 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 spray. “Parenteral” administration may be subcutaneous injection, or intradermal, intra-articular, intravenous, intramuscular, intravascular, intrasternal or intrathecal injection or infusion. The pharmaceutical composition will typically be formulated in a format suitable for administration to the subject, for example, as a syrup, elixir, tablet, troche, lozenge, hard or soft capsule, pill, suppository, oily or aqueous suspension, dispersible powder or granule, emulsion, injectable or solution. Pharmaceutical compositions may be provided as unit dosage formulations.

[0294] In certain embodiments, pharmaceutical compositions comprising the antibody constructs or ADCs may be formulated for parenteral administration by infusion or injection, or in a unit dosage injectable form, for example as lyophilized formulations or aqueous solutions.

[0295] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed. Examples of such carriers include, but are not limited to, buffers such as phosphate, citrate, and other organic acids; antioxidants such as ascorbic acid and methionine; preservatives such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl alcohol, benzyl alcohol, alkyl parabens (such as methyl or propyl paraben), catechol, resorcinol, cyclohexanol, 3 -pentanol and m-cresol; low molecular weight (less than about 10 residues) polypeptides; 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 dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes such as Zn-protein complexes, and non-ionic surfactants such as polyethylene glycol (PEG).

[0296] In certain embodiments, pharmaceutical compositions comprising the antibody constructs or ADCs may be in the form of a sterile injectable aqueous or oleaginous solution or suspension. Such suspensions may be formulated using suitable dispersing or wetting agents and / or suspending agents that are known in the art. The sterile injectable solution or suspension may comprise the antibody construct or ADC in a non-toxic parentally acceptable diluent or solvent. Acceptable diluents and solvents that may be employed include, for example, 1,3-butanediol, water, Ringer’s solution or isotonic sodium chloride solution. In addition, sterile, fixed oils may be employed as a solvent or suspending medium. For this purpose, various bland fixed oils may be employed, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables. Adjuvants such as local anesthetics, preservatives and / or buffering agents may also be included in the injectable solution or suspension.

[0297] In certain embodiments, pharmaceutical compositions comprising the antibody constructs or ADCs may be formulated for intravenous administration to a subject, for example a human. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and / or a local anaesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as adry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[0298] 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).METHODS OF USE

[0299] Certain aspects of the present disclosure relate to the therapeutic or diagnostic use of the bispecific FRa x NaPi2b antibody constructs, anti-FRa antibody constructs and ADCs. A wide variety of cancers express FRa and / or NaPi2b and certain embodiments of the present disclosure thus relate to the methods of using the antibody constructs and ADCs in the treatment or diagnosis of cancers expressing FRa, NaPi2b, or both FRa and NaPi2b.

[0300] Certain embodiments relate to methods of inhibiting the growth of tumor cells expressing FRa, NaPi2b, or both FRa and NaPi2b, comprising contacting the cells with an FRa x NaPi2b antibody construct or ADC described herein. The cells may be in vitro or in vivo. In certain embodiments, the FRa x NaPi2b antibody constructs and ADCs may be used in methods of treating a subject having a cancer or tumor expressing FRa, NaPi2b, or both FRa and NaPi2b.

[0301] Cancers that express FRa and / or NaPi2b are typically solid tumors. Examples include, but are not limited to, gynecological cancers (such as ovarian cancer, endometrial cancer, cervical cancer and uterine cancer), lung cancers (such as non-small cell lung cancer (NSCLC)), mesothelioma, breast cancer (including triple negative breast cancer (TNBC)), colorectal cancer, biliary tract cancer, pancreatic cancer and esophageal cancer. Certain embodiments of the present disclosure relate to methods of treating a FRa and / or NaPi2b-positive cancer with an FRa x NaPi2b antibody construct or ADC as described herein, where the cancer is ovarian cancer, endometrialcancer, lung cancer (such as non-small cell lung cancer (NSCLC)), mesothelioma, breast cancer, colorectal cancer, biliary tract cancer, pancreatic or esophageal cancer.

[0302] Certain embodiments of the present disclosure relate to methods of treating a FRa and / or NaPi2b-positive gynecological cancer, such as ovarian cancer, endometrial cancer, cervical cancer or uterine cancer, with an FRa x NaPi2b antibody construct or ADC as described herein. Some embodiments of the present disclosure relate to methods of treating a FRa and / or NaPi2b-positive ovarian cancer with an FRa x NaPi2b antibody construct or ADC as described herein.

[0303] Treatment of an FRa and / or NaPi2b-positive cancer may result in one or more of: alleviation of symptoms, shrinking the size of the tumor, inhibiting growth of the tumor, diminishing one or more direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, improving survival, increasing progression-free survival, remission and / or improving prognosis.

[0304] In certain embodiments, when used in the treatment of cancer, the antibody constructs or ADCs may be administered systemically to the subject to be treated, for example, by bolus injection or continuous infusion into the subject’s bloodstream. In certain embodiments, when used in the treatment of cancer, the antibody constructs or ADCs may be administered to the subject locally at the site to be treated.

[0305] It is contemplated that the antibody constructs or ADCs may be used alone or in combination with one or more known chemotherapeutic or immunotherapeutic agents typically used in the treatment of cancer. Combinations of the antibody constructs or ADCs with standard chemotherapeutics or immunotherapeutics may act to improve the efficacy of the chemotherapeutic or immunotherapeutic and, therefore, may improve standard cancer therapies. This application can be important in the treatment of drug-resistant cancers which are not responsive to standard treatment. When used in conjunction with one or more known chemotherapeutic or immunotherapeutic agents, the antibody construct or ADC may be administered prior to, or after, administration of the chemotherapeutic or immunotherapeutic agents, or they may be administered concomitantly.

[0306] The dosage of the antibody construct or ADC to be administered is not subject to defined limits, but it will a therapeutically effective amount. A “therapeutically effective amount” refers to that amount of an antibody construct or ADC described herein which, when administered to a subject, is sufficient to effect a treatment of the particular indication. A therapeutically effective amount of the antibody construct or ADC in respect of cancer treatment may, for example, have one or more of the following effects: reduce the number of cancer cells, reduce the tumor size, inhibit cancer cell infiltration into peripheral organs, inhibit tumor metastasis, inhibit tumor growth, increase survival time and / or relieve to some extent one or more of the symptoms associated with the cancer. For cancer therapy, efficacy may alternatively be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0307] Certain embodiments relate to methods of detecting the presence of FRa and / or NaPi2b in a biological sample, such as a sample comprising cells or tissue, using an antibody construct described herein. In some embodiments, the biological sample may have been taken from a patient, for example, a patient known or suspected to have cancer. Some embodiments relate to methods of detecting the presence of FRa and / or NaPi2b in a biological sample that comprise contacting the sample with an antibody construct described herein.

[0308] Certain embodiments relate to methods of diagnosing a disorder associated with increased expression of FRa and / or NaPi2b, such as a cancer, using an antibody construct described herein. The method of diagnosis may be an in vivo method in which the antibody construct is administered to the subject, or it may be an in vitro method in which a sample taken from the subject is contacted with the antibody construct. For in vivo methods, administration may be systemic or local.

[0309] In methods of detecting the presence of FRa and / or NaPi2b or diagnosing a disorder associated with increased expression of FRa and / or NaPi2b, the antibody construct may be labelled with a detectable label, such as a fluorescent, luminescent, chromophoric, chemiluminescent, radioactive or enzymatic label as is known in the art.PHARMACEUTICAL KITS

[0310] Certain embodiments relate to pharmaceutical kits (or articles of manufacture) comprising a bispecific FRa x NaPi2b antibody construct, anti-FRa antibody construct or ADC as described herein.

[0311] The kit typically will comprise a container holding the antibody construct or ADC and a label and / or package insert on or associated with the container. The label or package insert contains instructions customarily included in commercial packages of therapeutic products, providing information about the indications, usage, dosage, administration, contraindications and / or warnings concerning the use of such therapeutic products. The label or package insert may further include a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, for use or sale for human or animal administration. In some embodiments, the container may have a sterile access port. For example, the container may be an intravenous solution bag or a vial having a stopper that may be pierced by a hypodermic injection needle.

[0312] In addition to the container holding the antibody construct or ADC, the kit may optionally comprise one or more additional containers comprising 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 dextrose solution), other buffers or diluents.

[0313] Suitable containers include, for example, bottles, vials, syringes, intravenous solution bags, and the like. The containers may be formed from a variety of materials such as glass or plastic. If appropriate, one or more components of the kit may be lyophilized or provided in a dry form, such as a powder or granules, and the kit can additionally contain a suitable solvent for reconstitution of the lyophilized or dried component(s).

[0314] The kit may further include other materials desirable from a commercial or user standpoint, such as filters, needles, and syringes.

[0315] The following Examples are provided for illustrative purposes and are not intended to limit the scope of the invention in any way.EXAMPLES

[0316] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within the ordinary skill in the art. Such techniques are explained fully in the literature. See, e.g., T. E. Creighton, Proteins: Structures and Molecular Properties (W. H. Freeman and Company, 1993); A. L. Lehninger, Biochemistry (Worth Publishers, Inc., current addition); Sambrook, et al., Molecular Cloning: A Laboratory Manual (2nd Edition, 1989); Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.); Remington's Pharmaceutical Sciences, 18th Edition (Easton, Pennsylvania: Mack Publishing Company, 1990); Carey and Sundberg Advanced Organic Chemistry 3rdEd. (Plenum Press) Vols A and B(1992).EXAMPLE 1: ANTI-FRa ANTIBODY 76 - PREPARATION, HUMANIZATION, PRODUCTION AND CHARACTERIZATION1.1 Antibody Generation

[0317] Antibody constructs that specifically bind human FRa were generated by immunizing mice with human cells over-expressing FRa as summarized below.

[0318] HEK293-6E cells (National Research Council of Canada) were transiently transfected with a pTT5-based expression plasmid (National Research Council of Canada) encoding human FRa (pTT5- FRa, expressing the sequence of FRa as set forth in SEQ ID NO: 1), according to manufacturer’s instructions for Lipofectamine™ 2000 (Thermo Fisher Scientific, Waltham, MA).Human FRa Amino Acid Sequence [SEQ ID NO:1] MAQRMTTQLLLLLVWVAWGEAQTRIAWARTELLNVCMNAKHHKEKPGPEDKLHEQ CRPWRKNACCSTNTSQEAHKDVSYLYRFNWNHCGEMAPACKRHFIQDTCLYECSPNL GPWIQQVDQSWRKERVLNVPLCKEDCEQWWEDCRTSYTCKSNWHKGWNWTSGFNKC AVGAACQPFHFYFPTPTVLCNEIWTHSYKVSNYSRGSGRCIQMWFDPAQGNPNEEVAR FYAAAMSGAGPWAAWPFLLSLALMLLWLLS

[0319] Five CD1 and five B6xl29 mice were subcutaneously immunized with transfected HEK293-6E cells over 63 days, after which blood was drawn and spleens harvested. Anti-human FRa antibody titers were determined by flow cytometry using CHO-S cells expressing human FRa. All ten mice mounted a significant response against human FRa.

[0320] Splenocytes from the five strongest mice were subsequently pooled and used for hybridoma generation. Sp2 / 0 cells (ATCC) were mixed with IgG+ B cells isolated from spleens and fused using an ECM® 2001 electrofusion instrument (BTX, Harvard Bioscience, Holliston, MA) with optimized settings. Following overnight recovery, hybridomas were diluted and plated in selection media containing the following final concentrations of substituents: 100 pM hypoxanthine, 0.4 pM aminopterin and 16 pM thymidine. Following 14 days of selection, hybridomas were diluted to an average of one cell per well and plated into 96-well plates. Cell supernatants containing secreted antibodies were assessed for binding on CHO-S cells transfected with the same plasmid used to transfect HEK293-6E cells (pTT5-huFRa). Confirmed wells were pooled and subcloned into 384-well plates. Cell supernatants containing secreted antibodies were subsequently assessed for binding. Hybridoma cells from wells containing supernatants having antibodies that bound FRa were harvested for sequencing.

[0321] The murine VH and VL sequences for the anti-human FRa antibody 76 were used to prepare a mouse-human chimeric IgGl / kappa antibody construct, v31625, as follows. Coding sequences for antibody variable regions were cloned in frame into a human IgGl expression vector (with human IgGl constant region starting with alanine 118 according to Kabat numbering) or a human C kappa expression vector (with human C kappa constant region starting at arginine 108 according to Kabat numbering), both expression vectors based on pTT5. The activities of the resultant recombinant chimeric antibody construct were confirmed in specificity binding assays and were found comparable to the parental antibody.

[0322] The CDR sequences of v31625 are provided in Table 1.1, and the mouse VH and VL sequences are provided in Table 1.2.Table 1.1: CDR Sequences of Anti-FRa Antibody v31625Numbering Heavy Chain CDR1 SEQ Heavy Chain CDR2 SEQ Heavy Chain CDR3 SEQ System ID NO ID NO ID NO IMGT GYRFTDYE 2 IDPETGGT 3 STYSYYSYDRFAY 4 Rabat DYEMH 5 RIDPETGGTANNQNFRG 6 YSYYSYDRFAY 7 Chothia GYRFTDY 8 DPETGG 9 YSYYSYDRFAY 7 AbM GYRFTDYEMH 10 RIDPETGGTA 11 YSYYSYDRFAY 7 Contact TDYEMH 12 WIGRIDPETGGTA 13 STYSYYSYDRFA 14 Numbering Light Chain CDR1 SEQ Light Chain CDR2 SEQ Light Chain CDR3 SEQ System ID NO ID NO ID NO IMGT QSLLHSNGNTY 15 RVS 16 FQGTHVPHT 17 Rabat RSSQSLLHSNGNTYLY 18 RVSNRFS 19 FQGTHVPHT 17 Chothia RSSQSLLHSNGNTYLY 18 RVSNRFS 19 FQGTHVPHT 17 AbM RSSQSLLHSNGNTYLY 18 RVSNRFS 19 FQGTHVPHT 17 Contact LHSNGNTYLYWY 20 LLIYRVSNRF 21 FQGTHVPH 22Table 1.2: VH and VL Sequences of Anti-FRa Antibody v31625Sequence SEQ ID NO VH EVQLQQSGAELARPGASVKLSCKASGYRFTDYEMHWVKQTPVHGLEWIG 23 RIDPETGGTANNQNFRGKATLTADKSSNTVYMELRSLTSEDSAVYFCSTYS YYSYDRFAYWGQGTLVTVSA VL DVLMTQTPLSLPVSLGNQVSISCRSSQSLLHSNGNTYLYWYLQKPGQSPRL 24 LIYRVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYFCFQGTHVPHTFGSGTKLELK1.2 Humanization

[0323] The chimeric anti-human FRa antibody construct, variant v31625, was humanized as described below.

[0324] Sequence alignment of the mouse VH and VL sequences of v31625 to respective human germline sequences identified IGHV1 -46*01 and IGKV4-l*01 as the closest, as well as frequent, human germline sequences. These sequences, together with respectively IGHJ4*01 and IGKJ2*01 joining region germline sequences, were selected. CDR sequences according to the AbM definition (see Table 1.1) were ported onto the framework of these selected human germline sequences. Back mutations to mouse residues in the resultant sequences at positions judged likelyto be important for the retention of binding affinity to antigen, FRa, were included creating several humanized sequences in which generated sequences were built on the previous sequence, and where the first humanized sequence contained no back mutations. None of the variants modified the CDRs of the parent antibody as defined by the AbM method.

[0325] This process resulted in four variable heavy chain humanized sequences and three variable light chain humanized sequences. Full light chain sequences containing humanized light chain variable domain (VL) and human kappa light chain constant domain (kappa CL) were assembled. Two full heavy chains for each humanized sequence were assembled as follows:

[0326] Chain A: full heavy chain sequences containing humanized heavy chain variable domain (VH) and hlgGl heavy chain constant domains (CHI, hinge, CH2, CH3) containing the mutations T350V_L351Y_F405A_Y407V in the CH3 domain for heterodimeric pairing, and

[0327] Chain B: full heavy chain sequences containing humanized heavy chain variable domain (VH) and hlgGl heavy chain constant domains (CHI, hinge, CH2, CH3) containing the mutations T350V T366L K392L T394W (complementary to the chain A mutations) in the CH3 domain for heterodimeric pairing.

[0328] Monoclonal antibody (mAb) variants were then assembled such that each of the four humanized heavy chain pairs (chain A and B) was paired with each of the three humanized light chains to provide 12 humanized variants that were evaluated experimentally.1.3 Production and QC of Humanized Antibody Constructs

[0329] Each of the 12 humanized antibody constructs was produced in full-size antibody (FSA) format containing full-length heavy chains (A and B) and two identical kappa light chains.

[0330] The full-length heavy chain A contained the human CHl-hinge-CH2-CH3 domain sequence of IGHGl*01 with T350V_L351Y_F405A_Y407V mutations ([SEQ ID NO:25]; see Table 1.3) and full-length heavy chain B contained the human CHl-hinge-CH2-CH3 domain sequence of IGHGl*01 with T350V T366L K392L T394W mutations ([SEQ ID NO:26]; see Table 1.3). The light chain contained human kappa CL sequence of IGKC*01 [SEQ ID NO:27]; see Table 1.3).Table 1.3: Human Constant Heavy and Light Chain SequencesSequence SEQ ID NO CHl-hinge-CH2-CH3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS 25 (IGHGl*01) \chain A WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYV YPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSF ALVSKLTVDKSRWQQGNVF SC S V MHEALHNHYTQKSLSLSPG CHl-hinge-CH2-CH3 ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS 26 (IGHGl*01) \chain B WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVL PPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQPENN YLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPG CL (kappa) RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ 27 (IGKC*01) WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0331] Each of the humanized VH domain sequences was appended to a human CH1-hinge-CH2-CH3 domain sequence of IGHGl*01 with T350V_L351Y_F405A_Y407V mutations (chain A) and to a human CHl-hinge-CH2-CH3 domain sequence of IGHGl*01 with T350V_T366L_K392L_T394W mutations (chain B) to provide eight humanized full heavy chain sequences (four chain As and four chain Bs). Each of the humanized VL domain sequences was appended to the human kappa CL sequence of IGKC*01 to provide three humanized light chain sequences. All sequences were reverse translated to DNA, codon optimized for mammalian expression and gene synthesized.

[0332] Heavy chain vector inserts comprising a signal peptide (artificially designed sequence: MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO:28) (Barash et al., 2002, Biochem and Biophys Res. Comm., 294:835-842)) and the heavy chain clone terminating atresidue G446 (EU numbering) of the CH3 domain were ligated into a pTT5 vector to produce heavy chain expression vectors. Light chain vector inserts comprising the same signal peptide were ligated into a pTT5 vector to produce light chain expression vectors. The resulting heavy and light chain expression vectors were sequenced to confirm correct reading frame and sequence of the coding DNA. Sequences of the humanized VH and VL sequences are provided in Table 1.4 below.Table 1.4: Amino Acid Sequences of Humanized VH and VL SequencesName Amino Acid Sequence SEQ ID NOHl QVQLVQSGAEVKKPGASVKVSCKASGYRFTDYEMHWVRQAPGQGLE 30 WMGRIDPETGGTAYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVY YC ARYS YYS YDRF AYWGQGTL VT VS SH2 QVQLVQSGAEVKKPGASVKVSCKASGYRFTDYEMHWVRQAPGQGLE 29 WMGRIDPETGGTAYAQKFQGRVTMTADTSTSTVYMELSSLRSEDTAV YYCSTYSYYS YDRF AYWGQGTL VTVS SH3 QVQLVQSGAEVKKPGASVKLSCKASGYRFTDYEMHWVRQAPGQGLE 31 WIGRIDPETGGTAYAQKFQGRATLTADTSTSTVYMELSSLRSEDTAVYY CSTYSYYSYDRF AYWGQGTL VTVSSH4 QVQLVQSGAEVKKPGASVKLSCKASGYRFTDYEMHWVRQAPGQGLE 32 WIGRIDPETGGTAYAQKFQGRATLTADKSTSTVYMELSSLRSEDTAVY YCSTYSYYSYDRF AYWGQGTL VTVSS LI DIVMTQSPDSLAVSLGERATINCRSSQSLLHSNGNTYLYWYQQKPGQPP 33 KLLIYRVSNRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGTHV PHTFGQGTKLEIKL2 DIVMTQSPDSLAVSLGERVTINCRSSQSLLHSNGNTYLYWYQQKPGQPP 34 KLLIYRVSNRFSGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCFQGTH VPHTFGQGTKLEIKL3 DVVMTQSPDSLAVSLGERVTINCRSSQSLLHSNGNTYLYWYQQKPGQP 35 PKLLIYRVSNRFSGVPDRFSGSGSGTDFTLTISSVQAEDVAVYYCFQGTH VPHTFGQGTKLELK

[0333] Cloning and Transfection Conditions: Gene products were cloned (by GenScript BioTech Corporation, Piscataway, NJ) into the pTT5™ mammalian expression vector and transiently expressed in CHO 3E7 cells.

[0334] CHO-3E7 cells were cultured in FreeStyle™ F17 expression medium (Gibco, Cat # A-1383501) supplemented with 4 mM L-glutamine (HyClone / Cytivia, Cat # SH30034.01) and 0.1% Pluronic F-68 ™ (Gibco, Cat# 24040-032) at 37°C. A transfection mixture of 200ug DNA (lOOug DNA coding for the Ab and lOOug of Salmon Sperm DNA, pTT22-AKTdd, GFP) was complexed with PEI MAX® 40,000 (Polyscience Inc., Cat # 24765-1) at a 1:4 (w / w) DNA / PEI ratio. Transfections were performed with DNA / PEI complexes in 200mL cell culture volumes supplemented with IX Antibiotic / Antimycotic Solution (HyClone, Cat # SV30079.01) and incubated at 37°C for 24 hrs. This was followed by a temperature shift to 32°C for 6 days with 0.5 mM valproic acid (Sigma, Cat # P4543) and 1.0% (w / v) peptone (Tryptone Nl, Organotechnie, Cat # 19553). GFP measurements were taken 1 day post transfection to determine transfection efficiency.

[0335] Productions were harvested 7 days post transfection. Prior to filtration on 0.22uM Millipore filter, cells were centrifuged. Clarified supernatants were analyzed by small scale HPLC-PA for titer measurements.

[0336] Protein A Affinity Chromatography and Preparatory Size Exclusion Chromatography: Clarified culture medium was loaded onto a MabSelect™SuRe™ (Cytiva) Protein A column and washed with Dulbecco’s phosphate buffered saline (DPBS) (pH 7.0-7.2). Protein was eluted with 1 OOmM citrate buffer at pH 3.0 and neutralized with 10% (v / v) IM HEPES solution to yield a final pH of 6-7. Samples then underwent preparatory SEC chromatography (Superdex 200 16 / 60 or 26 / 60, Cytiva) in DPBS mobile phase. All samples were concentrated using Vivaspin® TURBO units (Sartorius). Protein was quantified based on absorbance at 280nm (A280nm).

[0337] Caliper: Purity of samples was assessed by electrophoresis under non-reducing and reducing conditions using the High Throughput Protein Express assay and Caliper LabChip® GXII or GXII Touch HT (Perkin Elmer, Waltham, MA). Procedures were carried out according to HT Protein Express LabChip® User Guide version 2 with the following modifications. Antibody samples, at either 2pl or 5 pl (concentration range 5-2000 ng / pl), were added to separate wells in 96 well plates (BioRad, Hercules, CA, Cat # HSP9601B) along with 7pl of HT Protein Express Sample Buffer (Perkin Elmer, Cat # 760328). Antibody samples were then denatured at 70°C for15 mins. The LabChip® instrument was operated using the HT Protein Express Chip (Perkin Elmer, Waltham, MA) and the Ab-200 assay setting.

[0338] The yields following preparatory SEC for most of the 12 humanized antibody variants were similar, ranging from approximately 8-14 mg (from 200mL culture). The Caliper results for these antibody samples are shown in Fig. 3, which shows that non-reducing (NR) and reducing (R) Caliper reflected predominantly a single species corresponding to full-size antibody and intact heavy and light chains.

[0339] UPLC-SEC: Species homogeneity of the humanized antibody variants and parental mouse-human chimeric antibody variant samples was assessed by UPLC-SEC.

[0340] UPLC-SEC was performed using a Waters Acquity BEH200 SEC column (2.5 mL, 4.6 x 150 mm, stainless steel, 1.7 pm particles) (Waters Ltd., Mississauga, ON) set to 30°C and mounted on a Waters Acquity UPLC™ H-Class Bio system with a photodiode array (PDA) detector. The mobile phase was Dulbecco’s phosphate buffered saline (DPBS) with 0.02% Tween 20 pH 7.4 and the flow rate was 0.4 mL / min. Total run time for each injection was 7 min with a total injection of lOug of protein sample. Elution was monitored by UV absorbance in the range 210-500 nm and chromatograms were extracted at 280 nm. Peak integration was performed using Waters Empower® 3 software employing the Apex Track™ and detect shoulders features.

[0341] UPLC-SEC profiles for representative humanized antibody variants are shown in Fig. 4. The profiles are reflective of high species homogeneity. The samples from the remaining humanized antibody variants had similar profiles to those shown in Fig. 4.1.4 Binding Assessment by Octet and Thermal Stability by Differential Scanning Calorimetry

[0342] Octet / DSC: Antigen binding was assessed using the ForteBio Octet™ RED96 system by cycling through the following steps: loading of antibodies (5 pg / mL) onto AHC biosensors over 500s; stabilization of baseline for 60s; association to recombinant His-tagged human FRa (Acrobiosystem) at multiple relevant concentrations spanning the expected KD for 400s. Dissociation was recorded for 550s and regeneration was performed by cycling 3 times between 10 mM glycine pH 1.5 (5s) and the assay buffer (5s) before proceeding to the next antibody. The assay buffer used was KB buffer (kinetics buffer, composed of PBS pH 7.4, 0.1 %BSA, 0.02 % Tween 20, 0.05% sodium azide) supplemented with 0.06% Tween 20 and 1% BSA. The experiment was conducted at 30°C with a shake speed of 1000 rpm.

[0343] Data analysis was performed using the ‘Data analysis software 9.0’ (ForteBio, Fremont, CA). The reference-subtracted binding curves were globally fitted to the 1:1 interaction model to generate the binding kinetic parameters kon, koff, and the dissociation constant KD.

[0344] Humanized antibody variants based on Hl (HILI, H1L2 and H1L3) did not exhibit any antigen binding. All humanized antibody variants based on H2 and H4 exhibited similar binding to the parental mouse-human chimeric antibody (v31625), specifically within a range of ~2x higher KD. Humanized antibody variants based on H3 showed antigen binding that was impacted by more than 2x (i.e >2x higher KD).

[0345] DSC: The thermal stability of the humanized antibody variants was assessed by differential scanning calorimetry (DSC) as described below.

[0346] 400 mL of purified samples primarily at concentrations of 0.4 mg / mL in PBS were used for DSC analysis with 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 placed before each sample injection for referencing. Each sample was scanned from 20°C to 100°C at a 60°C / hr rate, with low feedback, 8 sec filter, 3 min pre-scan thermostat and 70 psi nitrogen pressure. The resulting thermograms were referenced and analyzed using Origin 7 software (OriginLab Corporation, Northampton, MA) to determine melting temperature (Tm) as an indicator of thermal stability.

[0347] The Fab Tm values for selected humanized variants are shown in Table 1.4. These variants exhibited increased thermal stability compared to the parental antibody, v31625 (Fab Tm of ~69.5°C), with Fab Tm values ranging from ~82-86°C.Table 1.4: Binding Affinity to Human FRa and Thermal Stability of Selected Humanized VariantsVariant Heavy & Light KD (M) Fab Tm (°C) Chain Compositionv31625 (parental) Chimera 9.8E-09 69.5 v32977 H2L1 1.7E-08 82.3 v32978 H2L2 1.8E-08 84.6 v32979 H2L3 1.9E-08 84.1 v32983 H4L1 2.0E-08 82.0 v32984 H4L2 1.9E-08 86.0 v32985 H4L3 1.6E-08 85.8EXAMPLE 2: FUNCTIONAL CHARACTERIZATION OF ANTI-FRa AND ANTI-NAPI2B MONOSPECIFIC ANTIBODIES - CELLULAR BINDING AND INTERNALIZATION

[0348] Three anti-FRa monospecific antibodies (10L18, 76 and 2L16) and one anti-NaPi2b monospecific antibody (12A10) in full-sized antibody and one-armed antibody formats were assessed for binding to their target antigen and internalization as described below. The anti-FRa antibody 10L18 is described in International Publication No. WO 2023 / 178451 and the anti-FRa antibody 2L16 is described in International Publication No. WO 2021 / 207827. The anti-NaPi2b antibody 12A10 is described in International Patent Application No. PCT / CA2023 / 051386. The variant numbers, CDR (by AbM numbering), VH and VL sequences of each of the antibodies are provided in Table 2.1. The CDR sequences for as defined by IMGT, Kabat, Chothia and Contact numbering systems are provided in Table 2.1 A, 2. IB and 2.1C (antibodies 10L18, 2L16 and 12A10, respectively) and in Table 1.1 (antibody 76).Table 2.1: CDR (by AbM), VH and VL Sequences for Anti-FRa and Anti-NaPi2b Antibodies Variant Target Format1Sequences2SEQ Antigen ID NO FRa FSA HCDR1: GFSLSSYGVS 36HCDR2: AVNSGGSAY 37Variant Target Format1Sequences2SEQ Antigen ID NO v36675 HCR3: SGSGYPMDYLAI 38 (humanizedLCDR1: QASQSIGDWLA 39 10L18)LCDR2: EASTLAS 40 LCDR3: QQGYGRSNVDNI 41 VH: 42 EVQLLESGGGLVQPGGSLRLSCAVSGFSLS SYGVSWVRQAPGKGLEWIGAVNSGGSAY YADSVKGRSTISRDNSKNTVYLQMNSLRA EDTAVYYCARSGSGYPMDYLAIWGQGTL VTVSS VL: 43 DYQMTQSPSSVSASVGDRVTITCRASQSIG DWLAWYQQKPGKAPKLLIYEASTLASGV PSRFSGSGSGTDFTLTISSVQPEDAATYYC QQGYGRSNVDNIFGGGTKVEVKv32597 FRa OAA As for v36675(basedonv36675)v32977 FRa FSA HCDR1: GYRFTDYEMH 10 (humanized HCDR2: RIDPETGGTA 11 76)HCR3: YSYYSYDRFAY 7 LCDR1: RSSQSLLHSNGNTYLY 18 LCDR2: RVSNRFS 19 LCDR3: FQGTHVPHT 17 VH: 29 Q VQLVQ SGAEVKKPGAS VKVSCKASGYR FTDYEMHWVRQAPGQGLEWMGRIDPETG GTAYAQKFQGRVTMTADTSTSTVYMELSS LRSEDTAVYYC STYS YYS YDRFAYWGQGT LVTVSS VL: 33 DIVMTQSPDSLAVSLGERATINCRSSQSLL HSNGNTYLYWYQQKPGQPPKLLIYRVSNR FSGVPDRFSGSGSGTDFTLTISSLQAEDVAV YYCFQGTHVPHTFGQGTKLEIKVariant Target Format1Sequences2SEQ Antigen ID NO v40456 FRa OAA As for v32977(basedonv32977)v33283 FRa FSA HCDR1: GIDFSSYYDMS 44 (humanized HCDR2: AIYTGSGSTY 45 2L16)HCR3: GDANSGWGLKL 46 LCDR1: RASEDIYSLLA 47 LCDR2: RASTLES 48 LCDR3: QSTYGGSSNSYGRNG 49 VH: 50 EVQLVESGGGLVQPGGSLRLSCAASGIDF S SYYDMSWVRQAPGKGLEWIGAIYTGSGS TYYAYSVKGRFTISKDNSKNTVYLQMNSL RAEDTAVYYCARGDANSGWGLKLWGQG TLVTVSS VL: 51 DIQMTQSPSSLSASVGDRVTITCRASEDIYS LLAWYQQKPGKAPKLLIYRASTLESGVPS RFSGSGSGTDFTLTISSLQPEDFATYYCQST YGGSSNSYGRNGFGGGTKVEIKv40455 FRa OAA As for v33283(basedonv33283)v38591 NaPi2b FSA HCDR1: GFTFTSYNVH 52 (humanizedHCDR2: AISPGNGDLS 53 12A10)HCR3: GRTGMGAVDY 54 LCDR1: RASQDVYSYLN 55 LCDR2: YTSRLQS 56 LCDR3: QQYNIFPWT 57 VH: 58 Q VQLVQ SGAEVKKPGAS VKVSCKASGFTF TSYNVHWVRQAPGQGLEWMGAISPGNGDLSYAQKFQGRVTMTRDTSTSTVYMELSSVariant Target Format1Sequences2SEQ Antigen ID NO LRSEDTAVYYCARGRTGMGAVDYWGQGT LVTVSS VL: 59 DVQMTQSPSSLSASVGDRVTITCRASQDV YSYLNWYQQKPGKAVKLLIYYTSRLQ SG VPSRFSGSGSGTDYTLTISSLQPEDFATYYC QQYNIFPWTFGQGTKLEIKv32164 NaPi2b OAA As for v38591(basedonv38591)1FSA = full-sized antibodyOAA = one-armed antibody2CDR sequences defined by AbM numberingTable 2.1A: CDR Sequences of Anti-FRa Antibody 10L18Numbering Heavy Chain CDR1 SEQ ID Heavy Chain CDR2 SEQ Heavy Chain CDR3 SEQ System NO ID NO ID NO IMGT GFSLSSYG 196 VNSGGSA 197 ARSGSGYPMDYLAI 198 Rabat SYGVS 199 AVNSGGSAYYANWARS 200 SGSGYPMDYLAI 38 Chothia GFSLSSY 201 NSGGS 202 SGSGYPMDYLAI 38 AbM GFSLSSYGVS 36 AVNSGGSAY 37 SGSGYPMDYLAI 38 Contact SSYGVS 203 WIGAVNSGGSAY 204 ARSGSGYPMDYLA 205 Numbering Light Chain CDR1 SEQ ID Light Chain CDR2 SEQ Light Chain CDR3 SEQ System NO ID NO ID NO IMGT QSIGDW 206 EAS 207 QQGYGRSNVDNI 41 Rabat QASQSIGDWLA 39 EASTLAS 40 QQGYGRSNVDNI 41 Chothia QASQSIGDWLA 39 EASTLAS 40 QQGYGRSNVDNI 41 AbM QASQSIGDWLA 39 EASTLAS 40 QQGYGRSNVDNI 41 Contact GDWLAWY 208 LLIYEASTLA 209 QQGYGRSNVDN 210Table 2.1B: CDR Sequences of Anti-FRa Antibody 2L16Numbering Heavy Chain CDR1 SEQ ID Heavy Chain CDR2 SEQ ID Heavy Chain CDR3 SEQ System NO NO ID NO IMGT GIDFSSYYD 211 IYTGSGST 212 ARGDANSGWGLRL 213 Rabat SYYDMS 214 AIYTGSGSTYYAYSVRG 215 GDANSGWGLRL 46 Chothia GIDFSSYY 216 YTGSGS 217 GDANSGWGLRL 46AbM GIDFSSYYDMS 44 AIYTGSGSTY 45 GDANSGWGLKL 46 Contact SSYYDMS 218 WIGAIYTGSGSTY 219 ARGDANSGWGLK 220 Numbering Light Chain CDR1 SEQ ID Light Chain CDR2 SEQ ID Light Chain CDR3 SEQ System NO NO ID NO IMGT EDIYSL 221 RAS 222 QSTYGGSSNSYGRNG 49 Kabat RASEDIYSLLA 47 RASTLES 48 QSTYGGSSNSYGRNG 49 Chothia RASEDIYSLLA 47 RASTLES 48 QSTYGGSSNSYGRNG 49 AbM RASEDIYSLLA 47 RASTLES 48 QSTYGGSSNSYGRNG 49 Contact YSLLAWY 223 LLIYRASTLE 224 QSTYGGSSNSYGRN 225Table 2.1C: CDR Sequences of Anti-NaPi2b Antibody 12A10Numbering Heavy Chain CDR1 SEQ Heavy Chain CDR2 SEQ Heavy Chain CDR3 SEQ System ID NO ID NO ID NO IMGT GFTFTSYN 226 ISPGNGDL 227 ARGRTGMGAVDY 228 Kabat SYNVH 229 AISPGNGDLSYAQKFQG 230 GRTGMGAVDY 54 Chothia GFTFTSY 231 SPGNGD 232 GRTGMGAVDY 54 AbM GFTFTSYN VH 52 AISPGNGDLS 53 GRTGMGAVDY 54 Contact TSYNVH 233 WMGAISPGNGDLS 234 ARGRTGMGAVD 235 Numbering Light Chain CDR1 SEQ Light Chain CDR2 SEQ Light Chain CDR3 SEQ System ID NO ID NO ID NO IMGT QDVYSY 236 YTS 237 QQYNIFPWT 57 Kabat RASQDVYSYLN 55 YTSRLQS 56 QQYNIFPWT 57 Chothia RASQDVYSYLN 55 YTSRLQS 56 QQYNIFPWT 57 AbM RASQDVYSYLN 55 YTSRLQS 56 QQYNIFPWT 57 Contact YSYLNWY 238 LLIYYTSRLQ 239 QQYNIFPW 240

[0349] The ability of humanized antibody variants v36675, v32597, v32977, v40456, v33283, v40455, v38591, and v32164 to bind to, and internalize with, FRa and NaPi2b expressed on the surface of tumor cell lines IGROV-1 (ovarian adenocarcinoma), HCC-78 (lung carcinoma), JEG-3 (placental choriocarcinoma), H441 (lung adenocarcinoma), T-47D (breast carcinoma), and TOV-21G (ovarian carcinoma) was evaluated by flow cytometry. IGROV-1 expresses endogenous FRa and NaPi2b at high levels; HCC-78 and H441 express FRa and NaPi2b at moderate-to-low levels; JEG-3 and T-47D express endogenous FRa at moderate levels and do not express NaPi2b; TOV-21G expresses endogenous NaPi2b at moderate levels and does not express FRa. Levels ofexpression of FRa and NaPi2b were determined by surface protein quantification as described in Example 3.2.1 Cell Binding

[0350] Cells were maintained under standard culture conditions (37°C / 5% CO2) until assay set-up; IGROV-1, HCC-78, H441, and T-47D cells were cultured in RPMI 1640, ATCC modification (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA); JEG-3 cells were cultured in Minimum Essential Medium (MEM) (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS. TOV-21G cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA) supplemented with 15% FBS.

[0351] For cellular binding, cells were removed from culture vessels using Cell Dissociation Buffer (Invitrogen, Waltham, MA), seeded at 50,000 cells / well in coni cal -bottom 96-well plates, and treated with test antibody for 24 hours at 4°C to prevent internalization. Palivizumab (anti-RSV antibody, v21995) was included as a negative control. Following incubation, cells were washed and stained with anti-human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-605-098) at 4°C for 30 minutes. Following incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1,000 minimum events collected per well. The AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-human AF647 binding) was calculated for the live cell population using FlowJo™ Version 10.8.1 (BD Biosciences, Franklin Lake, NJ) and plotted for each test antibody using GraphPad Prism Version 9 (GraphPad Software, La Jolla, CA).

[0352] Cellular binding results are shown in Table 2.2. Anti-FRa full-sized antibodies (FSA) demonstrated cellular binding on FRa-expressing cell lines IGROV-1, JEG-3, minimally on HCC-78, and no binding on FRa-negative TOV-21G cells. Similarly, anti -FRa one-armed antibodies (OAA) demonstrated cellular binding on IGROV-1, JEG-3, minimally on HCC-78, and no binding on TOV-21G cells, with overall greater Ka values than their paratope-matched FSA. Anti-NaPi2b FSA, v38591, demonstrated cellular binding on NaPi2b-expressing cell lines IGROV-1, HCC-78, and TOV-21G, and no binding on NaPi2b-negative JEG-3 cells. Similarly,anti-NaPi2b OAA, v32164, demonstrated cellular binding on NaPi2b-expressing cell lines IGROV-1, HCC-78, and TOV-21G, with overall greater Ka and Bmax values than its paratopematched FSA, and no binding on NaPi2b-negative JEG-3 cells. Negative control palivizumab (v21995) showed no cellular binding, as expected.Table 2.2: Cellular BindingCell LineTargetVariant Format1IGROV-1 HCC-78 JEG-3 TOV-21G AntigenKd(nM)v36675 (10L18) FRa FSA 0.17 0.24 0.29 NB2v32597 (10L18) FRa OAA 11.10 12.74 8.45 NB v32977 (76) FRa FSA 0.16 not assayed not assayed not assayedV4O456 (76) FRa OAA 1.53 6.96 3.29 NB v33283 (2L16) FRa FSA 0.51 0.95 0.56 NB v40455 (2L16) FRa OAA 2.23 37.25 5.78 NB v38591 (12A10) NaPi2b FSA 0.38 2.55 NR30.70 v32164 (12A10) NaPi2b OAA 1.47 4.59 NR 3.35 v21995 (palivizumab) RSV FSA NB NB NB NB Bmaxv36675 (10L18) FRa FSA 8,053 2,215 9,335 NB v32597 (10L18) FRa OAA 5,667 1,200 7,855 NB v32977 (76) FRa FSA 10,551 not assayed not assayed not assayedV4O456 (76) FRa OAA 10,561 1,679 10,670 NB v33283 (2L16) FRa FSA 9,198 1,633 9,755 NB v40455 (2L16) FRa OAA 11,413 2,371 11,453 NB v38591 (12A10) NaPi2b FSA 17,882 9,723 397 6,971 v32164 (12A10) NaPi2b OAA 27,637 11,613 230 11,293v21995 (palivizumab) RSV FSA NB NB NB NB1FSA = full-sized antibodyOAA = one-armed antibody2NB = no binding (apparent Kd value greater than the highest antibody testing concentration >200 nM)3NR = calculated Kd not reliable. Poor curve -fitting (low Hill slope) due to low binding.2.2 Internalization

[0353] For internalization, antibodies were fluorescently labeled by coupling to a Fab fragment AF488 conjugate targeting human IgG Fc (Jackson ImmunoResearch Labs, West Grove, PA; Cat. No. 109-547-008) at a 1:1 molar ratio in PBS pH 7.4 (Thermo Fisher Scientific, Waltham,MA; Cat. No. 10010-023), for 24 hours at 4°C. Cells were seeded at 50,000 cells / well in their standard culture medium in 48-well plates and incubated overnight under standard culturing conditions (37°C / 5% CO2) to allow attachment. Coupled antibodies were added to cells the following day at 10 nM and incubated under standard culturing conditions for 24 hours to allow for internalization. Following incubation, cells were dissociated, washed, and surface AF488 fluorescence was quenched using an anti-AF488 antibody (Life Technologies, Carlsbad, CA; Cat. No. A-11094) at 100 nM for 30 minutes at 4°C. Quenched AF488 fluorescence (internalized fluorescence) was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1,000 minimum events collected per well. The AF488 / FITC-A GeoMean (fluorescence signal geometric mean, proportional to anti -human Fab AF488 labelling) was calculated for the live single cell population using FlowJo™ Version 10.8.1 (BD Biosciences, Franklin Lake, NJ), normalized to the fluorescence signal of the untreated control, and plotted using GraphPad Prism Version 9 (GraphPad Software, La Jolla, CA).

[0354] Internalization results are shown in Table 2.3. Anti-FRa FSAs demonstrated internalization in FRa-expressing cell lines IGROV-1, HCC-78, H441, and T-47D, with the degree of internalized fluorescence ranking being v36675 greater than v32977, which in turn was greater than v33283, across all FRa-expressing cell lines. Similarly, anti-FRa OAAs demonstrated internalization in FRa-expressing cell lines IGROV-1 and T-47D, with the degree of internalized fluorescence ranking being v40456 greater or comparable to v32597, which in turn were both greater than v40455. Anti-FRa OAAs v 40456 and v32597 also showed internalization in cell lines HCC-78 and H441. Anti-FRa OAAs demonstrated a lower degree of internalization than their paratope-matched FSA in all FRa-expressing cell lines. Anti-FRa FSAs and OAAs demonstrated no internalization in FRa-negative TOV-21G cells. Anti-NaPi2b FSA and OAA demonstrated internalization in NaPi2b-expressing cell lines IGROV-1, HCC-78, H441, and TOV-21G, with the OAA demonstrating a lower degree of internalization than its paratope-matched FSA in all NaPi2b-expressing cell lines. Anti-NaPi2b FSA and OAA did not show internalization into NaPi2b-negative T-47D cells.Table 2.3: InternalizationCell LineVariant TargetFormat1IGROV-1 HCC-78 H441 T-47D TOV-21G (10 nM) AntigenInternalized Fluorescence Fold-Over Untreated v36675 (10L18) FRa FSA 56.15 10.11 14.78 26.50 1.94 v32597 (10L18) FRa OAA 6.66 2.02 2.58 3.44 1.75 v32977 (76) FRa FSA 28.52 7.18 7.55 22.17 1.83V4O456 (76) FRa OAA 8.05 2.05 2.51 6.23 1.64 v33283 (2L16) FRa FSA 18.21 2.93 4.77 8.33 1.72 v40455 (2L16) FRa OAA 4.21 1.29 1.74 2.90 1.55 v38591 (12A10) NaPi2b FSA 48.47 36.54 3.08 1.32 21.07 v32164 (12A10) NaPi2b OAA 25.57 23.66 2.62 1.24 14.79 v21995RSV FSA 1.65 1.21 1.85 1.34 1.65(palivizumab)1FSA = full-sized antibodyOAA = one-armed antibodyEXAMPLE 3: FRa AND NAPI2B QUANTIFICATION BY FLOW CYTOMETRY

[0355] The average number of FRa and NaPi2b proteins per cell for the cancer cell lines used in Example 2 and listed in Table 3.1 was determined by flow cytometry as described below. Quantum™ Simply Cellular® (QSC) anti-Human IgG Beads (Bangs Laboratories, Cat. No. 816C) containing 5 bead populations were used: 1 blank and 4 with increasing levels of Fc-specific capture antibody (Antibody Binding Capacity (ABC)).

[0356] Briefly, cells were seeded at 50,000 cells / well and 20 pL of each QSC anti-human IgG bead population was added to 96-well plates. Cells and bead populations 1-4 were then pelleted by centrifugation, washed, and resuspended with 100 pL / well of anti -FRa antibody V17716, anti-NaPi2b antibody vl8992, or negative control anti-RSV antibody v21995, conjugated to Alexa Fluor® 647 at saturating concentration (15 pg / mL). Blank bead population was resuspended in blank buffer only (PBS pH 7.4 and 2% FBS). The 96-well plate was incubated for 30 minutes on ice, in the dark. Following incubation, cell fluorescence was analyzed by flow cytometry with BD LSR Fortessa™ (BD Biosciences, Franklin Lakes, NJ, USA). Each treatment sample captured 10,000 events.

[0357] For data analysis and FRa / cell and NaPi2b / cell calculation, AF647 mean fluorescence intensity (MFI) values for all bead populations were plotted against relevant ABC values using Bangs Laboratories QuickCal® v 2.3 calibration line template for QSC anti-human IgG Lot # 14490. The AF647 MFI values for all cell samples stained with either V17716-AF647 or V18992-AF647 were applied to their respective calibration line by linear regression to obtain ABC values for all cell samples. Surface FRa and NaPi2b expression for cell lines was calculated based on a monovalent binding model and therefore equivalent to the background- subtracted ABC (SABC). The AF647 MFI values of v21995-AF647 (negative control antibody)-stained cells from each of the respective cell lines were subtracted from the vl7716-AF647 and vl8992-AF647-stained cells for determination of SABC (FRa / cell and NaPi2b / cell, respectively). Average FRa / cell and NaPi2b / cell values determined are provided in Table 3.1.Table 3.1: Quantification of FRa and NaPilb Protein on Cancer Cell LinesAverage Average Cell Line Source OriginFRa / cellaNaPi2b / cellaNCI-DCTD RepositoryOvarian 2,019,143 ± 1,936,043 ± IGROV-1 0509653; Bethesda,adenocarcinoma 1,323,247 484,056 MD ATCC® HTB-36™; Placental 1,360,788 ±JEG-3 40,340Manassas, VA choriocarcinoma 643,734DSMZ ACC-563; 839,042 ± HCC-78 Lung carcinoma 599,220 ± 32,413Braunschweig, GER 420,676 ATCC® HTB-133™;T-47D Breast carcinoma 564, 139 ± 114,962 20,684Manassas, VAATCC® CRL-5924™;H2110 Lung carcinoma 427,961 ± 262,139 70,080Manassas, VAATCC® CRM-HTB- LungH441 194,063 ± 10,124 38,601 ± 4,945174™; Manassas, VA adenocarcinomaATCC® CRL-5894™; Lung 248,968 ± Hl 781 15,203Manassas, VA adenocarcinoma 148,852 ATCC® CRL-3577™; 347,050 ± TOV-21G Ovarian carcinoma 6,896Manassas, VA 93,616aAverage of 1 to 8 independent biological experimentsEXAMPLE 4: PRODUCTION OF HALF-ANTIBODIES4.1 Half-Antibody Construct Design

[0358] To allow for production of FRa x NaPi2b bispecific antibodies as described in Example 5, various “half-antibody” constructs were prepared from the anti-NaPi2b antibody 12A10 (see Example 2) and the anti-FRa antibodies 10L18, 76 and 2L16 (see Example 2) as described below.

[0359] A total of 56 half-antibody constructs as shown in Table 4.1 were prepared. The half-antibodies had several formats, which are defined by the particular combination of domains in the heavy chain (as outlined in Table 4.1) and either the absence of a light chain or the presence of one or two copies of light chain. The sequences of the constant domains, hinges and linkers used in the half-antibody constructs are provided in Table 4.2. The CH3 domain of each heavy chain construct contained either the mutations T350V_L351Y_F405A_Y407V (chain A) or the mutations T350V_T366L_K392L_T394W (chain B) to facilitate bispecific molecule generation. The sequences of the VL and VH domains of each antibody are provided in Table 2.1 (see Example 2). In Table 4.1, “scFvl” refers to an scFv that comprises a VH connected from its C-terminus to the N-terminus of a VL and “scFv2” refers to an scFv that comprises a VL connected through its C-terminus to the N-terminus of a VH.Table 4.1: Half-Antibody ConstructsName Description Domain CompositionFormat lb 10L18 (Fab N) chain B H: VH-CHl-hinge-CH2-CH310L18 L: VL-CLFormat 1 12A10 (Fab N) chain A H: VH-CHl-hinge-CH2-CH312A10 L: VL-CLFormat lb 12A10 (Fab N) chain B H: VH-CHl-hinge-CH2-CH312A10 L: VL-CLFormat 2 12A10 (scFvl N) chain A H: VH-linker-VL-mod_hinge-CH2-CH3 12A10Format 2 12A10 (scFv2 N) chain A H: VL-linker-VH-mod_hinge-CH2-CH312A10bName Description Domain CompositionFormat 2 76 (scFv N) chain A H: VH-linker-VL-mod_hinge-CH2-CH3 76Format 2 10L18 (scFv N) chain A H: VL-linker-VH-mod_hinge-CH2-CH3 10L18Format 2 2L16 (scFvl N) chain A H: VH-linker-VL-mod_hinge-CH2-CH3 2L16Format 2 2L16 (scFv2 N) chain A H: VL-linker-VH-mod_hinge-CH2-CH3 2L16bFormat 3 2xl2A10 (Fab-Fab N) H: VH-CH1 -hinge-linker- VH-CHl-hinge-CH2- 12A10 chain A CH32xL: VL-CLFormat 3 2xlOL18 (Fab-Fab N) H: VH-CH1 -hinge-linker- VH-CHl-hinge-CH2- 10L18 chain A CH32xL: VL-CLFormat 3 2x76 (Fab -Fab N) chain A H: VH-CH1 -hinge-linker- VH-CHl-hinge-CH2- 76 CH32xL: VL-CLFormat 3 2x2L16 (Fab-Fab N) H: VH-CH1 -hinge-linker- VH-CHl-hinge-CH2- 2L16 chain A CH32xL: VL-CLFormat 3b 2xl2A10 (Fab-Fab N) H: VH-CH1 -hinge-linker- VH-CHl-hinge-CH2- 12A10 chain B CH32xL: VL-CLFormat 3b 2xlOL18 (Fab-Fab N) H: VH-CH1 -hinge-linker- VH-CHl-hinge-CH2- 10L18 chain B CH32xL: VL-CLFormat 3b 2x76 (Fab -Fab N) chain B H: VH-CH1 -hinge-linker- VH-CHl-hinge-CH2- 76 CH32xL: VL-CLFormat 3b 2x2L16 (Fab-Fab N) H: VH-CH1 -hinge-linker- VH-CHl-hinge-CH2- 2L16 chain B CH32xL: VL-CLFormat 3b 2xl2A10 (Fab-Fab N+C) H: VH-CH1 -hinge-CH2-CH3 -linker- VH-CH 1 - 12A10 chain B hinge2xL: VL-CLName Description Domain CompositionFormat 4b 2xlOL18 (Fab-Fab N+C) H: VH-CH1 -hinge-CH2-CH3 -linker- VH-CH 1 - 10L18 chain B hinge2xL: VL-CLFormat 4b 2x76 (Fab-Fab N+C) H: VH-CH 1 -hinge-CH2-CH3 -linker- VH-CH 1 - 76 chain B hinge2xL: VL-CLFormat 4b 2x2L16 (Fab-Fab N+C) H: VH-CH 1 -hinge-CH2-CH3 -linker- VH-CH 1 - 2L16 chain B hinge2xL: VL-CLFormat 4 2xl2A10 (Fab-Fab N+C) H: VH-CH 1 -hinge-CH2-CH3 -linker- VH-CH 1 - 12A10 chain A hinge2xL: VL-CLFormat 4 2xlOL18 (Fab-Fab N+C) H: VH-CH 1 -hinge-CH2-CH3 -linker- VH-CH 1 - 10L18 chain A hinge2xL: VL-CLFormat 4 2x76 (Fab-Fab N+C) H: VH-CH 1 -hinge-CH2-CH3 -linker- VH-CH 1 - 76 chain A hinge2xL: VL-CLFormat 4 2x2L16 (Fab-Fab N+C) H: VH-CH 1 -hinge-CH2-CH3 -linker- VH-CH 1 - 2L16 chain A hinge2xL: VL-CLFormat 5 2xl2A10 (scFvl-Fab N) H: VH-linker-VL-linker-VH-CHl-hinge-CH2- 12A10 chain A CH3L: VL-CLFormat 5 2xl2A10 (scFv2-Fab N) H: VL-linker-VH-linker-VH-CHl-hinge-CH2- 12A10b chain A CH3L: VL-CLFormat 5 2xlOL18 (scFv-Fab N) H: VL-linker-VH-linker-VH-CHl-hinge-CH2- 10L18 chain A CH3L: VL-CLFormat 5 2x76 (scFv-Fab N) chain H: VH-linker-VL-linker-VH-CHl-hinge-CH2- 76 A CH3L: VL-CLFormat 5 2x2L16 (scFv2-Fab N) H: VL-linker-VH-linker-VH-CHl-hinge-CH2- 2L16b chain A CH3L: VL-CLName Description Domain CompositionFormat 5b 2xl2A10 (scFvl-Fab N) H: VH-linker-VL-linker-VH-CHl-hinge-CH2- 12A10 chain B CH3L: VL-CLFormat 5b 2xl2A10 (scFv2-Fab N) H: VL-linker-VH-linker-VH-CHl-hinge-CH2- 12A10b chain B CH3L: VL-CLFormat 5b 2xlOL18 (scFv-Fab N) H: VL-linker-VH-linker-VH-CHl-hinge-CH2- 10L18 chain B CH3L: VL-CLFormat 5b 2x76 (scFv-Fab N) chain H: VH-linker-VL-linker-VH-CHl-hinge-CH2- 76 B CH3L: VL-CLFormat 5b 2x2L16 (scFv2-Fab N) H: VL-linker-VH-linker-VH-CHl-hinge-CH2- 2L16b chain B CH3L: VL-CLFormat 6 2xl2A10 (FabN+ scFvl H: VH-CH1 -hinge-CH2-CH3 -linker- VH-linker- 12A10 C) chain A VLL: VL-CLFormat 6 2xl2A10 (Fab N+ scFv2 H: VH-CH1 -hinge-CH2-CH3 -linker- VL-linker- 12A10b C) chain A VHL: VL-CLFormat 6 2xlOL18 (Fab N+ scFv C) H: VH-CH1 -hinge-CH2-CH3 -linker- VL-linker- 10L18 chain A VHL: VL-CLFormat 6 2x76 (Fab N+ scFv C) H: VH-CH1 -hinge-CH2-CH3 -linker- VH-linker- 76 chain A VLL: VL-CLFormat 6 2x2L16 (Fab N+scFv2 C) H: VH-CH1 -hinge-CH2-CH3 -linker- VL-linker- 2L16b chain A VHL: VL-CLFormat 6b 2xl2A10 (Fab N+scFvl H: VH-CH1 -hinge-CH2-CH3 -linker- VH-linker- 12A10 C) chain B VLL: VL-CLFormat 6b 2xl2A10 (Fab N+scFv2 H: VH-CH1 -hinge-CH2-CH3 -linker- VL-linker- 12A10b C) chain B VHL: VL-CLName Description Domain CompositionFormat 6b 2xlOL18 (Fab N+scFv C) H: VH-CH1 -hinge-CH2-CH3 -linker- VL-linker- 10L18 chain B VHL: VL-CLFormat 6b 2x76 (Fab N+scFv C) H: VH-CH1 -hinge-CH2-CH3 -linker- VH-linker- 76 chain B VLL: VL-CLFormat 6b 2x2L16 (Fab N+scFv2 C) H: VH-CH1 -hinge-CH2-CH3 -linker- VL-linker- 2L16b chain B VHL: VL-CLFormat 7 10L18 scFv N-12A10Fab H: VL-linker-VH-linker-VH-CHl-hinge-CH2- 12A10+10L18 N chain A CH3L: VL-CLFormat 7 76 scFv N-12A10 Fab N H: VH-linker-VL-linker-VH-CHl-hinge-CH2- 12A10+76 chain A CH3L: VL-CLFormat 7 2L16 scFv2 N- 12A10 H: VL-linker-VH-linker-VH-CHl-hinge-CH2- 12A10+2L16b Fab N chain A CH3L: VL-CLFormat 8 2xl2A10 (Fab-Fab N+C) H: VL-linker-VH-linker-VH-CHl-hinge-CH2- 12A10+10L18 + 10L18 (scFvN) chain A CH3 -linker- VH-CH 1 -hinge2xL: VL-CLFormat 8 2xl2A10 (Fab-Fab N+C) H: VH-linker-VL-linker-VH-CHl-hinge-CH2- 12A10+76 + 76( scFv N) chain A CH3 -linker- VH-CH 1 -hinge2xL: VL-CLFormat 8 2xl2A10 (Fab-Fab N+C) H: VL-linker-VH-linker-VH-CHl-hinge-CH2- 12A10+2L16b + 76 (scFv N) chain A CH3 -linker- VH-CH 1 -hinge2xL: VL-CLFormat 1 76 (Fab N) chain A H: VH-CHl-hinge-CH2-CH376 L: VL-CLFormat 1 2L16 (Fab N) chain A H: VH-CHl-hinge-CH2-CH32L16 L: VL-CLFormat 1 10L18 (Fab N) chain A H: VH-CHl-hinge-CH2-CH310L18 L: VL-CLName Description Domain CompositionFormat lb 76 (Fab N) chain B H: VH-CHl-hinge-CH2-CH376 L: VL-CLFormat lb 2L16 (Fab N) chain B H: VH-CHl-hinge-CH2-CH32L16 L: VL-CLTable 4.2: Amino Acid Sequences of CH Domains, CL Domains, Hinges and LinkersDomain Sequence SEQID NO CHI (IGHG1*O1) ASTKGP S VFPLAPS SKSTSGGT AALGCLVKD YF 60PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLS S VVTVPS S SLGTQTYICNVNHKPSNTKVDKKVHinge (IGHG1*O1) EPKSCDKTHTCPPCP 61 Modified hinge (mod-hinge) AAEPKSSDKTHTCPPCP 62 CH2 (IGHG1*O1) APELLGGPSVFLFPPKPKDTLMISRTPEVTCVV 63VDVSHEDPEVI< FNWYVDGVEVHNAI< TI< PREE QYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNI< ALPAPIEI< TISI< AI<CH3 (IGHG1*O1)_ GQPREPQVYVYPPSRDELTKNQVSLTCLVKGF 64 T350V_L351 Y_F405A_Y407V YPSDIAVEWESNGQPENNYKTTPPVLDSDGSF (chain A) ALVSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG CH3 (IGHG1*O1)_ GQPREPQVYVLPPSRDELTKNQVSLLCLVKGF 65 T350V_T366L_K392L_T394W YPSDIAVEWESNGQPENNYLTWPPVLDSDGSF (chain B) FLYSKLTVDKSRWQQGNVFSCSVMHEALHNH YTQKSLSLSPG CL kappa RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY 66 (IGKC*01) PREAKVQWKVDNALQSGNSQESVTEQDSKDS TYSLSSTLTLSKADYEKHKVYACEVTHQGLSS PVTKSFNRGECLinkers (G4S)2 67(G4S)368 (G4S)469

[0360] The protein sequences for the heavy and light chains of each half-antibody were assembled according to Table 4.1. All sequences were reverse translated to DNA, codon optimized for mammalian expression and gene synthesized.

[0361] Heavy chain vector inserts comprising a signal peptide (artificially designed sequence: MRPTWAWWLFLVLLLALWAPARG (SEQ ID NO: 28) (Barash et al., 2002, Biochem and Biophys Res. Comm., 294:835-842)) were ligated into a pTT5 vector to produce heavy chain expression vectors. Light chain vector inserts comprising the same signal peptide were ligated into a pTT5 vector to produce light chain expression vectors. The resulting heavy and light chain expression vectors were sequenced to confirm the correct reading frame and sequence of the coding DNA.4.2 Production and QC of Half-Antibody Constructs

[0362] The 56 half-antibody constructs were produced in CHO-3E7 cells, protein A purified and further purified by preparatory SEC if the criteria of <3% HMW species presence (as determined by UPC-SEC) was not met. Resultant samples were characterized for homogeneity and chain intactness.

[0363] Cloning and Transfection Conditions'. Gene products were cloned (by GenScript BioTech Corporation, Piscataway, NJ) into the pTT5™ mammalian expression vector and transiently expressed in CHO 3E7 cells as follows.

[0364] CHO-3E7 cells were cultured in FreeStyle™ F17 expression medium (Gibco, Cat # A-1383501) supplemented with 4 mM L-glutamine (HyClone / Cytiva, cat# SH30034.01) and 0.1% Pluronic® F-68 (Gibco, Cat # 24040-032) at 37°C. A transfection mixture of 500ug (or lOOOug) DNA (250ug (or 500ug) DNA coding for the Ab construct and 250ug (or 500ug) of Salmon Sperm DNA, pTT22-AKTdd, GFP) was complexed with PEI MAX® 40,000 (Polysciences Inc., Cat # 24765-1) at a 1:4 (w / w) DNA / PEI ratio. Transfections were performed with DNA / PEI complexes in 500mL (or lOOOmL) cell culture volumes supplemented with IX Antibiotic / Antimycotic Solution (HyClone, Cat # SV30079.01) and incubated at 37°C for 24 hrs. This was followed by a temperature shift to 32°C for 6 days with 0.5 mM valproic acid and 1.0% (w / v) peptone. GFP measurements were taken 1 day post transfection to determine transfection efficiency.

[0365] Cells were harvested 7 days post transfection. Cells were centrifuged then filtered on a 0.22uM Millipore® filter. Clarified supernatants were analyzed by small scale HPLC-PA for titer measurements.

[0366] Protein A Affinity and Preparatory SEC: Clarified culture medium was loaded onto a MabSelect™SuRe™ (Cytiva) Protein A column and washed with DPBS buffer (pH 7.0-7.2). Protein was eluted with 1 OOmM citrate buffer at pH 3.0 and neutralized with 10% (v / v) IM HEPES solution to yield a final pH of 6-7. Protein samples not undergoing preparatory SEC were subsequently buffer-exchanged into DPBS using Zeba™ Spin columns (Thermo Fisher Scientific, Waltham, MA). Protein samples that required further purification underwent preparatory SEC chromatography (Superdex 200 16 / 60 or 26 / 60, Cytiva) in DPBS mobile phase. All samples were concentrated using Vivaspin® TURBO units (Sartorius). Protein was quantified based on absorbance at 280nm (A280nm).

[0367] Caliper. Purity of samples was assessed by electrophoresis under non-reducing and reducing conditions using the High Throughput Protein Express assay and Caliper LabChip® GXII or GXII Touch HT (Perkin Elmer, Waltham, MA). Procedures were carried out according to HT Protein Express LabChip® User Guide version 2 with the following modifications. Antibody samples, at either 2pl or 5 pl (concentration range 5-2000 ng / pl), were added to separate wells in 96 well plates (BioRad, Hercules, CA, Cat # HSP9601B) along with 7pl of HT Protein Express Sample Buffer (Perkin Elmer, Cat # 760328). Antibody samples were then denatured at 70°C for 15 mins. The LabChip® instrument was operated using the HT Protein Express Chip (Perkin Elmer, Waltham, MA) and the Ab-200 assay setting.

[0368] The final yields for the 56 half-antibody constructs varied, ranging from approximately 10-100 mg (from 500-1000ml culture), except for two variants for which the yield was ~l-2 mg. The Caliper results for selected half-antibody constructs are shown in Fig. 5. Electrophoresis under non-reducing conditions (as shown in Fig. 5(A)) reflects the presence of both monomeric and dimeric species. Non-covalent and / or covalent dimeric species can form as a result of the presence of the mutations in the CH3 domain which drive heterodimer formation. Electrophoresis under reducing conditions (as shown in Fig. 5(B)) shows intact heavy chains, as well as intact light chains (where present).

[0369] UPLC-SEC'. Species homogeneity of the final half-antibody constructs was assessed by UPLC-SEC.

[0370] UPLC-SEC was performed using a Waters Acquity BEH200 SEC column (2.5 mL, 4.6 x 150 mm, stainless steel, 1.7 pm particles) (Waters Ltd., Mississauga, ON) set to 30°C and mounted on a Waters Acquity UPLC™ H-Class Bio system with a photodiode array (PDA) detector. The mobile phase was Dulbecco’s phosphate buffered saline (DPBS) with 0.02% Tween 20 pH 7.4 and the flow rate was 0.4 mL / min. Total run time for each injection was 7 min with a total injection of lOug of protein sample. Elution was monitored by UV absorbance in the range 210-500 nm, and chromatograms were extracted at 280 nm. Peak integration was performed using Waters Empower® 3 software employing the Apex Track™ and detect shoulders features.

[0371] UPLC-SEC profiles for the majority of the variants reflected expected species homogeneity, with peaks corresponding to monomeric and dimeric species, as can be seen in Fig.6. UPLC-SEC for some of the variants showed additional peaks reflective of HMW species. Those variants with amounts of HMW species >3-5% were deprioritized for the generation of bispecific antibodies.EXAMPLE 5: PREPARATION OF BISPECIFIC ANTIBODIES

[0372] The FRa x NaPi2b bispecific antibodies shown in Table 5.1 were prepared from the half-antibodies generated in Example 4. An exemplary protocol is provided below.

[0373] Solutions of each of the corresponding half-antibody variants (0.7 to 1.4 mg) in PBS pH 7.4 were mixed in equimolar amounts and diluted to a protein concentration of 2.5 to 3.5 mg / mL. The solution was reduced for 180 minutes at 37°C in the presence of excess reducing agent. Buffer exchange was performed by passage over Zeba™ Spin Desalting Columns (40 KDa MWCO; Pierce) pre-equilibrated with phosphate buffered saline pH 7.4. Half of the eluate was set aside for conjugation to Drug-Linker 001 (see Example 10). The remaining material was reoxidized at 25°C for 18 hours in the presence of excess oxidant.

[0374] Formats of the bispecific antibodies are illustrated schematically in Figs. 1 A-E and 2A-F. The formats shown in Fig. 1 A and Fig. 2A are also referred to as “1+1 formats,” the formatsshown in Fig. IB, 1C and Fig. 2B, 2C are also referred to as “2+1 formats,” and the formats shown in Fig. ID, IE and Fig. 2D, 2E, 2F are also referred to as “2+2 formats.”

[0375] Amino acid sequences for exemplary bispecific antibodies are provided in TableTable 5.1: Bispecific AntibodiesName Chain A Chain B Format1Fab 1 12A10 (Fab N) chain A 10L18 (Fab N) chain B 1A Fab 2 76 (Fab N) chain B 1A Fab 3 2L16 (Fab N) chain B 1A Fab 4 2 x 10L18 (Fab-Fab N) chain B IB Fab 5 2 x 76 (Fab-Fab N) chain B IB Fab 6 2 x 2L16 (Fab-Fab N) chain B IB Fab 7 10L18 (Fab N) chain A 2 x 12A10 (Fab-Fab N) chain B IB Fab 8 76 (Fab N) chain A IB Fab 9 2L16 (Fab N) chain A IB Fab 10 12A10 (Fab N) chain A 2 x 10L18 (Fab-Fab N+C) chain B 1C Fab 11 2 x 76 (Fab-Fab N+C) chain B 1C Fab 12 2 x 2L16 (Fab-Fab N+C) chain B 1C Fab 13 10L18 (Fab N) chain A 2 x 12A10 ((Fab-Fab N+C) chain B 1C Fab 14 76 (Fab N) chain A 1C Fab 15 2L16 (Fab N) chain A 1C Fab 16 2 x 12A10 (Fab-Fab N) chain A 2 x 10L18 (Fab-Fab N) chain B ID Fab 17 2 x 76 (Fab-Fab N) chain B ID Fab 18 2 x 2L16 (Fab-Fab N) chain B ID Fab 19 2 x 12A10 (Fab-Fab N+C) chain A 2 x 10L18 (Fab-Fab N+C) chain B IE Fab 20 2 x76 (Fab-Fab N+C) chain B IE Fab 21 2 x 2L16 (Fab-Fab N+C) chain B IE scFv 1 76 (scFv N) chain A 12A10 ((Fab N) chain B 2A scFv 2 2L16 (scFv N) chain A 2A scFv 3 2 x 2L16 (scFv2-Fab N) chain A 2B scFv 4 2 x 12A10 (scFvl-Fab N) chain A 10L18 (Fab N) chain B 2BName Chain A Chain B Format1scFv 5 76 (Fab N) chain B 2B scFv 6 2L16 (Fab N) chain B 2B scFv 7 76 scFv N-12A10 Fab N chain A 76 (Fab N) chain B 2B scFv 8 2L16 scFv2 N-12A10 Fab N chain A 2L16 (Fab N) chain B 2B scFv 9 2 x 10L18 (Fab N + scFv C) chain A 12A10 (Fab N) chain B 2C scFv 10 2 x 76 (Fab N + scFv C) chain A 2C scFv 11 2 x 2L16 (Fab N + scFv2 C) chain A 2C scFv 12 2 x 12A10 (Fab N + scFvl C) chain A 10L18 (Fab N) chain B 2C scFv 13 76 (Fab N) chain B 2C scFv 14 2L16 (Fab N) chain B 2C scFv 15 2 x 2L16 (scFv2-Fab N) chain A 2 x 12A10 (Fab-Fab N) chain B 2D scFv 16 2 x 12A10 (scFvl-Fab N) chain A 2 x 10L18 (Fab-Fab N) chain B 2D scFv 17 2 x 76 (Fab-Fab N) chain B 2D scFv 18 2 x2L16 (Fab-Fab N) chain B 2D scFv 19 2 x 10L18 (Fab N + scFv C) chain A 2 x 12A10 (Fab-Fab N+C) chain B 2E scFv 20 2 x 76 (Fab N + scFv C) chain A 2E scFv 21 2 x 2L16 (Fab N + scFv2 C) chain A 2E scFv 22 2 x 12A10 (Fab N + scFvl C) chain A 2 x 10L18 (Fab-Fab N+C) chain B 2E scFv 23 2 x 76 (Fab-Fab (N+C) chain B 2E scFv 24 22L16 (Fab-Fab N+C) chain B 2E scFv 25 2 x 12A10 (Fab-Fab N+C) + 10L18 10L18 (Fab N) chain B 2F (scFv N) chain AscFv 26 2 x 12A10 (Fab-Fab N+C) + 76 (scFv N) 76 (Fab N) chain B 2F chain AscFv 27 2 x 12A10 (Fab-Fab N+C) + 2L16 (scFv 2L16 (Fab N) chain B 2F N) chain A1Format descriptions are with reference to Figs. 1 and 2. For example, “1 A” refers to the format shown in Fig. 1A, and “2A” refers to the format shown in Fig. 2A.Table 5.2: Amino Acid Sequences of Exemplary Bispecific AntibodiesName Amino Acid Sequence SEQ ID NOFab 2 Hl:QVQLVQSGAEVKKPGASVKVSCKASGFTFTSYNVHWVRQAPGQGLEWMGA 70 ISPGNGDLSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGRTGMGAVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEName Amino Acid Sequence SEQ ID NO PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG LI:DVQMTQSPSSLSASVGDRVTITCRASQDVYSYLNWYQQKPGKAVKLLIYYTS 71 RLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNIFPWTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GECH2:QVQLVQSGAEVKKPGASVKVSCKASGYRFTDYEMHWVRQAPGQGLEWMG 72 RIDPETGGTAYAQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYYCSTYSY YSYDRFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTK NQVSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGL2:DIVMTQSPDSLAVSLGERATINCRSSQSLLHSNGNTYLYWYQQKPGQPPKLLI 73 YRVSNRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGTHVPHTFGQG TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECFab 4 Hl:QVQLVQSGAEVKKPGASVKVSCKASGFTFTSYNVHWVRQAPGQGLEWMGA 74 ISPGNGDLSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGRTG MGAVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG LI:DVQMTQSPSSLSASVGDRVTITCRASQDVYSYLNWYQQKPGKAVKLLIYYTS 75 RLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNIFPWTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GECName Amino Acid Sequence SEQ ID NOH2:EVQLLESGGGLVQPGGSLRLSCAVSGFSLSSYGVSWVRQAPGKGLEWIGAVN 76 SGGSAYYADSVKGRSTISRDNSKNTVYLQMNSLRAEDTAVYYCARSGSGYP MDYLAIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCA VSGFSLSSYGVSWVRQAPGKGLEWIGAVNSGGSAYYADSVKGRSTISRDNSK NTVYLQMNSLRAEDTAVYYCARSGSGYPMDYLAIWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQ PENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG2xL2:DYQMTQSPSSVSASVGDRVTITCRASQSIGDWLAWYQQKPGKAPKLLIYEAS 77 TLASGVPSRFSGSGSGTDFTLTISSVQPEDAATYYCQQGYGRSNVDNIFGGGT KVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECFab 5 Hl:QVQLVQSGAEVKKPGASVKVSCKASGFTFTSYNVHWVRQAPGQGLEWMGA 78 ISPGNGDLSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGRTG MGAVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG LI:DVQMTQSPSSLSASVGDRVTITCRASQDVYSYLNWYQQKPGKAVKLLIYYTS 79 RLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNIFPWTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GECH2:QVQLVQSGAEVKKPGASVKVSCKASGYRFTDYEMHWVRQAPGQGLEWMG 80 RIDPETGGTAYAQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYYCSTYSY YSYDRFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTGGGGSGGGGSQVQLVQSGAEVKKPGASVK VSCKASGYRFTDYEMHWVRQAPGQGLEWMGRIDPETGGTAYAQKFQGRVT MTADTSTSTVYMELS SLRSEDTAVYYCSTYSYYSYDRFAYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPName Amino Acid Sequence SEQ ID NO AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEW ESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG2xL2:DIVMTQSPDSLAVSLGERATINCRSSQSLLHSNGNTYLYWYQQKPGQPPKLLI 81 YRVSNRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGTHVPHTFGQG TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECFab 8 Hl:QVQLVQSGAEVKKPGASVKVSCKASGYRFTDYEMHWVRQAPGQGLEWMG 82 RIDPETGGTAYAQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYYCSTYSY YSYDRFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG LI:DIVMTQSPDSLAVSLGERATINCRSSQSLLHSNGNTYLYWYQQKPGQPPKLLI 83 YRVSNRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGTHVPHTFGQG TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECH2:QVQLVQSGAEVKKPGASVKVSCKASGFTFTSYNVHWVRQAPGQGLEWMGA 84 ISPGNGDLSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGRTG MGAVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTGGGGSGGGGSQVQLVQSGAEVKKPGASVKVS CKASGFTFTSYNVHWVRQAPGQGLEWMGAISPGNGDLSYAQKFQGRVTMT RDTSTSTVYMELSSLRSEDTAVYYCARGRTGMGAVDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWES NGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG2xL2:Name Amino Acid Sequence SEQ ID NO DVQMTQSPSSLSASVGDRVTITCRASQDVYSYLNWYQQKPGKAVKLLIYYTS 85 RLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNIFPWTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GECFab 10 Hl:QVQLVQSGAEVKKPGASVKVSCKASGFTFTSYNVHWVRQAPGQGLEWMGA 86 ISPGNGDLSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGRTG MGAVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG LI:DVQMTQSPSSLSASVGDRVTITCRASQDVYSYLNWYQQKPGKAVKLLIYYTS 87 RLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNIFPWTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GECH2:EVQLLESGGGLVQPGGSLRLSCAVSGFSLSSYGVSWVRQAPGKGLEWIGAVN 88 SGGSAYYADSVKGRSTISRDNSKNTVYLQMNSLRAEDTAVYYCARSGSGYP MDYLAIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQ VSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSEVQLLESGGG LVQPGGSLRLSCAVSGFSLSSYGVSWVRQAPGKGLEWIGAVNSGGSAYYADS VKGRSTISRDNSKNTVYLQMNSLRAEDTAVYYCARSGSGYPMDYLAIWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHT2xL2:DYQMTQSPSSVSASVGDRVTITCRASQSIGDWLAWYQQKPGKAPKLLIYEAS 89 TLASGVPSRFSGSGSGTDFTLTISSVQPEDAATYYCQQGYGRSNVDNIFGGGT KVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECFab 16 Hl:QVQLVQSGAEVKKPGASVKVSCKASGFTFTSYNVHWVRQAPGQGLEWMGA 90ISPGNGDLSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGRTGName Amino Acid Sequence SEQ ID NO MGAVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTGGGGSGGGGSQVQLVQSGAEVKKPGASVKVS CKASGFTFTSYNVHWVRQAPGQGLEWMGAISPGNGDLSYAQKFQGRVTMT RDTSTSTVYMELSSLRSEDTAVYYCARGRTGMGAVDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG2xLl:DVQMTQSPSSLSASVGDRVTITCRASQDVYSYLNWYQQKPGKAVKLLIYYTS 91 RLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNIFPWTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GECH2:EVQLLESGGGLVQPGGSLRLSCAVSGFSLSSYGVSWVRQAPGKGLEWIGAVN 92 SGGSAYYADSVKGRSTISRDNSKNTVYLQMNSLRAEDTAVYYCARSGSGYP MDYLAIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCA VSGFSLSSYGVSWVRQAPGKGLEWIGAVNSGGSAYYADSVKGRSTISRDNSK NTVYLQMNSLRAEDTAVYYCARSGSGYPMDYLAIWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQ PENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG2xL2:DYQMTQSPSSVSASVGDRVTITCRASQSIGDWLAWYQQKPGKAPKLLIYEAS 93 TLASGVPSRFSGSGSGTDFTLTISSVQPEDAATYYCQQGYGRSNVDNIFGGGT KVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECFab 17 Hl:QVQLVQSGAEVKKPGASVKVSCKASGFTFTSYNVHWVRQAPGQGLEWMGA 94 ISPGNGDLSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGRTG MGAVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSName Amino Acid Sequence SEQ ID NO CKASGFTFTSYNVHWVRQAPGQGLEWMGAISPGNGDLSYAQKFQGRVTMT RDTSTSTVYMELSSLRSEDTAVYYCARGRTGMGAVDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTC PPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFALVSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPG2xLl:DVQMTQSPSSLSASVGDRVTITCRASQDVYSYLNWYQQKPGKAVKLLIYYTS 95 RLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNIFPWTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GECH2:QVQLVQSGAEVKKPGASVKVSCKASGYRFTDYEMHWVRQAPGQGLEWMG 96 RIDPETGGTAYAQKFQGRVTMTADTSTSTVYMELSSLRSEDTAVYYCSTYSY YSYDRFAYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTGGGGSGGGGSQVQLVQSGAEVKKPGASVK VSCKASGYRFTDYEMHWVRQAPGQGLEWMGRIDPETGGTAYAQKFQGRVT MTADTSTSTVYMELS SLRSEDTAVYYCSTYSYYSYDRFAYWGQGTLVTVS SA STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTH TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKA LPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEW ESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEAL HNHYTQKSLSLSPG2xL2:DIVMTQSPDSLAVSLGERATINCRSSQSLLHSNGNTYLYWYQQKPGQPPKLLI 97 YRVSNRFSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCFQGTHVPHTFGQG TKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECFab 19 Hl:QVQLVQSGAEVKKPGASVKVSCKASGFTFTSYNVHWVRQAPGQGLEWMGA 98 ISPGNGDLSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGRTG MGAVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPE PVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHK PSNTKVDKKVEPKSCDKTHTCPPAPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKSName Amino Acid Sequence SEQ ID NO RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQVQLVQSGAE VKKPGASVKVSCKASGFTFTSYNVHWVRQAPGQGLEWMGAISPGNGDLSYA QKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGRTGMGAVDYWGQ GTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGA LTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKV EPKSCDKTHT2xLl:DVQMTQSPSSLSASVGDRVTITCRASQDVYSYLNWYQQKPGKAVKLLIYYTS 99 RLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNIFPWTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GECH2:EVQLLESGGGLVQPGGSLRLSCAVSGFSLSSYGVSWVRQAPGKGLEWIGAVN 100 SGGSAYYADSVKGRSTISRDNSKNTVYLQMNSLRAEDTAVYYCARSGSGYP MDYLAIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQ VSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSEVQLLESGGG LVQPGGSLRLSCAVSGFSLSSYGVSWVRQAPGKGLEWIGAVNSGGSAYYADS VKGRSTISRDNSKNTVYLQMNSLRAEDTAVYYCARSGSGYPMDYLAIWGQG TLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVE PKSCDKTHT2xL2:DYQMTQSPSSVSASVGDRVTITCRASQSIGDWLAWYQQKPGKAPKLLIYEAS 101 TLASGVPSRFSGSGSGTDFTLTISSVQPEDAATYYCQQGYGRSNVDNIFGGGT KVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECscFv 16 Hl:QVQLVQSGAEVKKPGASVKVSCKASGFTFTSYNVHWVRQAPGQGLEWMGA 102 ISPGNGDLSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGRTG MGAVDYWGQGTLVTVSSGGGGSGGGGSGGGGSGGGGSDVQMTQSPSSLSA SVGDRVTITCRASQDVYSYLNWYQQKPGKAVKLLIYYTSRLQSGVPSRFSGS GSGTDYTLTISSLQPEDFATYYCQQYNIFPWTFGQGTKLEIKGGGGSGGGGSQ VQLVQSGAEVKKPGASVKVSCKASGFTFTSYNVHWVRQAPGQGLEWMGAIS PGNGDLSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGRTGM GAVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPAPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHName Amino Acid Sequence SEQ ID NO QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTKNQ VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPG LI:DVQMTQSPSSLSASVGDRVTITCRASQDVYSYLNWYQQKPGKAVKLLIYYTS 103 RLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNIFPWTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GECH2:EVQLLESGGGLVQPGGSLRLSCAVSGFSLSSYGVSWVRQAPGKGLEWIGAVN 104 SGGSAYYADSVKGRSTISRDNSKNTVYLQMNSLRAEDTAVYYCARSGSGYP MDYLAIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCA VSGFSLSSYGVSWVRQAPGKGLEWIGAVNSGGSAYYADSVKGRSTISRDNSK NTVYLQMNSLRAEDTAVYYCARSGSGYPMDYLAIWGQGTLVTVSSASTKGP SVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS SGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYVLPPSRDELTKNQVSLLCLVKGFYPSDIAVEWESNGQ PENNYLTWPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT QKSLSLSPG2xL2:DYQMTQSPSSVSASVGDRVTITCRASQSIGDWLAWYQQKPGKAPKLLIYEAS 105 TLASGVPSRFSGSGSGTDFTLTISSVQPEDAATYYCQQGYGRSNVDNIFGGGT KVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECscFv 25 Hl:DYQMTQSPSSVSASVGDRVTITCRASQSIGDWLAWYQQKPGKAPKLLIYEAS 106 TLASGVPSRFSGSGSGTDFTLTISSVQPEDAATYYCQQGYGRSNVDNIFGGGT KVEVKGGGGSGGGGSGGGGSGGGGSEVQLLESGGGLVQPGGSLRLSCAVSG FSLSSYGVSWVRQAPGKGLEWIGAVNSGGSAYYADSVKGRSTISRDNSKNTV YLQMNSLRAEDTAVYYCARSGSGYPMDYLAIWGQGTLVTVSSGGGGSGGG GSQVQLVQSGAEVKKPGASVKVSCKASGFTFTSYNVHWVRQAPGQGLEWM GAISPGNGDLSYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGR TGMGAVDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYF PEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTV LHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVYPPSRDELTK NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFALVSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGGGGGSGGGGSQVQLVQSGAEVKKPGASVKVSCKASGFTFTSYNVHWVRQAPGQGLEWMGAISPGNGDLName Amino Acid Sequence SEQ ID NO SYAQKFQGRVTMTRDTSTSTVYMELSSLRSEDTAVYYCARGRTGMGAVDY WGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSW NSGALTSGVHTFPA VLQS SGLYSLS SVVTVPS S SLGTQTYICNVNHKPSNTKV DKKVEPKSCDKTHT2xLl:DVQMTQSPSSLSASVGDRVTITCRASQDVYSYLNWYQQKPGKAVKLLIYYTS 107 RLQSGVPSRFSGSGSGTDYTLTISSLQPEDFATYYCQQYNIFPWTFGQGTKLEI KRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GECH2:EVQLLESGGGLVQPGGSLRLSCAVSGFSLSSYGVSWVRQAPGKGLEWIGAVN 108 SGGSAYYADSVKGRSTISRDNSKNTVYLQMNSLRAEDTAVYYCARSGSGYP MDYLAIWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEP VTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYVLPPSRDELTKNQ VSLLCLVKGFYPSDIAVEWESNGQPENNYLTWPPVLDSDGSFFLYSKLTVDKS RWQQGNVFSCSVMHEALHNHYTQKSLSLSPGL2:DYQMTQSPSSVSASVGDRVTITCRASQSIGDWLAWYQQKPGKAPKLLIYEAS 109 TLASGVPSRFSGSGSGTDFTLTISSVQPEDAATYYCQQGYGRSNVDNIFGGGT KVEVKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNAL QSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGECEXAMPLE 6: CHARACTERIZATION OF BISPECIFIC ANTIBODIES

[0376] The concentration of each of the bispecific antibodies from Example 5 was determined by measurement of absorbance at 280 nm using extinction coefficients calculated from the primary amino acid sequence of the molecule. Bispecific antibodies were also characterized by hydrophobic interaction chromatography (HIC), size exclusion chromatography (SEC), capillary electrophoresis sodium dodecyl sulfate (CE-SDS) and intact liquid-chromatogram mass spectrometry (Intact LC-MS) as described below.

[0377] Hydrophobic Interaction Chromatography: Bispecific antibodies were analyzed by HIC to assess hydrophobicity of each antibody. Chromatography was performed on an Agilent Infinity II 1290 HPLC (Agilent Technologies, Santa Clara, CA) using a TSKgel® Butyl-NPRcolumn (2.5pm, 4.6 x 35mm; TOSOH Bioscience GmbH, Griesheim, Germany) and employing a gradient of 95 / 5% MPA / MPB to 5 / 95% MPA / MPB over a period of 12 minutes at a flow rate of 0.5 mL / min (MPA=1.5 M (NH4)2SO4, 25 mM NaxPO4, pH 7 and MPB=75% (v / v) 25 mM NaxPO4, pH 7, 25% (v / v) isopropanol). Detection was by absorbance at 280 nm.

[0378] Size Exclusion Chromatography: The extent of aggregation of the bispecific antibodies (~15 ug, 5 uL injection volume) was assessed by SEC on an Agilent Infinity II 1260 HPLC (Agilent Technologies, Santa Clara, CA) using either an Agilent AdvanceBio SEC column (300 angstroms, 2.7 pm, 7.8 x 150 mm) (Agilent, Santa Clara, CA) or using a Waters Acquity BEH200 SEC column (2.5 mL, 4.6 x 150 mm, stainless steel, 1.7 pm particles) (Waters Ltd., Mississauga, ON). A mobile phase consisting of 150 mM sodium phosphate, pH 6.95 was used with both columns and a flow rate of 1 mL / min was applied to the AdvanceBIO SEC column while a flow rate of 0.5 mL / min was applied to the Waters Acquity column. Detection was by absorbance at 280 nm.

[0379] Capillary Electrophoresis: Purity of the bispecific antibodies was assessed by electrophoresis under non-reducing and reducing conditions using the High Throughput Protein Express assay and Caliper LabChip® GXII or GXII Touch HT (Perkin Elmer, Waltham, MA). Procedures were carried out according to HT Protein Express LabChip® User Guide version 2 with the following modifications. Antibody samples, at either 2 pL or 5 pL (concentration range 5-2000 ng / pL), were added to separate wells in 96 well plates (BioRad, Hercules, CA) along with 7 pL of HT Protein Express Sample Buffer (Perkin Elmer, Cat # 760328). Antibody samples were then denatured at 70°C for 15 mins. The LabChip® instrument was operated using the HT Protein Express LabChip® (Perkin Elmer, Waltham, MA) and the Ab-200 assay setting.

[0380] Intact Liquid Chromatography - Mass Spectrometry: The mass of each bispecific antibody was identified by reversed phase liquid chromatography coupled to mass spectrometry (RPLC-MS). Antibodies were incubated with Endo S endoglycosidase for one hour at room temperature and then injected into a Waters™ BioSuite Phenyl Column, 1000A, 10 pm, 4.6 mm X 75 mm using an Agilent 1290 Infinity II LC System coupled with Agilent 6545 Quadrupole Time of Flight (Q-TOF) instrument with a column temperature of 70° C and a flow rate of 0.3 ml / min. Mobile phases consisted of A: LC-MS grade water with 0.1% v / v formic acid and 10%v / v isopropyl alcohol, andB: acetonitrile with 0.1% v / v formic acid and 10% v / v isopropyl alcohol. The column was pre-equilibrated in 10% mobile phase B before injection. Then, a 5 min, 10% to 60% mobile phase B gradient was applied, followed by a 2 min, 60% to 90% mobile phase B gradient and a column wash of 2 min at 99% mobile phase B. The column was re-equilibrated to 10% mobile phase B for 2 minutes between runs. ESI (electro spray ionization) was performed in a Dual Agilent Jet Stream (AJS) source in positive mode with 5kV of VCap voltage, 2kV Nozzle voltage, 170 V fragmentor voltage, gas temperature of 300°C, gas flow of 13 L / min, nebulizer pressure of 45 psig, sheath gas temperature of 400°C and a sheath gas flow of 12 L / min. Data format was continuum with analyzer set in sensitivity mode, with a m / z range from 500 to 7000 with a scan rate of 1 spectra per sec.

[0381] Peak integration, MS deconvolution and mass assignments and identification were performed in Protein Metrics Byos® v5 (Protein Metrics LLC, Cupertino, CA) using a deconvolution window of 120000-310000 Da with an m / z range of 600-4000. The expected mass of each bispecific antibody was determined using the average mass of each half-antibody component with one 2-acetamido-2-deoxy-beta-D-glucopyranose-(l-4)-[alpha-L-fucopyranose-(1-6)] stubs per heavy chain arising from Endo S activity on N-glycans, disulfide bonds and the formation of pyroglutamic acid, if applicable, at the N-terminus. Mass identifications had a mass tolerance of ±12 Da. Other adducts assigned based on mass shifts relative to the reference mass were the loss of one fucose unit, the addition of a hexose unit, oxidation, cysteinylation and glutathionylation.Results

[0382] The hydrophobicity of the bispecific antibodies was assessed by integration of the HPLC-HIC chromatogram. The main peak corresponding to the bispecific antibody was identified by comparison to the retention times of monospecific control antibodies. The hydrophobicity represented by the HIC retention time (RT) in minutes is shown in Table 6.1.

[0383] The extent of aggregation and monomer content was assessed by integration of the HPLC-SEC chromatogram. The monomer peak of each bispecific antibody was identified as the peak with the same retention time as monospecific control antibodies. All peaks with an earlierretention time relative to the monomer species were determined to be aggregated species. Percent monomer species determined for each ADC is shown in Table 6.1.

[0384] The purity and extent of reoxidation of the bispecific antibodies was assessed by CE-SDS. Formation of the desired bispecific antibody was identified by the presence of a band at the expected size on the non-reducing (NR) CE-SDS with the extent of reoxidation determined by the band density of the bispecific antibody relative to other expected antibody fragments. The percentage of fully oxidized bispecific antibody is shown in Table 6.1.

[0385] In cases where the desired heterodimeric bispecific antibody and homodimeric monospecific antibody could not be resolved by CE-SDS due to similarities in size, intact LC-MS was used to confirm the identity of the fully reoxidized antibody.Table 6.1: Characterization of Bispecific Antibodies% Fully Desired HPLC-HIC% Monomer by Oxidized Bispecific Sample Main Peak RTHPLC-SEC Antibody by NR Present by LC- (min)CE-SDS MS Fab 1 94.3 3.9 93.0 Yes Fab 2 94.7 4.1 91.0 Yes Fab 3 94.2 4.4 92.0 Yes Fab 4 93.6 3.9 97.0 Yes Fab 5 93.3 4.1 96.0 Yes Fab 6 92.0 4.5 93.0 Yes Fab 7 96.2 4.1 95.0 Yes Fab 8 95.4 4.2 95.0 Yes Fab 9 94.4 4.5 95.0 Yes Fab 10 94.3 3.6 95.0 Yes Fab 11 94.4 4.0 93.0 Yes Fab 12 94.4 4.5 94.0 Yes Fab 13 94.3 4.0 7.0 Yes Fab 14 94.6 4.1 4.0 Yes Fab 15 92.8 4.4 4.0 Yes% Fully Desired HPLC-HIC% Monomer by Oxidized Bispecific Sample Main Peak RTHPLC-SEC Antibody by NR Present by LC- (min)CE-SDS MS Fab 16 92.2 4.0 91.0 Yes Fab 17 92.7 4.2 97.0 Yes Fab 18 89.3 4.6 89.0 Yes Fab 19 94.9 3.7 3.0 Yes Fab 20 95.5 3.9 2.0 Yes Fab 21 93.5 4.4 4.0 Yes scFv 1 91.3 4.2 51.1 Yes scFv 2 94.2 4.7 47.2 Yes scFv 3 92.8 4.8 31.9 Yes scFv 4 95.3 6.0 2.4 Yes scFv 5 94.6 6.0 2.0 Yes scFv 6 94.4 6.2 2.6 Yes scFv 7 86.3 6.6 32.3 Yes scFv 8 94.9 4.9 36.3 Yes scFv 9 94.2 4.3 26.5 Yes scFv 10 83.5 6.1 30.9 Yes scFv 11 89.7 4.8 33.1 Yes scFv 12 95.0 5.7 2.4 Yes scFv 13 94.9 5.8 1.9 Yes scFv 14 94.2 6.0 2.0 Yes scFv 15 96.3 4.9 16.5 Yes scFv 16 94.3 5.9 0.7 Yes scFv 17 94.1 6.0 5.0 No scFv 18 91.0 6.2 4.1 No scFv 19 93.9 4.3 0.3 No scFv 20 84.8 5.8 4.4 Yes scFv 21 90.5 4.6 1.2 No scFv 22 92.9 5.3 0.9 Yes% Fully Desired HPLC-HIC% Monomer by Oxidized Bispecific Sample Main Peak RTHPLC-SEC Antibody by NR Present by LC- (min)CE-SDS MS scFv 23 93.8 5.5 1.2 Yes scFv 24 87.1 5.9 3.7 Yes scFv 25 95.1 4.2 15.0 Yes scFv 26 88.9 6.4 13.6 Yes scFv 27 95.3 4.8 12.6 YesEXAMPLE 7: CELLULAR BINDING OF BISPECIFIC ANTIBODIES

[0386] The ability of the bispecific antibodies from Example 5 to bind to FRa and NaPi2b expressed on the surface of tumor cell lines HCC-78 (lung carcinoma), TOV-21G (ovarian carcinoma), JEG-3 (placental choriocarcinoma) and T-47D (breast carcinoma) was evaluated by flow cytometry. HCC-78 expresses endogenous FRa and NaPi2b at moderate-to-low levels; TOV-21G expresses endogenous NaPi2b at moderate levels and does not express FRa; JEG-3 and T-47D express endogenous FRa at moderate levels and do not express NaPi2b, as determined by surface protein quantification described in Example 3.

[0387] Cells were maintained under standard culture conditions (37°C / 5% CO2) until assay set-up. HCC-78 and T-47D cells were cultured in RPMI 1640, ATCC modification (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA). TOV-21G cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) (Thermo Fisher Scientific, Waltham, MA) supplemented with 15% FBS. JEG-3 cells were cultured in Minimum Essential Medium (MEM) (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS. Bispecific Fab constructs were evaluated against HCC-78, TOV-21G and JEG-3. Bispecific scFv-containing constructs were evaluated against HCC-78, TOV-21G and T-47D. Palivizumab (anti-RSV antibody, v21995) was included as a negative control against all cell lines.

[0388] Cells were removed from culture vessels using Cell Dissociation Buffer (Thermo Fisher Scientific, Waltham, MA), seeded at 50,000 cells / well in coni cal -bottom 96-well plates,and treated with test article for 24 hours at 4°C to prevent internalization. Following incubation, cells were washed and stained with anti-Human IgG Fc AF647 conjugate (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-605-098) at 4°C for 30 minutes. Following incubation and washing, fluorescence was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1,000 minimum events collected per well. The AF647 / APC-A GeoMean (fluorescence signal geometric mean, proportional to anti-Human AF647 binding) was calculated for the live cell population using FlowJo™ Version 10.8.1 (BD Biosciences, Franklin Lake, NJ) and plotted for each test antibody or antibody-drug conjugate using GraphPad Prism Version 9 (GraphPad Software, La Jolla, CA).

[0389] Results are shown in Tables 7.1 and 7.2. On FRa- and NaPi2b-expressing HCC-78 cells, all bispecific antibodies demonstrated a range in cellular binding with Ka values between 1.49 and 5.81 nM. On FRa-negative, NaPi2b-expressing TOV-21G cells, all bispecific antibodies demonstrated a range in cellular binding with Ka values between 0.84 and 5.34 nM, dependent on anti-NaPi2b valency. On FRa-expressing, NaPi2b-negative JEG-3 cells, Fab bispecific antibodies demonstrated a range of cellular binding with Ka values between 0.36 and 11.09 nM, dependent on anti-FRa paratope and valency. On FRa-expressing, NaPi2b-negative T-47D cells, scFv bispecific antibodies evaluated demonstrated a range in cellular binding with Ka values between 0.10 and 4.18 nM, dependent on anti-FRa paratope and valency. Similar trends were observed between Fab and scFv bispecific antibodies. Negative control palivizumab (v21995) showed no cellular binding, as expected.Table 7.1: Cellular Binding of FRa and NaPilb Fab Bispecific AntibodiesHCC-78 TOV-21G JEG-3 HCC-78 TOV-21G JEG-3 SampleBmax Ka (nM)Fab 1 10,788 10,670 7,404 3.09 3.84 3.65 Fab 2 11,002 10,540 8,120 3.29 3.08 2.33 Fab 3 10,660 11,116 9,478 4.72 3.19 5.14 Fab 4 11,288 12,340 7,480 3.27 4.76 0.61 Fab 5 10,658 11,433 9,356 3.34 3.66 0.36 Fab 6 11,940 11,945 9,633 5.56 3.75 1.36 Fab 7 8,942 8,694 8,247 1.66 2.97 4.66HCC-78 TOV-21G JEG-3 HCC-78 TOV-21G JEG-3 SampleBmax Ka (nM)Fab 8 9,247 8,109 8,582 2.84 2.45 3.21 Fab 9 8,187 8,056 10,907 4.64 2.36 7.31 Fab 10 9,152 11,705 7,026 3.67 4.29 0.43 Fab 11 9,805 12,207 8,034 3.53 5.26 0.38 Fab 12 10,591 11,732 9,291 5.44 5.34 2.02 Fab 13 7,171 6,781 11,836 2.49 2.77 4.56 Fab 14 6,974 6,713 14,586 2.98 3.12 4.86 Fab 15 6,368 5,529 14,599 4.01 1.87 11.09 Fab 16 9,425 8,009 9,087 3.47 3.80 0.83 Fab 17 11,737 7,620 9,374 5.16 2.71 0.45 Fab 18 11,082 7,713 12,325 5.81 1.78 3.69 not not Fab 19 8,569 5,903 3.40 2.46assayed assayed Fab 20 7,820 6,423 8,080 3.77 2.70 0.48 Fab 21 7,695 5,990 7,517 5.36 2.18 2.24 v21995 NB1NB NB NB NB NB1NB = no binding (apparent Kd value greater than the highest antibody testing concentration >200 nM) Table 7.2: Cellular Binding of FRa and NaPilb scFv Bispecific AntibodiesHCC-78 TOV-21G T-47D HCC-78 TOV-21G T-47D SampleBmax Ka (nM)scFv 1 8,379 1,521 2,163 3.34 1.64 3.94 scFv 2 7,785 1,563 4,548 3.35 1.83 1.01 scFv 3 8,136 1,513 3,517 3.51 1.94 1.42 scFv 4 6,487 1,110 3,221 3.06 0.98 3.33 scFv 5 6,257 1,066 4,417 3.16 0.95 2.35 scFv 6 5,448 1,071 4,205 3.23 1.01 4.18 scFv 7 6,661 1,354 3,472 3.16 3.94 0.22 scFv 8 6,458 1,544 4,204 3.44 4.43 1.27 scFv 9 8,796 1,557 2,140 3.25 2.18 0.41HCC-78 TOV-21G T-47D HCC-78 TOV-21G T-47D SampleBmax Ka (nM)scFv 10 8,719 1,537 2,703 3.35 2.45 0.20 scFv 11 7,132 1,519 2,224 3.48 2.15 0.59 scFv 12 5,753 1,042 2,901 2.58 0.92 3.36 scFv 13 5,772 981 3,301 3.14 0.84 1.79 scFv 14 5,210 1,006 3,629 3.14 0.87 3.27 scFv 15 6,773 1,262 3,168 3.29 0.94 1.25 scFv 16 6,405 1,117 2,265 3.02 1.05 0.20 scFv 17 6,364 986 3,095 3.04 1.39 0.12 scFv 18 6,238 1,005 2,955 3.14 1.15 0.52 scFv 19 5,289 980 2,224 2.21 1.06 0.51 scFv 20 5,264 996 2,890 3.00 1.37 0.25 scFv 21 4,645 1,366 2,361 3.18 2.60 1.02 scFv 22 5,626 1,072 2,150 3.04 1.21 0.30 scFv 23 5,706 1,189 2,713 3.09 1.60 0.15 scFv 24 5,453 1,184 2,975 3.22 1.47 0.90 scFv 25 5,120 929 2,642 1.49 3.16 0.18 scFv 26 5,371 899 3,143 2.51 3.02 0.10 scFv 27 4,850 971 2,855 3.36 3.33 0.45 v21995 NB1NB NB NB NB NB1NB = no binding (apparent Kd value greater than the highest antibody testing concentration >200 nM)EXAMPLE 8: INTERNALIZATION OF BISPECIFIC ANTIBODIES

[0390] The ability of the bispecific antibodies targeting FRa and NaPi2b from Example 5 to internalize with FRa and NaPi2b expressed on the surface of tumor cell lines was evaluated by flow cytometry following 24 hours treatment at 5 nM of antibody. The anti-RSV antibody palivizumab (v21995) was used as a negative control.

[0391] Cell lines used were IGROV-1 (ovarian adenocarcinoma), TOV-21G (ovarian carcinoma) and T-47D (breast carcinoma). IGROV-1 express endogenous FRa and NaPi2b at highlevels, T0V-21G express NaPi2b at moderate levels and T-47D express FRa at moderate levels, as determined by surface protein quantification described in Example 3.

[0392] Briefly, antibodies were fluorescently labeled by coupling to a Fab fragment AF488 conjugate targeting anti-human IgG Fc (Jackson Immuno Research Labs, West Grove, PA; Cat. No. 109-547-008) at a 1:1 molar ratio in PBS pH 7.4 (Thermo Fisher Scientific, Waltham, MA; Cat. No. 10010-023), for 24 hours at 4°C. Cells were seeded at 50,000 cells / well in RPMI 1640, ATCC modification (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA) in 48-well plates and incubated overnight under standard culturing conditions (37°C / 5% CO2) to allow attachment. Coupled antibodies were added to cells the following day at 5 nM and incubated under standard culturing conditions for 24 hours to allow for internalization. An untreated control (cells incubated with complete growth medium only, no test article) was included for each cell line at each treatment time point. Following incubation, cells were dissociated, washed, and surface AF488 fluorescence was quenched using an anti-AF488 antibody (Life Technologies, Carlsbad, CA; Cat. No. A-11094) at 100 nM for 30 minutes at 4°C. Quenched AF488 fluorescence (internalized fluorescence) was detected by flow cytometry on a BD LSRFortessa™ Cell Analyzer (BD Biosciences, Franklin Lake, NJ) with 1,000 minimum events collected per well. The AF488 / FITC-A GeoMean (fluorescence signal area geometric mean, proportional to anti-Human Fab AF488 labelling) was calculated for the live single cell population using FlowJo™ Version 10.8.1 (BD Biosciences, Franklin Lake, NJ), normalized to the fluorescence signal of the untreated control, and plotted using GraphPad Prism Version 9 (GraphPad Software, La Jolla, CA).

[0393] Results are shown in Table 8.1 and plotted in Fig. 7 (Fab bispecific antibodies) and Fig. 8 (scFv bispecific antibodies). Across all three cell lines, bispecific antibodies demonstrated a range in degree of internalization dependent on the level of FRa and NaPi2b protein expressed on the cell lines. Against FRa-expressing T-47D, bispecific antibodies showed a large differentiation in internalization dependent on the anti -FRa paratope. In NaPi2b-expressing TOV-21G, all bispecific antibodies showed comparable degree of internalization. In FRa- andNaPi2b-expressing IGROV-1, bispecific antibodies showed a varying degree of internalization dependent on the anti -FRa paratope and number of FRa and NaPi2b binding sites per antibody. Similar trends were observed between Fab and scFv bispecific antibodies across all three cell lines evaluated.Negative control antibody v21995 demonstrated a negligible degree of internalization comparable to the untreated control (cells incubated with complete growth medium only, no test article). Table 8.1: Internalization of Bispecific AntibodiesInternalized Fluorescence Fold-Over UntreatedSampleIGROV-1 TOV-21G T-47DFab 1 54.5 15.4 9.1Fab 2 56.5 17.5 21.5Fab 3 49.5 14.6 4.9Fab 4 50.2 14.5 33.0Fab 5 38.6 12.0 22.7Fab 6 39.7 13.9 12.5Fab 7 52.0 18.7 6.0Fab 8 55.8 17.0 11.7Fab 9 47.8 16.0 4.0Fab 10 46.5 14.2 31.0Fab 11 40.0 12.9 18.2Fab 12 43.9 14.0 13.9Fab 13 61.6 16.0 13.5Fab 14 61.6 13.8 20.5Fab 15 40.0 15.2 6.1Fab 16 53.3 18.0 29.1Fab 17 48.0 16.5 23.4Fab 18 44.2 17.0 12.7Fab 19 41.5 14.5 26.6Fab 20 37.9 13.6 16.9Fab 21 34.6 14.5 13.5scFv 1 57.7 9.8 5.4scFv 2 58.0 9.5 10.8scFv 3 51.9 9.6 15.6scFv 4 57.7 11.5 10.8Internalized Fluorescence Fold-Over Untreated SampleIGROV-1 TOV-21G T-47D scFv 5 58.5 11.2 18.7 scFv 6 50.9 11.0 5.9 scFv 7 42.5 7.3 32.7 scFv 8 43.1 7.2 17.5 scFv 9 53.9 9.7 23.7 scFv 10 55.9 9.3 26.7 scFv 11 46.5 10.2 12.5 scFv 12 68.9 9.8 8.4 scFv 13 63.8 9.0 18.0 scFv 14 48.7 9.6 5.2 scFv 15 59.6 11.7 15.8 scFv 16 54.0 10.9 34.5 scFv 17 55.6 10.9 33.1 scFv 18 53.2 10.5 17.5 scFv 19 58.1 9.5 20.8 scFv 20 47.7 8.9 25.0 scFv 21 42.9 9.2 10.0 scFv 22 53.6 9.7 19.5 scFv 23 47.0 8.9 20.0 scFv 24 46.7 9.3 17.8 scFv 25 54.1 8.3 32.6 scFv 26 42.9 8.1 22.3 scFv 27 41.1 8.6 16.9 v21995 1.4 1.5 1.6EXAMPLE 9: COMPLEMENT-DEPENDENT CYTOTOXICITY (CDC) ACTIVITY OF BISPECIFIC ANTIBODIES

[0394] The ability of selected bispecific antibodies targeting FRa and NaPi2b from Example 5 to mediate complement-dependent cytotoxicity (CDC) was assessed in the tumor cell line IGROV-1 (ovarian adenocarcinoma). Monospecific antibodies v36675 and v32977 targeting FRa, monospecific antibody v38591 targeting NaPi2b, and palivizumab (anti -RS V antibody, v21995) were included as controls. IGROV-1 expresses endogenous FRa and NaPi2b at high levels as determined by surface protein quantification described in Example 3.

[0395] Cells were maintained under standard culture conditions (37°C / 5% CO2) until assay set-up; IGROV-1 cells were cultured in RPMI 1640, ATCC modification (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA). For assay set-up, cells were seeded in 384-well tissue culture-treated microtiter plates in serum-free medium, RPMI 1640, ATCC modification, and treated with a titration of test article prepared in serum-free medium with or without human complement serum (Innovative Research, Inc, Novi, MI) to a final 25% v / v. Cells were incubated for 24 hours under standard culturing conditions. After incubation, CellTiter-Glo® reagent (Promega Corporation, Madison, WI) was spiked in all wells and luminescence corresponding to ATP present in each well was measured using a Synergy™ Hl plate reader (BioTek Instruments, Winooski, VT). Percent cytotoxicity values were calculated based on blank wells (no test article added) and plotted against test article concentration using GraphPad Prism 9 software (GraphPad Software, La Jolla, CA). EC50 values were calculated based on a non-linear regression log(agonist) versus response, variable slope (four parameters) by GraphPad Prism 9.

[0396] Results are shown in Table 9.1. Bispecific antibody Fab 2 demonstrated the greatest degree of CDC activity against IGROV-1 cells of the bispecific antibodies tested. Several bispecific antibodies, including Fab 4, Fab 5, Fab 8, Fab 16 and scFv 9, demonstrated varying moderate degrees of CDC activity. Monospecific controls against FRa and NaPi2b demonstrated minimal and no activity, respectively. Negative control palivizumab demonstrated no activity, as expected.Table 9.1: CDC Activity Mediated by Bispecific Antibodies25% Normal HumanNo Serum Bispecific SerumSampleFormat1ECso Maximum % ECso Maximum % (nM) Cytotoxicity (nM) Cytotoxicity Fab 2 1A 37.77 78.95 >300 no activity Fab 4 IB 37.76 23.95 >300 no activity Fab 5 IB 40.58 24.42 >300 no activity Fab 8 IB 61.57 14.80 >300 no activity Fab 10 IC >300 no activity >300 no activity Fab 16 ID 66.89 11.83 >300 no activity Fab 17 ID >300 no activity >300 no activity Fab 19 IE >300 no activity >300 no activity scFv 9 2C 12.80 26.51 >300 no activity scFv 16 2D >300 no activity >300 no activity v36675 (10L18) — 71.12 16.68 >300 no activity v32977 (76) — IC2IC >300 no activity v38591 (12A10) — >300 no activity >300 no activity v21995 (palivizumab) — >300 no activity >300 no activity1Format descriptions are with reference to Figs. 1 and 2. For example, “1 A” refers to the format shown in Fig. 1A, and “2C” refers to the format shown in Fig. 2C.2IC = incomplete curveEXAMPLE 10: PREPARATION OF BISPECIFIC ANTIBODY-DRUG CONJUGATES

[0397] Antibody drug conjugates (ADCs) comprising the FRa x NaPi2b bispecific antibodies described in Example 5 (Fab 1-21 and scFv 1-27) conjugated to Drug-Linker 001 were prepared. Drug -Linker 001 (shown below) may be prepared as described in International Publication No. WO 2019 / 173911 or WO 2023 / 141714.Drug-Linker 001

[0398] An exemplary protocol for the ADC preparation is provided below.

[0399] A solution of corresponding half antibody variants (0.7 to 1.4 mg) in PBS pH 7.4 were mixed in equimolar amounts and diluted to a protein concentration of 2.5 to 3.5 mg / mL. The solution was reduced for 180 minutes at 37°C in the presence of excess reducing agent. Buffer exchange was performed by passage over Zeba™ Spin Desalting Columns (40 KDa MWCO; Pierce) pre-equilibrated with phosphate buffered saline pH 7.4. To the antibody solution was added a limiting amount of Drug-Linker 001 from a 10 mM DMSO stock solution. The conjugation reaction proceeded at room temperature for 60 minutes.EXAMPLE 11: CHARACTERIZATION OF BISPECIFIC ANTIBODY-DRUG CONJUGATES

[0400] Following purification, the concentration of each of the bispecific ADCs prepared as described in Example 10 was determined by measurement of absorption at 280 nm using extinction coefficients determined experimentally. Bispecific ADCs were also characterized for aggregation by size exclusion chromatography (SEC) and the drug-to-antibody ratio (DAR) of the ADCs was determined by reduced reverse-phase liquid-chromatography mass spectrometry (rRP-LC-MS) as described below.

[0401] Size Exclusion Chromatography (SEC): The extent of aggregation of the bi specific antibodies (~15 ug, 5 uL injection volume) was assessed by SEC on an Agilent Infinity II 1260 HPLC (Agilent Technologies, Santa Clara, CA) using a Waters Acquity BEH200 SEC column (2.5 mL, 4.6 x 150 mm, stainless steel, 1.7 pm particles) (Waters Ltd., Mississauga, ON). A mobile phase consisting of 150 mM phosphate, pH 6.95 was used with a flow rate of 0.5 mL / min. Detection was by absorbance at 280 nm.

[0402] Reduced Reverse-Phase Liquid-Chromatography Mass Spectrometry (rRP-LC-MS): ADCs were incubated with Endo S endoglycosidase for one hour at room temperature followed by reduction with TCEP to a final concentration of 50 mM for 1 hour at room temperature before injection. 1 pL of sample was injected into a Waters™ BioSuite Phenyl Column, 1000 A, 10 pm, 4.6 mm X 75 mm using an Agilent 1290 Infinity II LC System coupled with Agilent 6545 Quadrupole Time of Flight (Q-TOF) instrument with a column temperature of 70°C and a flow rate of 0.3 ml / min. Mobile phases consisted of A: LC-MS grade water with 0.1% v / v formic acid, and 10% v / v isopropyl alcohol, and B: acetonitrile with 0.1% v / v formic acid and 10% v / v isopropyl alcohol. The column was pre-equilibrated in 10% mobile phase B before injection. Then, a 5 min, 10 to 60% mobile phase B gradient was applied, followed by a 2 min, 27 to 90% mobile phase B gradient and a column wash of 2 min at 99% mobile phase B. The column was reequilibrated to 10% mobile phase B for 2 minutes between runs. ESI (electro spray ionization) was performed in a Dual Agilent Jet Stream (AJS) source in positive mode with 5kV of VCap voltage, 2kV Nozzle voltage, 170 V fragmentor voltage, gas temperature of 300°C, gas flow of 13 L / min, nebulizer pressure of 45 psig, sheath gas temperature of 400°C and a sheath gas flow of 12 L / min. Data format was continuum with analyser set in sensitivity mode, with a m / z range from 500 to 7000 with a scan rate of 1 spectra per sec.

[0403] Peak integration, MS deconvolution and mass assignments were performed in Protein Metrics Byos® v5 (Protein Metrics LLC, Cupertino, CA). Reference masses for unconjugated light chain (LC) and heavy chain (HC) species were determined using the average mass of each component for relevant antibodies while accounting for expected stubs arising from Endo S activity on N-glycans. LC and HC species conjugated to Drug-Linker 001 were identified by mass shifts to reference masses equal to the molecular weight of Drug-Linker 001 as well as mass shifts equal to the molecular weight of Drug-Linker 001 and H2O due to hydrolysis of the drug-linker maleimide.Results

[0404] The extent of aggregation and monomer content was assessed by integration of the HPLC-SEC chromatogram. The monomer peak of each bispecific antibody was identified as the peak with the same retention time as monospecific control antibodies. All peaks with an earlierretention time relative to the monomer species were determined to be aggregated species. Percent (%) monomer species determined for each ADC is shown in Table 11.1.

[0405] The DAR for each bispecific ADC was calculated using the peak intensities observed in the deconvoluted mass spectra of LC and HC species based on the following equation: average DAR 2' i\ ' — - — — - - - 1 - — -:— — - -:

[0406] The DAR determined for each bispecific ADC is shown in Table 11.1.Table 11.1: Percent Monomer and DAR of Bispecific ADC Preparations% Monomer by DAR by LC- ADC HPLC-SEC MSFab 1 -Drug Linker 001 90.9 3.2Fab 2-Drug Linker 001 90.6 3.1Fab 3 -Drug Linker 001 93.1 3.1Fab 4-Drug Linker 001 88.3 3.5Fab 5 -Drug Linker 001 88.8 2.3Fab 6-Drug Linker 001 85.7 2.6Fab 7-Drug Linker 001 94.3 3.3Fab 8-Drug Linker 001 92.3 3.5Fab 9-Drug Linker 001 93.0 2.9Fab 10-Drug Linker 001 91.6 3.1Fab 11 -Drug Linker 001 91.5 2.7Fab 12-Drug Linker 001 91.4 3.6Fab 13 -Drug Linker 001 92.5 2.6Fab 14-Drug Linker 001 91.9 4.1Fab 15 -Drug Linker 001 90.8 3.3Fab 16-Drug Linker 001 91.1 3.0Fab 17-Drug Linker 001 90.3 3.0Fab 18-Drug Linker 001 88.6 3.3Fab 19-Drug Linker 001 93.1 3.1Fab 20-Drug Linker 001 91.8 3.2% Monomer by DAR by LC- ADC HPLC-SEC MS Fab 21 -Drug Linker 001 91.3 3.3 scFv 1-Drug Linker 001 91.1 3.5 scFv 2-Drug Linker 001 91.8 3.4 scFv 3 -Drug Linker 001 90.3 3.1 scFv 4-Drug Linker 001 95.5 3.6 scFv 5-Drug Linker 001 95.5 3.6 scFv 6-Drug Linker 001 95.8 3.6 scFv 7-Drug Linker 001 88.2 3.7 scFv 8-Drug Linker 001 94.3 3.7 scFv 9-Drug Linker 001 92.7 3.3 scFv 10-Drug Linker 001 82.6 3.5 scFv 11-Drug Linker 001 88.8 3.2 scFv 12-Drug Linker 001 95.9 3.7 scFv 13 -Drug Linker 001 95.8 3.6 scFv 14-Drug Linker 001 95.6 3.4 scFv 15-Drug Linker 001 92.0 3.4 scFv 16-Drug Linker 001 93.8 4.1 scFv 17-Drug Linker 001 93.5 3.9 scFv 18-Drug Linker 001 90.7 4.2 scFv 19-Drug Linker 001 92.6 4.0 scFv 20-Drug Linker 001 85.3 3.6 scFv 21-Drug Linker 001 89.0 4.0 scFv 22-Drug Linker 001 93.0 3.8 scFv 23 -Drug Linker 001 93.4 4.2 scFv 24-Drug Linker 001 88.4 3.0 scFv 25-Drug Linker 001 93.4 3.4 scFv 26-Drug Linker 001 88.8 3.8 scFv 27-Drug Linker 001 93.7 3.7EXAMPLE 12: CYTOTOXICITY OF BISPECIFIC ANTIBODY-DRUG CONJUGATES

[0407] The cell growth inhibition (cytotoxicity) capabilities of ADCs comprising FRa x NaPi2b bispecific antibodies conjugated to Drug-Linker 001 as described in Examples 10 and 11 was assessed in a panel of FRa- and NaPi2b-expressing cell lines in 2D monolayer and 3D spheroid formats as described below. The anti-RSV antibody palivizumab, conjugated to Drug-Linker 001 at a DAR of 4, was included as a non-targeted control.

[0408] Cell lines used were IGROV-1 (ovarian adenocarcinoma), JEG-3 (placental choriocarcinoma), H2110 (lung carcinoma), and H441 (lung adenocarcinoma). IGROV-1 expresses endogenous FRa and NaPi2b at high levels; H2110 and H441 express FRa and NaPi2b at moderate-to-low levels; Hl 781 (lung adenocarcinoma) expresses FRa at low levels and NaPi2b and moderate levels; JEG-3 expresses endogenous FRa at moderate levels and does not express NaPi2b, as determined by surface protein quantification described in Example 3.

[0409] Cells were maintained under standard culture conditions (37°C / 5% CO2) until assay set-up; IGROV-1, H2110, and H441 cells were cultured in RPMI 1640, ATCC modification (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% fetal bovine serum (FBS) (Thermo Fisher Scientific, Waltham, MA); H1781 cells were cultured in RPMI 1640, ATCC modification supplemented with 15% FBS; JEG-3 cells were cultured in Minimum Essential Medium (MEM) (Thermo Fisher Scientific, Waltham, MA) supplemented with 10% FBS.

[0410] Briefly, for spheroid generation, cells were seeded into 384-well round bottom ultra-low attachment (ULA) microtiter plates and incubated for 3 days at 37°C / 5% CO2 prior to test article addition. Test articles were serially diluted in growth medium to create a 9-point doseresponse curve, including growth medium-only blank controls, and added to cells in monolayer and spheroid formats in 384-well microtiter plates. Monolayer and spheroid plates were incubated at 37°C / 5% CO2 for 4 and 6 days, respectively. After incubation, cell viability was measured by addition of ATP metabolism detection reagents, CellTiter-Glo® or CellTiter-Glo® 3D (Promega Corporation, Madison, WI)), to monolayer or spheroid plates, respectively. Resulting luminescence signal was measured using the Synergy™ Hl plate reader (BioTek Instruments, Winooski, VT). Percent cytotoxicity values were calculated based on blank wells (no test article added) and plotted against test article concentration using GraphPad Prism 9 software (GraphPadSoftware, La Jolla, CA). EC50 values were calculated based on a non-linear regression log(agonist) versus response, variable slope (four parameters) by GraphPad Prism 9.

[0411] 2D cytotoxicity results are shown in Table 12.1. All bispecific ADCs demonstrated significant potency against high FRa- and NaPi2b-expressing IGROV-1 cells, with EC 50 values ranging between 0.05 and 0.29 nM. Bispecific ADCs demonstrated a range in potency against FRa-expressing, NaPi2b-negative JEG-3 cells, with EC 50 values ranging between 0.06 and 8.79 nM, dependent on anti-FRa paratope and valency. Bispecific ADCs demonstrated a range of modest potency against moderate-to-low FRa- and NaPi2b-expressing H2110 cells, with EC 50 values ranging between 0.32 and >70 nM, dependent on anti-FRa paratope. Negative control ADC, v21995 (palivizumab)-Drug-Linker 001, demonstrated no targeted killing, with EC50 values >70 nM across the cell lines tested.

[0412] 3D cytotoxicity results are shown in Table 12.2. All bispecific ADCs assessed against Hl 781 spheroids demonstrated modest targeted potency, with EC 50 values ranging between 0.17 and 4.33 nM and some yielding incomplete curves within the concentration range tested (>70 nM). Bispecific ADCs assessed against H441 spheroids demonstrated moderate targeted potency, with EC50 values ranging between 0.33 and 4.86 nM. Negative control ADC, v21995-Drug-Linker 001, demonstrated no targeted killing, with EC 50 values >70 nM, across both cell line spheroids tested.Table 12.1: In Vitro Cytotoxicity of FRa x NaPi2b Bispecific ADCs in 2D Monolayer Format EC50 (nM)Variant DAR IGROV-1 JEG-3 H2110 Monolayer Monolayer Monolayer Fab 1 -Drug-Linker 001 3.2 0.14 0.54 1.44Fab 2-Drug-Linker 001 3.1 0.13 0.56 3.07Fab 3 -Drug-Linker 001 3.1 0.29 4.03 ICFab 4-Drug-Linker 001 3.5 0.10 0.08 0.39Fab 5 -Drug-Linker 001 2.3 0.18 0.12 3.51Fab 6-Drug-Linker 001 2.6 0.19 0.72 13.16Fab 7-Drug-Linker 001 3.3 0.06 1.39 7.34EC50 (nM) Variant DAR IGROV-1 JEG-3 H2110Monolayer Monolayer Monolayer Fab 8-Drug-Linker 001 3.5 0.08 1.15 26.60 Fab 9-Drug-Linker 001 2.9 0.09 4.66 15.48 Fab 10-Drug-Linker 001 3.1 0.09 0.10 0.39 Fab 11 -Drug-Linker 001 2.7 0.11 0.12 5.41 Fab 12-Drug-Linker 001 3.6 0.16 0.65 9.72 Fab 13 -Drug-Linker 001 2.6 0.09 1.20 6.70 Fab 14-Drug-Linker 001 4.1 0.07 0.76 8.83 Fab 15 -Drug-Linker 001 3.3 0.18 5.49 11.83 Fab 16-Drug-Linker 001 3.0 0.06 0.10 0.42 Fab 17-Drug-Linker 001 3.0 0.07 0.10 9.61 Fab 18-Drug-Linker 001 3.3 0.10 0.56 8.43 Fab 19-Drug-Linker 001 3.1 0.08 0.20 0.94 Fab 20-Drug-Linker 001 3.2 0.10 0.20 7.16 Fab 21 -Drug-Linker 001 3.3 0.15 1.08 6.34 scFv 1 -Drug-Linker 001 3.5 0.09 2.15 7.63 scFv 2-Drug-Linker 001 3.4 0.12 2.35 IC scFv 3 -Drug-Linker 001 3.1 0.25 1.79 IC scFv 4-Drug-Linker 001 3.6 0.06 2.03 3.97 scFv 5 -Drug-Linker 001 3.6 0.09 1.12 8.85 scFv 6-Drug-Linker 001 3.6 0.23 8.79 IC scFv 7-Drug-Linker 001 3.7 0.10 0.09 2.44 scFv 8-Drug-Linker 001 3.7 0.22 0.54 5.53 scFv 9-Drug-Linker 001 3.3 0.08 0.17 0.43 scFv 10-Drug-Linker 001 3.5 0.09 0.12 12.68 scFv 11 -Drug-Linker 001 3.2 0.23 0.90 3.48 scFv 12-Drug-Linker 001 3.7 0.05 1.19 5.23 scFv 13 -Drug-Linker 001 3.6 0.08 0.84 IC scFv 14-Drug-Linker 001 3.4 0.24 5.10 IC scFv 15 -Drug-Linker 001 3.4 0.13 0.64 8.70EC50 (nM)Variant DAR IGROV-1 JEG-3 H2110 Monolayer Monolayer Monolayer scFv 16-Drug-Linker 001 4.1 0.05 0.08 0.34scFv 17-Drug-Linker 001 3.9 0.08 0.10 18.34 scFv 18-Drug-Linker 001 4.2 0.12 0.49 ICscFv 19-Drug-Linker 001 4.0 0.08 0.19 0.71scFv 20-Drug-Linker 001 3.6 0.11 0.31 10.22 scFv 21 -Drug-Linker 001 4.0 0.16 1.04 ICscFv 22-Drug-Linker 001 3.8 0.09 0.14 1.29scFv 23 -Drug-Linker 001 4.2 0.08 0.09 14.84 scFv 24-Drug-Linker 001 3.0 0.15 0.65 ICscFv 25-Drug-Linker 001 3.4 0.06 0.07 0.32scFv 26-Drug-Linker 001 3.8 0.07 0.06 2.75scFv 27-Drug-Linker 001 3.7 0.18 0.49 16.66 v21995-Drug-Linker 001 4.0 >70 >70 >70Table 12.2: In Vitro Cytotoxicity of FRa x NaPi2b Bispecific ADCs in 3D Spheroid Format EC50 (nM)Variant DARH1781 Spheroid H441 SpheroidFab 4-Drug-Linker 001 3.5 2.10 0.50Fab 6-Drug-Linker 001 2.6 4.33 4.22Fab 8-Drug-Linker 001 3.5 IC11.20Fab 16-Drug-Linker 001 3.0 0.24 ICFab 17-Drug-Linker 001 3.0 IC 0.57Fab 19-Drug-Linker 001 3.1 0.79 ICscFv 3 -Drug-Linker 001 3.1 IC 1.68scFv 9-Drug-Linker 001 3.3 2.09 0.33scFv 16-Drug-Linker 001 4.1 0.45 4.86scFv 25-Drug-Linker 001 3.4 0.95 1.42scFv 26-Drug-Linker 001 3.8 0.17 4.15EC50 (nM)Variant DARH1781 Spheroid H441 Spheroid v21995-Drug-Linker 001 4.0 >70 >701IC = incomplete curveEXAMPLE 13: ADDITIONAL FUNCTIONAL CHARACTERIZATION OF BISPECIFIC ANTIBODIES AND ANTIBODY-DRUG CONJUGATES

[0413] The cellular binding and internalization abilities of the bi specific antibodies shown in Table 5.1 were tested in additional cell lines following the protocols described in Examples 7 and 8, respectively. Specifically, the additional cell line used for testing cellular binding was IGROV-1 (ovarian adenocarcinoma), and the additional cell lines used for testing internalization were HCC-78 (lung carcinoma) and H441 (lung adenocarcinoma).

[0414] In addition, the cytotoxicity of ADCs comprising the bispecific FRa x NaPi2b antibodies conjugated to Drug-Linker 001 (as described in Example 10) was assessed in additional cell lines in 2D monolayer following the protocol described in Example 12. Specifically, in the cell lines HCC-78 (lung carcinoma), TOV-21G (ovarian carcinoma), T-47D (breast carcinoma) and H441 (lung adenocarcinoma).

[0415] The average number of FRa and NaPi2b proteins per cell for the cancer cell lines noted above were determined as described in Example 3 and are shown in Table 3.1 and summarized in Fig. 9.

[0416] The results of the cellular binding, internalization and cytotoxicity testing overall are summarized in Figs. 10 to 19 as described below. In these figures, the formats designated as Bsp 1-21 correspond to the formats designated as Fab 1-21, respectively, in Table 5.1, and the formats designated as Bsp 22-48 correspond to the forma...

Claims

CLAIMS1. A bispecific antibody construct comprising one or more NaPi2b antigen-binding domains that specifically bind to NaPi2b, one or more folate receptor alpha (FRa) antigen-binding domains that specifically bind to FRa, and an immunoglobulin (IgG) Fc region comprising a first Fc polypeptide and a second Fc polypeptide, wherein at least two of the antigen-binding domains are linked to the IgG Fc region.

2. The bispecific antibody construct according to claim 1, wherein the antibody construct is bivalent, trivalent or tetravalent.

3. The bispecific antibody construct according to claim 1 or 2, wherein the antibody construct is bivalent and comprises one NaPi2b antigen-binding domain and one FRa antigen-binding domain, wherein (a) both antigen-binding domains are in Fab format, or (b) one antigen-binding domain is in Fab format and the other is in scFv format.

4. The bispecific antibody construct according to claim 3, wherein one antigen-binding domain is linked to the N-terminus of one Fc polypeptide and the other antigen-binding domain is linked to the N-terminus of the other Fc polypeptide.

5. The bispecific antibody construct according to claim 1, wherein the antibody construct is trivalent and comprises one NaPi2b antigen-binding domain and two FRa antigen-binding domains.

6. The bispecific antibody construct according to claim 1, wherein the antibody construct is trivalent and comprises two NaPi2b antigen-binding domains and one FRa antigen-binding domain.

7. The bispecific antibody construct according to claim 5 or 6, wherein (a) all three antigenbinding domains are in Fab format, or (b) two antigen-binding domains are in Fab format and one antigen-binding domain is in scFv format.

8. The bispecific antibody construct according to any one of claims 5 to 7, wherein:(a) a first antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a second antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, and a third antigen-binding domain is linked to the C-terminus of one of the Fc polypeptides, or(b) a first antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a second antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, and a third antigen-binding domain is linked to the N-terminus of either the first or the second antigen-binding domain.

9. The bispecific antibody construct according to claim 1, wherein the antibody construct is trivalent and comprises:(a) two FRa antigen-binding domains in Fab format and one NaPi2b antigen-binding domain in Fab format, wherein a first FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide, the NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide and a second FRa antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain, or(b) one FRa antigen-binding domain in Fab format and two NaPi2b antigen-binding domains in Fab format, wherein the FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide and a second NaPi2b antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain, or(c) two FRa antigen-binding domains in Fab format and one NaPi2b antigen-binding domain in Fab format, wherein a first FRa antigen-binding domain is linked to the N-terminus of the first Fc polypeptide, a second FRa antigen-binding domain is linked to the C-terminus of the first Fc polypeptide, and the NaPi2b antigen-binding domain is linked to the N-terminus of the second Fc polypeptide, or(d) one FRa antigen-binding domain in Fab format and two NaPi2b antigen-binding domains in Fab format, wherein the FRa antigen-binding domain is linked to the N-terminus of the first Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of thesecond Fc polypeptide, and a second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide, or(e) one FRa antigen-binding domain in Fab format, a first NaPi2b antigen-binding domain in Fab format and a second NaPi2b antigen-binding domain in scFv format, wherein the FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide, the first NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, and the second NaPi2b antigen-binding domain is linked to the N-terminus of the first NaPi2b antigen-binding domain, or(f) a first FRa antigen-binding domain in Fab format, a second FRa antigen-binding domain in scFv format and one NaPi2b antigen-binding domain in Fab format, wherein the first FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide, the second FRa antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain, and the NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, or(g) one FRa antigen-binding domain in Fab format, a first NaPi2b antigen-binding domain in Fab format and a second NaPi2b antigen-binding domain in scFv format, wherein the FRa antigen-binding domain is linked to the N-terminus of the first Fc polypeptide, the first NaPi2b antigen-binding domain is linked to the N-terminus of the second Fc polypeptide, and the second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide, or(h) a first FRa antigen-binding domain in Fab format, a second FRa antigen-binding domain in scFv format and one NaPi2b antigen-binding domain in Fab format, wherein the first FRa antigen-binding domain is linked to the N-terminus of the first Fc polypeptide, the second FRa antigen-binding domain is linked to the C-terminus of the first Fc polypeptide, and the NaPi2b antigen-binding domain is linked to the N-terminus of the second Fc polypeptide.

10. The bispecific antibody construct according to claim 1, wherein the antibody construct is tetravalent and comprises two NaPi2b antigen-binding domains and two FRa antigen-binding domains.

11. The bispecific antibody construct according to claim 10, wherein (a) all four antigenbinding domains are in Fab format, or (b) three antigen-binding domains are in Fab format and one antigen-binding domain is in scFv format.

12. The bispecific antibody construct according to claim 10 or 11, wherein:(a) a first antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a second antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, a third antigen-binding domain is linked to the C-terminus of one of the Fc polypeptides and a fourth antigen-binding domain is linked to the C-terminus of the other Fc polypeptide, or(b) a first antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a second antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, a third antigen-binding domain is linked to the C-terminus of one of the Fc polypeptides and a fourth antigen-binding domain is linked to the N-terminus of either the first or the second antigen-binding domain, or(c) a first antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a second antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, a third antigen-binding domain is linked to the N-terminus of either the first or the second antigen-binding domain and a fourth antigen-binding domain is linked to the N-terminus of the other of the first or second antigen-binding domain.

13. The bispecific antibody construct according to claim 10, wherein the antibody construct is tetravalent and comprises:(a) two FRa antigen-binding domains in Fab format and two NaPi2b antigen-binding domains in Fab format, wherein a first FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, a second FRa antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain and a second NaPi2b antigen-binding domain is linked to the N-terminus of the first NaPi2b antigen-binding domain, or(b) two FRa antigen -binding domains in Fab format and two NaPi2b antigen-binding domains in Fab format, wherein a first FRa antigen-binding domain is linked to the N-terminus of the first Fc polypeptide, a second FRa antigen-binding domain is linked to the C-terminus of the first Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the second Fc polypeptide, and a second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide, or(c) two FRa antigen-binding domains in Fab format and two NaPi2b antigen-binding domains in Fab format, wherein a first FRa antigen-binding domain is linked to the N-terminus of the first Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the second Fc polypeptide, a second FRa antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain and a second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide, or(d) two FRa antigen-binding domains in Fab format, a first NaPi2b antigen-binding domain in Fab format and a second NaPi2b antigen-binding domain in scFv format, wherein a first FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide, a second FRa antigenbinding domain is linked to the N-terminus of the first FRa antigen-binding domain, the first NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, and the second NaPi2b antigen-binding domain is linked to the N-terminus of the first NaPi2b antigenbinding domain, or(e) a first FRa antigen-binding domain in Fab format, a second FRa antigen-binding domain in scFv format and two NaPi2b antigen-binding domains in Fab format, wherein the first FRa antigen-binding domain is linked to the N-terminus of one Fc polypeptide, the second FRa antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, and a second NaPi2b antigen-binding domain is linked to the N-terminus of the first NaPi2b antigenbinding domain, or(f) two FRa antigen-binding domains in Fab format, a first NaPi2b antigen-binding domain in Fab format and a second NaPi2b antigen-binding domain in scFv format, wherein a first FRa antigen-binding domain is linked to the N-terminus of the first Fc polypeptide, a second FRaantigen-binding domain is linked to the C-terminus of the first Fc polypeptide, the first NaPi2b antigen-binding domain is linked to the N-terminus of the second Fc polypeptide, and the second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide, or(g) a first FRa antigen-binding domain in Fab format, a second FRa antigen-binding domain in scFv format and two NaPi2b antigen-binding domains in Fab format, wherein the first FRa antigen-binding domain is linked to the N-terminus of the first Fc polypeptide, the second FRa antigen-binding domain is linked to the C-terminus of the first Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the second Fc polypeptide, and a second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide, or(h) two FRa antigen-binding domains in Fab format, a first NaPi2b antigen-binding domain in Fab format and a second NaPi2b antigen-binding domain in scFv format, in which a first FRa antigen-binding domain is linked to the N-terminus of the first Fc polypeptide, a second FRa antigen-binding domain is linked to the C-terminus of the first Fc polypeptide, the first NaPi2b antigen-binding domain is linked to the N-terminus of the second Fc polypeptide, and the second NaPi2b antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain, or(i) a first FRa antigen-binding domain in Fab format, a second FRa antigen-binding domain in scFv format and two NaPi2b antigen-binding domains in Fab format, wherein the first FRa antigen-binding domain is linked to the N-terminus of the first Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the second Fc polypeptide, a second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide, and the second FRa antigen-binding domain is linked to the N-terminus of the first NaPi2b antigen-binding domain.

14. The bispecific antibody construct according to any one of claims 1 to 13, wherein the one or more NaPi2b antigen-binding domains comprise the heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) of the VH domain as set forth in SEQ ID NO: 58, and the light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) of the VL domain as set forth in SEQ ID NO: 59.

15. The bispecific antibody construct according to claim 14, wherein the one or more NaPi2b antigen-binding domains comprise heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 52, 53 and 54, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 55, 56 and 57.

16. The bispecific antibody construct according to any one of claims 1 to 13, wherein the one or more NaPi2b antigen-binding domains comprise a VH domain comprising the sequence as set forth in SEQ ID NO: 58, and a VL domain comprising the sequence as set forth in SEQ ID NO: 59.

17. The bispecific antibody construct according to any one of claims 1 to 16, wherein the one or more FRa antigen-binding domains comprise:(a) the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) of the VH domain as set forth in SEQ ID NO: 50, and the light chain CDRs (LCDR1, LCDR2 and LCDR3) of the VL domain as set forth in SEQ ID NO: 51, or(b) the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) of the VH domain as set forth in SEQ ID NO: 42, and the light chain CDRs (LCDR1, LCDR2 and LCDR3) of the VL domain as set forth in SEQ ID NO: 43, or(c) the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) of the VH domain as set forth in SEQ ID NO:29, and the light chain CDRs (LCDR1, LCDR2 and LCDR3) of the VL domain as set forth in SEQ ID NO: 33.

18. The bispecific antibody construct according to claim 17, wherein the one or more FRa antigen-binding domains comprise:(a) heavy chain CDRs (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 44, 45 and 46, and light chain CDRs (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 47, 48 and 49, or(b) heavy chain CDRs (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 36, 37 and 38, and light chain CDRs (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 39, 40 and 41, or(c) heavy chain CDRs (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 10, 11 and 7, and light chain CDRs (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 18, 19 and 17.

19. The bispecific antibody construct according to any one of claims 1 to 16, wherein the one or more FRa antigen-binding domains comprise:(a) a VH domain comprising the sequence as set forth in SEQ ID NO: 50, and a VL domain comprising the sequence as set forth in SEQ ID NO: 51, or(b) a VH domain comprising the sequence as set forth in SEQ ID NO: 42, and a VL domain comprising the sequence as set forth in SEQ ID NO: 43, or(c) a VH domain comprising the sequence as set forth in SEQ ID NO: 29, and a VL domain comprising the sequence as set forth in SEQ ID NO: 33.

20. The bispecific antibody construct according to any one of claims 1 to 19, wherein the bispecific antibody construct comprises at least one FRa antigen-binding domain in Fab format and at least one NaPi2b antigen-binding domain in Fab format, and wherein the CHI and CL domains of the at least one FRa antigen-binding domain in Fab format and the CHI and CL domains of the at least one NaPi2b antigen-binding domain in Fab format comprise sets of amino acid mutations to drive correct pairing between the CHI and CL of the FRa antigen-binding domain and between the CHI and CL of the NaPi2b antigen-binding domain.

21. The bispecific antibody construct according to claim 20, wherein:(a) the CHI domain of the at least one FRa antigen-binding domain in Fab format comprises the amino acid mutations A139W / L143E / K145T / Q179E, the CL domain of the at least one FRa antigen-binding domain in Fab format comprises the amino acid mutations F116A / Q124R / L135V / T178R, the CH1 domain of the at least one NaPi2b antigen-binding domain in Fab format comprises the amino acid mutation Q179R, and the CL domain of the at least oneNaPi2b antigen-binding domain in Fab format comprises the amino acid mutations Q124E / L135W / T178E / T180E; or(b) the CHI domain of the at least one NaPi2b antigen-binding domain in Fab format comprises the amino acid mutations A139W / L143E / K145T / Q179E, the CL domain of the at least one NaPi2b antigen-binding domain in Fab format comprises the amino acid mutations F116A / Q124R / L135V / T178R, the CHI domain of the at least one FRa antigen-binding domain in Fab format comprises the amino acid mutation Q179R, and the CL domain of the at least one FRa antigen-binding domain in Fab format comprises the amino acid mutations Q124E / L135W / T178E / T180E; or(c) the CHI domain of the at least one FRa antigen-binding domain in Fab format comprises the amino acid mutation L143R, the CL domain of the at least one FRa antigen-binding domain in Fab format comprises the amino acid mutations Q124E / V133E, the CHI domain of the at least one NaPi2b antigen-binding domain in Fab format comprises the amino acid mutations L143E / K145T / Q179E, and the CL domain of the at least one NaPi2b antigen-binding domain in Fab format comprises the amino acid mutations Q124R / T178R; or(d) the CHI domain of the at least one NaPi2b antigen-binding domain in Fab format comprises the amino acid mutation L143R, the CL domain of the at least one NaPi2b antigenbinding domain in Fab format comprises the amino acid mutations Q124E / V133E, the CHI domain of the at least one FRa antigen-binding domain in Fab format comprises the amino acid mutations L143E / K145T / Q179E, and the CL domain of the at least one FRa antigen-binding domain in Fab format comprises the amino acid mutations Q124R / T178R.

22. The bispecific antibody construct according to any one of claims 1 to 21, wherein the IgG Fc region is an IgGl Fc region.

23. The bispecific antibody construct according to any one of claims 1 to 22, wherein the IgG Fc region is a heterodimeric Fc region comprising one or more amino acid substitutions to promote formation of the heterodimeric Fc region.

24. The bispecific antibody construct according to claim 23, wherein the amino acid substitutions comprised by the Fc region are:(a) the amino acid substitutions L351Y F405A Y407V in one Fc polypeptide and the amino acid substitutions T366L K392M T394W in the other Fc polypeptide, or(b) the amino acid substitutions L351Y F405A Y407V in one Fc polypeptide and the amino acid substitutions T366L K392L T394W in the other Fc polypeptide, or(c) the amino acid substitutions T350V L351 Y F405A Y407V in one Fc polypeptide and the amino acid substitutions T350V_T366L_K392L_T394W in the other Fc polypeptide, or (d) the amino acid substitutions T350V L351 Y F405A Y407V in one Fc polypeptide and the amino acid substitutions T350V_T366L_K392M_T394W in the other Fc polypeptide, or (e) the amino acid substitutions T350V_L351Y_S400E_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T350V_T366L_N390R_K392M_T394W in the other Fc polypeptide,wherein the numbering of amino acids is according to the EU index.

25. A bispecific antibody construct comprising one or more NaPi2b antigen-binding domains that specifically bind to NaPi2b, one or more folate receptor alpha (FRa) antigen-binding domains that specifically bind to FRa, and an immunoglobulin (IgG) Fc region comprising a first Fc polypeptide and a second Fc polypeptide, wherein at least two of the antigen-binding domains are linked to the IgG Fc region, and wherein the one or more NaPi2b antigen-binding domains comprise the heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) of the VH domain as set forth in SEQ ID NO: 58, and the light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) of the VL domain as set forth in SEQ ID NOs: 59.

26. The bispecific antibody construct according to claim 25, wherein the one or more NaPi2b antigen-binding domains comprise heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 52, 53 and 54, and light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 55, 56 and 57.

27. The bispecific antibody construct according to claim 25 or 26, wherein the one or more NaPi2b antigen-binding domains comprise a VH domain comprising the sequence as set forth in SEQ ID NO: 58, and a VL domain comprising the sequence as set forth in SEQ ID NO: 5928. The bispecific antibody construct according to any one of claims 25 to 27, wherein the one or more FRa antigen-binding domains comprise:(a) the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) of the VH domain as set forth in SEQ ID NO: 50, and the light chain CDRs (LCDR1, LCDR2 and LCDR3) of the VL domain as set forth in SEQ ID NO: 51, or(b) the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) of the VH domain as set forth in SEQ ID NO: 42, and the light chain CDRs (LCDR1, LCDR2 and LCDR3) of the VL domain as set forth in SEQ ID NO: 43, or(c) the heavy chain CDRs (HCDR1, HCDR2 and HCDR3) of the VH domain as set forth in SEQ ID NO: 29, and the light chain CDRs (LCDR1, LCDR2 and LCDR3) of the VL domain as set forth in SEQ ID NO: 33.

29. The bispecific antibody construct according to claim 28, wherein the one or more FRa antigen-binding domains comprise:(a) heavy chain CDRs (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 44, 45 and 46, and light chain CDRs (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 47, 48 and 49, or(b) heavy chain CDRs (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 36, 37 and 38, and light chain CDRs (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 39, 40 and 41, or(c) heavy chain CDRs (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 10, 11 and 7, and light chain CDRs (LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 18, 19 and 17.

30. The bispecific antibody construct according to any one of claims 25 to 29, wherein the one or more FRa antigen-binding domains comprise:(a) a VH domain comprising the sequence as set forth in SEQ ID NO: 50, and a VL domain comprising the sequence as set forth in SEQ ID NO: 51, or(b) a VH domain comprising the sequence as set forth in SEQ ID NO: 42, and a VL domain comprising the sequence as set forth in SEQ ID NO: 43, or(c) a VH domain comprising the sequence as set forth in SEQ ID NO: 29, and a VL domain comprising the sequence as set forth in SEQ ID NO: 33.

31. The bispecific antibody construct according to any one of claims 25 to 30, wherein the bispecific antibody construct is bivalent, trivalent or tetraval ent.

32. The bispecific antibody construct according to any one of claims 25 to 31, wherein:(a) the bispecific antibody construct is bivalent and comprises one FRa antigen-binding domain in Fab format and one NaPi2b antigen-binding domain in Fab format, wherein the FRa antigen-binding domain is linked to the N-terminus of one or the first or second Fc polypeptides and the NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide; or(b) the FRa x NaPi2b antibody constructs is trivalent and comprises two FRa antigenbinding domains in Fab format and one NaPi2b antigen-binding domain in Fab format, wherein a first FRa antigen-binding domain is linked to the N-terminus of one of the first or second Fc polypeptides, the NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide and a second FRa antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain; or(c) the bispecific antibody construct is trivalent and comprises one FRa antigen-binding domain in Fab format and two NaPi2b antigen-binding domains in Fab format, wherein the FRa antigen-binding domain is linked to the N-terminus of one of the first or second Fc polypeptides, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide and a second NaPi2b antigen-binding domain is linked to the N-terminus of the first NaPi2b antigenbinding domain; or(d) the bispecific antibody construct is trivalent and comprises two FRa antigen-binding domains in Fab format and one NaPi2b antigen-binding domain in Fab format, wherein a first FRa antigen-binding domain is linked to the N-terminus of the first Fc polypeptide, a second FRaantigen-binding domain is linked to the C-terminus of the first Fc polypeptide, and the NaPi2b antigen-binding domain is linked to the N-terminus of the second Fc polypeptide; or(e) the bispecific antibody construct is tetravalent and comprises two FRa antigen-binding domains in Fab format and two NaPi2b antigen-binding domains in Fab format, wherein a first FRa antigen-binding domain is linked to the N-terminus of one of the first or second Fc polypeptides, a first NaPi2b antigen-binding domain is linked to the N-terminus of the other Fc polypeptide, a second FRa antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain and a second NaPi2b antigen-binding domain is linked to the N-terminus of the first NaPi2b antigen-binding domain; or(f) the bispecific antibody construct is tetraval ent and comprises two FRa antigen-binding domains in Fab format and two NaPi2b antigen-binding domains in Fab format, wherein a first FRa antigen-binding domain is linked to the N-terminus of the first Fc polypeptide, a second FRa antigen-binding domain is linked to the C-terminus of the first Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the second Fc polypeptide, and a second NaPi2b antigen-binding domain is linked to the C-terminus of the second Fc polypeptide; or(g) the bispecific antibody construct is tetravalent and comprises two FRa antigen-binding domains in Fab format, a first NaPi2b antigen-binding domain in Fab format and a second NaPi2b antigen-binding domain in scFv format, wherein a first FRa antigen-binding domain is linked to the N-terminus of one of the first or second Fc polypeptides, a second FRa antigen-binding domain is linked to the N-terminus of the first FRa antigen-binding domain, the first NaPi2b antigenbinding domain is linked to the N-terminus of the other Fc polypeptide, and the second NaPi2b antigen-binding domain is linked to the N-terminus of the first NaPi2b antigen-binding domain; or(h) the bispecific antibody construct is tetravalent and comprises a first FRa antigenbinding domain in Fab format, a second FRa antigen-binding domain in scFv format and two NaPi2b antigen-binding domains in Fab format, wherein the first FRa antigen-binding domain is linked to the N-terminus of the first Fc polypeptide, a first NaPi2b antigen-binding domain is linked to the N-terminus of the second Fc polypeptide, a second NaPi2b antigen-binding domainis linked to the C-terminus of the second Fc polypeptide, and the second FRa antigen-binding domain is linked to the N-terminus of the first NaPi2b antigen-binding domain.

33. The bispecific antibody construct according to any one of claims 25 to 32, wherein the bispecific antibody construct comprises at least one FRa antigen-binding domain in Fab format and at least one NaPi2b antigen-binding domain in Fab format, and wherein the CHI and CL domains of the at least one FRa antigen-binding domain in Fab format and the CHI and CL domains of the at least one NaPi2b antigen-binding domain in Fab format comprise sets of amino acid mutations to drive correct pairing between the CHI and CL of the FRa antigen-binding domain and between the CHI and CL of the NaPi2b antigen-binding domain.

34. The bispecific antibody construct according to claim 33, wherein:(a) the CHI domain of the at least one FRa antigen-binding domain in Fab format comprises the amino acid mutations A139W / L143E / K145T / Q179E, the CL domain of the at least one FRa antigen-binding domain in Fab format comprises the amino acid mutations F116A / Q124R / L135V / T178R, the CH1 domain of the at least one NaPi2b antigen-binding domain in Fab format comprises the amino acid mutation Q179R, and the CL domain of the at least one NaPi2b antigen-binding domain in Fab format comprises the amino acid mutations Q124E / L135W / T178E / T180E; or(b) the CHI domain of the at least one NaPi2b antigen-binding domain in Fab format comprises the amino acid mutations A139W / L143E / K145T / Q179E, the CL domain of the at least one NaPi2b antigen-binding domain in Fab format comprises the amino acid mutations F116A / Q124R / L135V / T178R, the CHI domain of the at least one FRa antigen-binding domain in Fab format comprises the amino acid mutation Q179R, and the CL domain of the at least one FRa antigen-binding domain in Fab format comprises the amino acid mutations Q124E / L135W / T178E / T180E; or(c) the CHI domain of the at least one FRa antigen-binding domain in Fab format comprises the amino acid mutation L143R, the CL domain of the at least one FRa antigen-binding domain in Fab format comprises the amino acid mutations Q124E / V133E, the CHI domain of the at least one NaPi2b antigen-binding domain in Fab format comprises the amino acid mutationsL143E / K145T / Q179E, and the CL domain of the at least one NaPi2b antigen-binding domain in Fab format comprises the amino acid mutations Q124R / T178R; or(d) the CHI domain of the at least one NaPi2b antigen-binding domain in Fab format comprises the amino acid mutation L143R, the CL domain of the at least one NaPi2b antigenbinding domain in Fab format comprises the amino acid mutations Q124E / V133E, the CHI domain of the at least one FRa antigen-binding domain in Fab format comprises the amino acid mutations L143E / K145T / Q179E, and the CL domain of the at least one FRa antigen-binding domain in Fab format comprises the amino acid mutations Q124R / T178R.

35. The bispecific antibody construct according to any one of claims 25 to 34, wherein the IgG Fc region is an IgGl Fc region.

36. The bispecific antibody construct according to any one of claims 25 to 35, wherein the IgG Fc region is a heterodimeric Fc region comprising one or more amino acid substitutions to promote formation of the heterodimeric Fc region.

37. The bispecific antibody construct according to claim 36, wherein the amino acid substitutions comprised by the Fc region are:(a) the amino acid substitutions L351Y F405A Y407V in one Fc polypeptide and the amino acid substitutions T366L K392M T394W in the other Fc polypeptide, or(b) the amino acid substitutions L351Y F405A Y407V in one Fc polypeptide and the amino acid substitutions T366L K392L T394W in the other Fc polypeptide, or(c) the amino acid substitutions T350V L351 Y F405A Y407V in one Fc polypeptide and the amino acid substitutions T350V T366L K392L T394W in the other Fc polypeptide, or (d) the amino acid substitutions T350V L351 Y F405A Y407V in one Fc polypeptide and the amino acid substitutions T350V_T366L_K392M_T394W in the other Fc polypeptide, or (e) the amino acid substitutions T350V_L351Y_S400E_F405A_Y407V in one Fc polypeptide and the amino acid substitutions T350V_T366L_N390R_K392M_T394W in the other Fc polypeptide,wherein the numbering of amino acids is according to the EU index.

38. A polynucleotide or set of polynucleotides encoding the bispecific antibody construct according to any one of claims 1 to 37.

39. An expression vector or set of expression vectors comprising the polynucleotide or set of polynucleotides according to claim 38.

40. A host cell comprising the expression vector or set of expression vectors according to claim 39.

41. An antibody-drug conjugate comprising the bispecific antibody construct according to any one of claims 1 to 37 conjugated to one or more drug moieties.

42. The antibody-drug conjugate according to claim 41, wherein the antibody conjugate is conjugated to between 1 and about 8 drug moieties.

43. An antibody-drug conjugate having general Formula I:A-(L-(D)m)n(I)wherein:A is the bispecific antibody construct according to any one of claims 1 to 37;L is a linker;D is a drug moiety;m is between 1 and about 8, andn is 1 and about 12.

44. The antibody-drug conjugate according to claim 43, wherein m is 1 or 2.

45. The antibody-drug conjugate according to claim 43 or 44, wherein n is between about 2 and about 8.

46. The antibody-drug conjugate according to any one of claims 41 to 45, wherein the drug moiety is a maytansinoid, maytansinoid analogue, benzodiazepine, pyrrolobenzodiazepine, duocarmycin, calicheamicin, calicheamicin analogue, auristatin, auristatin analogue, hemiasterlin, hemiasterlin analogue, tubulysin, tubulysin analogue, amatoxin, amatoxin analogue, camptothecin, camptothecin analogue, eribulin, TLR agonist or STING agonist.

47. A pharmaceutical composition comprising the bispecific antibody construct according to any one of claims 1 to 37, or the antibody-drug conjugate according to any one of claims 41 to 46, and a pharmaceutically acceptable carrier or diluent.

48. A bispecific antibody construct according to any one of claims 1 to 37 or an antibody-drug conjugate according to any one of claims 41 to 46 for use in therapy.

49. The antibody-drug conjugate for use according to claim 48, or the antibody drug conjugate for use according to claim 48, wherein the therapy comprises treatment of cancer.

50. Use of a bispecific antibody construct according to any one of claims 1 to 37, or an antibody drug conjugate according to any one of claims 41 to 46, in the manufacture of a medicament for the treatment of cancer.

51. A method of inhibiting the growth of tumor cells that express FRa, NaPi2b or both FRa and NaPi2b, comprising contacting the cells with a bispecific antibody construct according to any one of claims 1 to 37 or an antibody-drug conjugate according to any one of claims 41 to 46.

52. A method of treating a subject having a cancer comprising administering to the subject an effective amount of a bispecific antibody construct according to any one of claims 1 to 37 or an antibody-drug conjugate according to any one of claims 41 to 46.

53. The method according to claim 52, wherein the cancer is a gynecological cancer.

54. The method according to claim 52, wherein the cancer is ovarian cancer.

55. An antibody construct comprising an antigen-binding domain that specifically binds to folate receptor alpha (FRa), the antigen-binding domain comprising the heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) of the VH sequence as set forth in SEQ ID NO: 29, and the light chain CDR amino acid sequences (LCDR1, LCDR2 and LCDR3) of the VL sequence as set forth in SEQ ID NO: 33.

56. The antibody construct according to claim 55, wherein the antigen-binding domain comprises heavy chain CDR amino acid sequences (HCDR1, HCDR2 and HCDR3) comprising the sequences as set forth in SEQ ID NOs: 10, 11 and 7, and light chain CDR amino acid sequences(LCDR1, LCDR2 and LCDR3) comprising the sequences as set forth in SEQ ID NOs: 18, 19 and 17.

57. The antibody construct according to claim 55 or 56, wherein the antigen-binding domain comprises:(a) a VH sequence having a sequence as set forth in SEQ ID NO: 29 and a VL sequence having a sequence as set forth in any one of SEQ ID NOs: 33, 34 or 35, or(b) a VH domain comprising the sequence as set forth in SEQ ID NO: 32 and a VL domain comprising the sequence as set forth in any one of SEQ ID NOs: 33, 34 or 35.

58. The antibody construct according to claim 55 or 56, wherein the antigen-binding domain comprises a VH domain comprising the sequence as set forth in SEQ ID NO: 29 and a VL domain comprising the sequence as set forth in SEQ ID NO: 33.

59. The antibody construct according to any one of claims 55 to 58 further comprising a scaffold, wherein the antigen-binding domain is operably linked to the scaffold.

60. The antibody construct according to claim 59, wherein the scaffold comprises an IgG Fc region.

61. A polynucleotide or set of polynucleotides encoding the antibody construct according to any one of claims 55 to 60.

62. An expression vector or set of expression vectors comprising the polynucleotide or set of polynucleotides according to claim 61.

63. A host cell comprising the expression vector or set of expression vectors according to claim 62.

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