EA043790B1 - NaPi2b-TARGETING ANTIBODY-DRUG CONJUGATES AND METHODS OF THEIR APPLICATION - Google Patents

Description

Related applications

This application claims priority to provisional patent application US 62/308,567, filed March 15, 2016; 62/323,068, filed April 15, 2016, and 62/383,324, filed September 2, 2016, pursuant to 35 USC 119(e). The contents of each of these applications are incorporated herein by reference in their entirety.

Disclosure area

This disclosure generally relates to NaPi2b-targeted antibody-drug conjugates (such as NaPi2b-targeted antibody-polymer-drug) that specifically bind to the extracellular region of SLC34A2, and to methods of using these conjugates as therapeutic and/or diagnostic agents .

State of the art

NaPi2b (SLC34A2, NaPiIIb, Npt2), a multitransmembrane, sodium-dependent phosphate transporter (Xu et al., Genomics 62:281-284 62:281-284 (1999)), is typically expressed in the brush border membrane of the mammalian small intestine and is involved in transcellular absorption of inorganic phosphate (Pi), helping to maintain phosphate homeostasis in the body. Expression of NaPi2b at the protein level was detected in the liver, in the apical surface of epithelial cells of the mammary and salivary glands, as well as in the lungs, testis, salivary gland, thyroid gland, small intestine and uterus. Mutations in NaPi2b have been associated with clinical syndromes of alveolar and testicular microlithiasis. NaPi2b is highly expressed in non-squamous non-small cell lung cancer (NSCLC), non-mucinous ovarian cancer and papillary thyroid cancer. NaPi2b-positive tissue immunoreactivity was present in 61% of NSCLC samples and 92% of ovarian cancer samples.

Ovarian cancer is one of the most common gynecological malignancies and the fifth leading cause of cancer death in women. The high mortality rates are partly a result of the frequent diagnosis of ovarian cancer at advanced stages, and the mortality rate is approximately 65% of the incidence rate. Epithelial tumors of the ovary comprise 58% of all ovarian tumors and more than 90% of malignant ovarian tumors. Surgery to debulk the tumor and platinum-based combination chemotherapy (including taxanes) are currently used treatment modalities; however, most patients with recurrent epithelial ovarian cancer ultimately die from the disease. There is a need for new treatments for ovarian cancer, including targeted therapies such as monoclonal antibody immunotherapy or cancer vaccine approaches.

NSCLC is any type of epithelial lung cancer other than small cell lung cancer (SCLC). NSCLC accounts for approximately 85% of all lung cancers. As a class, NSCLCs are relatively insensitive to chemotherapy compared with small cell carcinomas. If possible, they are primarily treated with surgical resection with curative intent, although chemotherapy is increasingly used, both preoperatively (neoadjuvant chemotherapy) and postoperatively (adjuvant chemotherapy). In metastatic or inoperable cases, chemotherapy and/or immunotherapy are used, although the disease at this stage is largely incurable and survival time remains short. There is a need for new treatments for NSCLC, including targeted therapies such as monoclonal antibody immunotherapy or cancer vaccine approaches.

Accordingly, there is a need for therapeutic agents targeting the biological activity of NaPi2b.

The essence of the invention

The present disclosure relates to a NaPi2b-targeted antibody-drug conjugate (such as a NaPi2b-targeted antibody-polymer-drug conjugate) that is biodegradable, biocompatible, and exhibits high drug loading as well as specific binding to the extracellular region of SLC34A2. The NaPi2b-targeting antibody-drug conjugates (eg, NaPi2b-targeting antibody-polymer drug conjugates) of the invention include an antibody that specifically recognizes NaPi2b, also known as sodium-dependent phosphate transport protein 2B. Antibodies used in the NaPi2b-targeting antibody-drug conjugates disclosed herein may also include antibodies that are capable of and useful for modulating, blocking, inhibiting, attenuating, counteracting, neutralizing, or otherwise interfering with at least one biological activity of NaPi2b . The antibodies disclosed herein also include antibodies that bind to soluble NaPi2b.

In some embodiments, a NaPi2b-targeting antibody disclosed herein may be linked to an agent to form a conjugate. In some embodiments, the agent is a therapeutic agent. In some embodiments, the agent is an antineoplastic agent. In some embodiments, the agent is a toxin or a fragment thereof. In some embodiments, the agent is (a) an auristatin compound; (b) a calicheamicin compound; (c) a duocarmycin compound; (d) SN3 8, (e) pyrrolobenzodiazepine; (f) connection bar

- 1 043790 wine; (g) a tubulisin compound; (h) a non-natural camptothecin compound; (i) a maytansinoid compound; (j) a DNA-binding drug; (k) a kinase inhibitor; (l) a MEK inhibitor; (m) a KSP inhibitor; (n) a topoisomerase inhibitor; (o) a DNA alkylating drug; (p) RNA polymerase inhibitor; or their analogues. In some embodiments, the agent is conjugated to a NaPi2b-targeting antibody via a linker. In some embodiments, the linker is a cleavable linker. In some embodiments, the linker is a non-cleavable linker. In some embodiments, the agent is any of the toxins described herein.

In one aspect, the NaPi2b-targeting antibody conjugate described herein includes an isolated NaPi2b-targeting antibody coupled directly or indirectly to one or more therapeutic or diagnostic agents (D). In some embodiments, the targeted antibody NaPi2b conjugate also includes one or more polymer backbones coupled to the antibody, with each one or more Ds independently linked to the antibody through one or more polymer backbones.

In some embodiments, each one or more polymer backbones associated with the isolated NaPi2b-targeting antibody independently comprises poly(1-hydroxymethylethylene hydroxymethyl formal) (PHF), e.g., PHF having a molecular weight ranging from about 2 kDa to about 40 kYes.

In some embodiments, each one or more polymer backbones independently has formula (Ic):

in which L ^d1 is a carbonyl-containing group;

each ^c in ^-cc=0)l3 4 ^d independently denotes a first linker containing a biodegradable bond such that when the bond is broken, D is released in an active form for its intended therapeutic effect; and in ^- between L ^d1 and D denotes direct or indirect attachment of D to L ^d1 ;

each - ) ι ^ρ independently denotes a second linker not yet attached to the isolated antibody, in which LP ² is a group containing a functional group that should form a covalent bond with the functional group of the isolated antibody, and between L ^d1 and LP ² denotes direct or indirect joining LP ² to L ^d1 and every second linker is different from every first linker;

each c(=on°—|-l—independently denotes a third linker connecting each D-supporting polymer backbone to the isolated antibody, in which the terminal , attached to LP ² , denotes direct or indirect attachment of LP ² to the isolated antibody after formation of a covalent bond between the functional group of LP ² and the functional group of the isolated antibody, and every third linker is different from every first linker;

m is an integer from 1 to about 300, m1 is an integer from 1 to about 140, m ₂ is an integer from 1 to about 40, m ₃ is an integer from 0 to about 18, m4 is an integer from 1 to about 18 10;

the sum of m, m1, _m2 , _m3 and _m4 is from about 15 to about 300;

the total number of LP ² associated with the isolated antibody is 10 or less.

The conjugate described herein may include one or more of the following features.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody has a molecular weight of 40 kDa or more (e.g., 60 kDa or more, 80 kDa or more, 100 kDa or more, 120 kDa or more, 140 kDa or more, 160 kDa or more than, 180 kDa or more, or 200 kDa or more, or approximately 40-200 kDa, 40-180 kDa, 40-140 kDa, 60-200 kDa, 60-180 kDa, 60-140 kDa, 80-200 kDa, 80-180 kDa, 80-140 kDa, 100-200 kDa, 100-180 kDa, 100-140 kDa or 140-150 kDa). In some embodiments, the isolated NaPi2b-targeting antibody or any antibody according to the disclosure, including, by way of non-limiting example, the XMT 1535 antibody and the 10H1.11.4B antibody described herein.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody specifically binds to an epitope of human NaPi2b. In some embodiments, the isolated antibody specifically binds to an epitope on the extracellular region of human NaPi2b.

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For example, in formula (Ic), the isolated NaPi2b-targeting antibody is the XMT 1535 antibody and/or the 10H1.11.4B antibody described herein, as well as their biosimilars. Also, a monoclonal antibody is an antibody that binds to the same epitope and/or cross-blocks the antibody of the disclosure or its biosimilars. These antibodies are respectively referred to herein as NaPi2b antibodies or NaPi2b-targeted antibodies. NaPi2b antibodies include fully human monoclonal antibodies, as well as humanized monoclonal antibodies and chimeric antibodies. These antibodies show specificity for human NaPi2b, and they can modulate, block, inhibit, reduce, counteract, neutralize or otherwise interfere with at least one activity of NaPi2b.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody is a monoclonal antibody.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody is a rabbit, mouse, chimeric, humanized or fully human monoclonal antibody.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody is an IgG isotype. In some embodiments, the isolated NaPi2b-targeting antibody is an IgG1 isotype.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody includes a complementary determining region heavy chain 1 (CDRH1) variable region comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97% 98%, 99% or more identical to the amino acid sequence of gytftgynih (seq id no: 5), complementarity determining region heavy chain 2 (CDRH2) variable region comprising at least 90%, 91% of the amino acid sequence , 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical amino acid sequence; and a complementarity determining region heavy chain 3 (CDRH3) variable region comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of getaratfay (seq id no: 7). For example, in formula (Ic), the isolated NaPi2b-targeting antibody includes a complementarity determining region light chain 1 (CDRL1) variable region comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97% 98%, 99% or more identical to the amino acid sequence sasqdignfln (seq id no: 8); complementarity determining region light chain 2 (CDRL2) variable region comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of ytsslys (seq id no: 9); and a complementarity determining region light chain 3 (CDRL3) variable region comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence QQYSKLPLT (seq id no: 10).

For example, in formula (Ic), the isolated NaPi2b-targeting antibody comprises CDRH1 comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99 % or more identical to the amino acid sequence gytftgynih (seq id NO: 5); CDRH2 comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence aiypgngdtsykqkfrg (seq id no: 6 ); CDRH3 comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of getaratfay (seq id no: 7 ); CDRL1 comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of sasqdignfln (seq id no: 8 ); CDRL2 comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of ytsslys (seq id no: 9 ); and CDRL3 comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of QQYSKLPLT (seq id no: io).

For example, in formula (Ic), the isolated NaPi2b-targeting antibody includes a complementary determining region heavy chain 1 (CDRH1) variable region comprising the amino acid sequence gytftgynih (seq id no: 5), a complementarity determining region heavy chain 2 (CDRH2) variable region comprising amino acid sequence aiypgngdtsykqkfrg (seq id no: 6); and a complementarity determining region heavy chain 3 (CDRH3) variable region comprising the amino acid sequence getaratfay (seq id NO: 7). For example, in formula (Ic), the isolated NaPi2b-targeting antibody includes a complementarity determining region light chain 1 (CDRL1) variable region comprising the amino acid sequence sasqdignfln (seq id no: 8); complementarity determining region light chain 2 (CDRL2) variable region comprising the amino acid sequence ytsslys (seq id NO: 9); and complementarity determining region light chain 3 (CDRL3) variable region comprising the amino acid sequence

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QQYSKLPLT (SEQ ID NO: 10) .

For example, in formula (Ic), the isolated NaPi2b-targeting antibody includes CDRH1 comprising the amino acid sequence gytftgynih (seq id no: 5); CDRH2, comprising the amino acid sequence aiypgngdtsykqkfrg (seq id no: 6); variable CDRH3, including the amino acid sequence getaratfay (seq id no: 7); CDRL1, including the amino acid sequence sasqdignfln (seq id no: 8); CDRL2, including the amino acid sequence ytsslys (seq id no: 9); and CDRL3, comprising the amino acid sequence QQYSKLPLT (SEQ ID NO: 10).

For example, in formula (Ic), the isolated NaPi2b-targeting antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 3. For example, in formula (Ic), the isolated NaPi2b-targeting antibody includes a light chain variable region (VL) comprising at least 90% of the amino acid sequence, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 4.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody comprises a VH region comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 3, and a VL region comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% , 99% or more identical to the amino acid sequence of SEQ ID NO: 4.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody includes a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 3. In some embodiments, the antibodies used in the antibody-drug conjugates disclosed herein include the variable region light chain (VL) comprising the amino acid sequence of SEQ ID NO: 4.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody includes a VH region comprising the amino acid sequence of SEQ ID NO: 3 and a VL region comprising the amino acid sequence of SEQ ID NO: 4.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody comprises a heavy chain comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 1. e.g., in formula (Ic), the isolated NaPi2b-targeting antibody comprises a light chain comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 2.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody comprises a heavy chain comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 1, and a light chain comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% , 99% or more identical to the amino acid sequence of SEQ ID NO: 2.

For example, in Formula (Ic), the isolated NaPi2b-targeting antibody includes a heavy chain comprising the amino acid sequence of SEQ ID NO: 1. For example, in Formula (Ic), the isolated NaPi2b-targeting antibody includes a light chain including the amino acid sequence of SEQ ID NO: 2.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody includes a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody includes a complementary determining region heavy chain 1 (CDRH1) variable region comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95% , 96%, 97% 98%, 99% or more identical to the amino acid sequence of GFSFSDFAMS (SEQ ID NO: 18); complementarity determining region heavy chain 2 (CDRH2) variable region comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence atigrvafhtyypdsmkg (seq id NO: 19); and a complementarity determining region heavy chain 3 (CDRH3) variable region comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence arhrgfdvghfdf (seq id no: 2 0). For example, in formula (Ic), the isolated NaPi2b targeting antibody comprises a complementarity determining region light chain 1 (CDRL1) variable region comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97% 98%, 99% or more identical amino acid sequence

- 4 043790 rssetlvhssgntyle (seq id NO: 21); complementarity determining region light chain 2 (CDRL2) variable region comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of RVSNRFS (seq id no: 22); and a complementarity determining region light chain 3 (CDRL3) variable region comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence fqgsfnplt (seq id no: 23).

For example, in formula (Ic), the isolated NaPi2b-targeting antibody comprises CDRH1 comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99 % or more identical to the amino acid sequence

GFSFSDFAMS (SEQ ID NO: 18); CDRH2 comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of atigrvafhtyypdsmkg (seq id no: 19 ); CDRH3 comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of arhrgfdvghfdf (seq id no: 20 ); CDRL1 comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence RSSETLVHSSGNTYLE (SEQ ID NO: 21 ) CDRL2 comprising an amino acid sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of RVSNRFS (SEQ ID NO: 22); and CDRL3 comprising an amino acid sequence at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of fqgsfnplt (seq id no: 23).

For example, in formula (Ic), the isolated NaPi2b-targeting antibody includes a complementary determining region heavy chain 1 (CDRH1) variable region comprising the amino acid sequence GFSFSDFAMS (SEQ ID NO: 18); complementarity determining region heavy chain 2 (CDRH2) variable region comprising the amino acid sequence atigrvafhtyypdsmkg (seq id NO: 19); and a complementarity determining region heavy chain 3 (CDRH3) variable region comprising the amino acid sequence arhrgfdvghfdf (seq id NO: 20). For example, in formula (Ic), the isolated NaPi2b-targeting antibody includes a complementarity determining region light chain 1 (CDRL1) variable region comprising the amino acid sequence rssetlvhssgntyle (seq id no: 21); complementarity determining region light chain 2 (CDRL2) variable region comprising the amino acid sequence RVSNRFS (SEQ ID NO: 22); and complementarity determining region light chain 3 (CDRL3) variable region comprising the amino acid sequence fqgsfnplt (SEQ ID NO: 23).

For example, in formula (Ic), the isolated NaPi2b-targeting antibody includes CDRH1 comprising the amino acid sequence GFSFSDFAMS (SEQ ID NO: 18); CDRH2, including the amino acid sequence atigrvafhtyypdsmkg (seq id no: 19); CDRH3, including the amino acid sequence arhrgfdvghfdf (seq id no: 2 0); CDRL1 comprising the amino acid sequence RSSETLVHSSGNTYLE (SEQ ID NO: 21); CDRL2, including the amino acid sequence RVSNRFS (seq id no: 22); and CDRL3, which includes the amino acid sequence fqgsfnplt (seq id no: 23).

For example, in formula (Ic), the isolated NaPi2b-targeting antibody comprises a heavy chain variable region (VH) comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 16. For example, in formula (Ic), the isolated NaPi2b-targeting antibody comprises a light chain variable region (VL) comprising at least 90% of the amino acid sequence, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 17.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody comprises a VH region comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 16, and a VL region comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% , 99% or more identical to the amino acid sequence of SEQ ID NO: 17.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody includes a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 16. for example, in formula (Ic), the isolated NaPi2b-targeting antibody includes a light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 17.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody includes a VH region comprising the amino acid sequence of SEQ ID NO: 16 and a VL region comprising the amino acid sequence of SEQ ID NO: 17.

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For example, in formula (Ic), the isolated NaPi2b-targeting antibody comprises a heavy chain comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 14. For example, in formula (Ic), the isolated NaPi2b-targeting antibody contains a light chain comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 15.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody comprises a heavy chain comprising an amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98%, 99% or more identical to the amino acid sequence of SEQ ID NO: 14, and a light chain comprising the amino acid sequence of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% 98% , 99% or more identical to the amino acid sequence of SEQ ID NO: 15.

For example, in Formula (Ic), the isolated NaPi2b-targeting antibody includes a heavy chain comprising the amino acid sequence of SEQ ID NO: 14. For example, in Formula (Ic), the isolated NaPi2b-targeting antibody includes a light chain including the amino acid sequence of SEQ ID NO: 15.

For example, in formula (Ic), the isolated NaPi2b-targeting antibody includes a heavy chain comprising the amino acid sequence of SEQ ID NO: 14 and a light chain comprising the amino acid sequence of SEQ ID NO: 15.

For example, in formula (Ic), an isolated NaPi2b-targeting antibody is an isolated antibody that competes for specific binding to human NaPi2b with an isolated antibody comprising (i) a complementary determining region heavy chain 1 (CDRH1) variable region comprising the amino acid sequence GYTFTGYNIH (SEQ ID no: 5); complementarity determining region heavy chain 2 (CDRH2) variable region comprising the amino acid sequence aiypgngdtsykqkfrg (seq id no: 6); complementarity determining region heavy chain 3 (CDRH3) variable region comprising the amino acid sequence getaratfay (seq id no: 7); complementarity determining region light chain 1 (CDRL1) variable region comprising the amino acid sequence sasqdignfln (seq id no: 8); complementarity determining region light chain 2 (CDRL2) variable region comprising the amino acid sequence ytsslys (seq id no: 9); and complementarity determining region light chain 3 (CDRL3) variable region comprising the amino acid sequence QQYSKLPLT (seq ID NO: 10) or (ii) CDRH1 comprising the amino acid sequence GFSFSDFAMS (SEQ ID NO: 18); CDRH2, including the amino acid sequence atigrvafhtyypdsmkg (seq id no: 19); CDRH3, including the amino acid sequence arhrgfdvghfdf (seq id no: 2 0); CDRL1 comprising the amino acid sequence RSSETLVHSSGNTYLE (SEQ ID NO: 21); CDRL2, including the amino acid sequence RVSNRFS (seq id no: 22); and CDRL3, which includes the amino acid sequence fqgsfnplt (seq id no: 23).

For example, in formula (Ic), m1 is an integer from 1 to about 120 (eg, about 1-90), and/or m ₃ is an integer from 1 to about 10 (eg, about 1-8).

For example, when the PHF in formula (Ic) has a molecular weight ranging from about 6 kDa to about 20 kDa (i.e., the sum of m, m1, _m2 , _m3 and _m4 is in the range from about 45 to about 150) , m2 is an integer from 2 to about 20, _m3 is an integer from 0 to about 9, m is an integer from 1 to about 10, and/or m1 is an integer from 1 to about 75 (for example, m1 is about 4-45).

For example, when the PHF in formula (Ic) has a molecular weight ranging from about 8 kDa to about 15 kDa (i.e., the sum of m, m1, _m2 , _m3 and m is in the range from about 60 to about 110), m ₂ is an integer from 2 to about 15, m ₃ is an integer from 0 to about 7, m4 is an integer from 1 to about 10, and/or m1 is an integer from 1 to about 55 (for example, m1 is about 4-30).

For example, when the PHF in formula (Ic) has a molecular weight ranging from about 2 kDa to about 20 kDa (i.e., the sum of m, m1, _m2 , _m3 and m4 is in the range from about 15 to about 150), m2 is an integer from 1 to about 20, _m3 is an integer from 0 to about 10 (e.g., m3 ranges from 0 to about 9), m4 is an integer from 1 to about 8, and/or m1 is an integer from 1 to about 70, and the total number of LP ² associated with the isolated antibody is from about 2 to about 8 (eg, about 2, 3, 4, 5, 6, 7 or 8).

For example, when the PHF in formula (Ic) has a molecular weight ranging from about 3 kDa to about 15 kDa (i.e., the sum of m, m1, _m2 , _m3 and m4 is in the range from about 20 to about 110), m ₂ is an integer from 2 to about 15, m ₃ is an integer from 0 to about 8 (for example, m3 ranging from 0 to about 7), m4 is an integer

- 6 043790 number from 1 to about 8, and/or m1 is an integer from 2 to about 50, and the total number of L ^P2 associated with the isolated antibody is from about 2 to about 8 (for example, about 2, 3, 4 , 5, 6, 7 or 8).

For example, when the PHF in formula (Ic) has a molecular weight ranging from about 5 kDa to about 10 kDa, (i.e., the sum of m, m _b m ₂ , m ₃ and m ₄ is from about 40 to about 75), m ₂ is an integer from about 2 to about 10 (for example, m ₂ is about 3-10), m ₃ is an integer from 0 to about 5 (for example, m ₃ is in the range from 0 to about 4), m ₄ is an integer from 1 to about 8 (for example, m ₄ is in the range from 1 to about 5), and/or m1 is an integer from about 2 to about 35 (for example, m 1 is about 5-35), and the total number The L ^P2 associated with the isolated antibody is from about 2 to about 8 (eg, about 2, 3, 4, 5, 6, 7 or 8).

For example, each D is independently a therapeutic agent, eg having a molecular weight <5 kDa.

For example, each D is independently a diagnostic or label.

For example, in some cases the Ds are independently therapeutic agents (eg, having a molecular weight <5 kDa), and in other cases the Ds are diagnostic agents or labels.

For example, each D is independently a cancer drug, for example selected from vinca alkaloids, auristatins, tubulisins, duocarmycins, unnatural camptothecin compounds, maitansinoids, calicheamicin compounds, topoisomerase inhibitors, pyrrolobenzodiazepines, DNA-binding drugs, DNA alkylating drugs, RNA polymerase inhibitors, kinase inhibitors, MEK inhibitors, KSP inhibitors and their analogues.

For example, each auristatin compound is auristatin, dolastatin (e.g., dolastatin 10 or dolastatin 15), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F hydroxypropyl amide (AF HPA), monomethyl auristatin F hydroxypropyl amide (AF HPA), or auristatin F phenylenediamine (AFP).

For example, each duocarmycin or analog thereof is duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, CC-1065, adozelesin, bizelesin, or carzelesin.

For example, each camptothecin compound is camptothecin, CPT-11 (irinotecan), SN-38, or topotecan.

For example, each pyrrolobenzodiazepine compound is a pyrrolobenzodiazepine monomer, a symmetric pyrrolobenzodiazepine dimer, or an unsymmetrical pyrrolobenzodiazepine dimer.

For example, each <, when not associated with an isolated antibody, independently includes a terminal group W _P , with each W ^P independently representing:

in which R ^1K is a leaving group (for example, a halide or RC(O)O-, in which R is hydrogen, an aliphatic, heteroaliphatic, carbocyclic or heterocycloalkyl group), R ^1a is a protecting group for sulfur, and ring A is cycloalkyl or heterocycloalkyl , and R ^1J is hydrogen, an aliphatic, heteroaliphatic, carbocyclic or heterocycloalkyl group.

- 7 043790

For example, each R ^1A independently represents Attcoor* 1 in which r is 1 or 2 and each of R ^s1 , R ^s2 and R ^s3 is hydrogen, an aliphatic, heteroaliphatic, carbocyclic or heterocycloalkyl group.

For example, a functional group LP ² that serves to form a covalent bond with a functional group of an isolated antibody (such as a functional group or reactive group on an amino acid residue of an antibody, such as a functional group on a cis, _ρ._ΥΫ residue of a thein, or a lysine residue of an antibody) , is selected from -SR, -SS-LG, □ and halogen, wherein LG is a leaving group, R ^p is H or a sulfur protecting group, and one of X _a and X _b is H and the other is a water-soluble maleimido protecting group, or _Xa and _Xb , together with the carbon atoms to which they are attached, form a carbon-carbon double bond, for example, the functional group L ^P2 , which serves to form a covalent bond, is a functional group that does not react with the functional group of the isolated antibody,

Y. . P, ..........

for example, ъ as the functional group L ^P2 , in which from Xa and X _b denotes H, and the other denotes the water-soluble protecting group of the maleimido group, or X _a and X _b .

For example, L ^D1 includes -X-(CH ₂ ) _v -C (=O) -, where X is directly bonded to the carbonyl group -c ₍ , in which X is CH ₂ , O or NH, and v is an integer from 1 until 6.

For example, each “=ο)-ι?'-|-ελ independently denotes -C(=O)-X-(CH ₂ ) _v -C(=O)-NH-(CH ₂ ) _u -NHC(=O )-(CH ₂ ) _w -(OSH ₂ ) _x -NHC(=O)-(CH ₂ ) _y -M, in which X represents CH ₂ , O or NH, each of v, u, w, x A, and y is independently an integer from 1 to 6, and M is ^x °, in which one of Xa and _Xb is H, and the other is a water-soluble maleimido protecting group, or Xa _{and Xb} , together with the carbon atoms to which they are attached, forming a carbon-carbon double bond.

For example, v, u, w, x and y each equal 2.

For example, the ratio between D and isolated NaPi2b-targeting antibody is approximately 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16 :1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1, b; 1, 5:1, 4:1, 3:1, 2:1 or 1:1.

For example, the ratio between D and the isolated NaPi2b-targeting antibody is approximately 20:1, 15:1, 10:1, 5:1, 2:1 or 1:1.

For example, the ratio between D and the isolated NaPi2b-targeting antibody is approximately 16:1, 15:1, 14:1, 13:1, 12:1, 11:1 or 10:1.

For example, the ratio between D and the isolated NaPi2b-targeting antibody is approximately 15:1, 14:1, 13:1, 12:1 or 11:1.

For example, the ratio between D and the isolated NaPi2b-targeting antibody is approximately 15:1, 14:1, 13:1 or 12:1.

For example, the ratio between D and the isolated NaPi2b-targeting antibody is approximately 6:1, 5:1, 4:1, 3:1, 2:1 or 1:1.

For example, each one or more D-supporting polymer backbones independently has the formula (Id):

in which m _3a is an integer from 0 to about 17, m _3b is an integer from 1 to about 8, and terminal ^: denotes the direct attachment of one or more polymer backbones to an isolated NaPi2b-targeting antibody having a molecular weight of 40 kDa or more and comprising (i) a complementarity determining region heavy chain 1 (CDRH1) variable region comprising the amino acid sequence gytftgynih (seq id no: 5); complementarity determining region heavy chain 2 (CDRH2) variable region comprising the amino acid sequence aiypgngdtsykqkfrg (seq id no: 6); complementarity determining region heavy chain 3 (CDRH3) variable region comprising the amino acid sequence getaratfay (seq id NO: 7); complementarity determining region light chain 1 (CDRL1) variable region comprising the amino acid sequence

- 8 043790 sasqdignfln (seq id no: 8); complementarity determining region light chain 2 (CDRL2) variable region comprising the amino acid sequence ytsslys (seq id no: 9); and complementarity determining region light chain 3 (CDRL3) variable region comprising the amino acid sequence QQYSKLPLT (seq id no: 10), or (ii) CDRH1 comprising the amino acid sequence gfsfsdfams (seq id no: 18); CDRH2, including the amino acid sequence atigrvafhtyypdsmkg (seq id no: 19); CDRH3, comprising the amino acid sequence arhrgfdvghfdf (seq id no: 20); CDRL1, including the amino acid sequence rssetlvhssgntyle (seq id no: 21); CDRL2, including the amino acid sequence RVSNRFS (seq id no: 22); and CDRL3, comprising the amino acid sequence FQGSFNPLT (SEQ ID NO: 23).

In some embodiments, the isolated NaPi2b-targeting antibody has a molecular weight of 40 kDa or greater and includes (i) CDRH1 comprising the amino acid sequence gytftgynih (seq id no: 5); CDRH2, comprising the amino acid sequence AIYPGNGDTSYKQKFRG (SEQ ID NO: b); CDRH3, including the amino acid sequence getaratfay (seq id NO: 7); CDRL1, including the amino acid sequence sasqdignfln (seq id no: 8); CDRL2, including the amino acid sequence ytsslys (seq id no: 9); and CDRL3, comprising the amino acid sequence QQYSKLPLT (SEQ ID NO: 10).

In some embodiments, the isolated NaPi2b-targeting antibody has a molecular weight of 40 kDa or greater and includes CDRH1 comprising the amino acid sequence GFSFSDFAMS (SEQ ID NO: 18); CDRH2 comprising the amino acid sequence ATIGRVAFHTYYPDSMKG (SEQ ID NO: 19); CDRH3, comprising the amino acid sequence arhrgfdvghfdf (seq id no: 20); CDRL1, comprising the amino acid sequence RSSETLVHSSGNTYLE (SEQ ID NO: 21); CDRL2, comprising the amino acid sequence RVSNRFS (seq id NO: 22); and CDRL3, comprising the amino acid sequence FQGSFNPLT (SEQ ID NO: 23).

The formula skeleton (Id) may include one or more of the following features.

Sum of m _3a and m _3b from 1 to 18.

When the PHF in formula (Id) has a molecular weight ranging from about 2 kDa to about 40 kDa, the sum of m, m1, _m2 , _m3a and _m3b is from about 15 to about 300, m1 is an integer from 1 to about 140, m2 is an integer from 1 to about 40, m _3a is an integer from 0 to about 17, m _3b is an integer from 1 to about 8, the sum of m _3a and m _3b is from 1 to about 18, and the ratio between The PHF and isolated NaPi2b-targeting antibody is 10 or less.

When the PHF in formula (Id) has a molecular weight ranging from about 2 kDa to about 20 kDa, the sum of m, m1, _m2 , _m3a and _m3b is from about 15 to about 150, m1 is an integer from 1 to about 70, m2 is an integer from 1 to about 20, m _3a is an integer from 0 to about 9, m _3b is an integer from 1 to about 8, the sum of m3a and m3b is from 1 to about 10, and the relationship between PHF and the isolated NaPi2b-targeting antibody is an integer from 2 to about 8.

When the PHF in formula (Id) has a molecular weight ranging from about 3 kDa to about 15 kDa, the sum of m, m1, _m2 , _m3a and _m3b is from about 20 to about 110, m1 is an integer from 2 to about 50, m2 is an integer from 2 to about 15, m _3a is an integer from 0 to about 7, m _3b is an integer from 1 to about 8, the sum of m _3a and m _3b is from 1 to about 8; and the ratio between PHF and the isolated NaPi2b-targeting antibody is an integer from 2 to about 8 (eg, from about 2 to about 6 or from about 2 to about 4).

When the PHF in formula (Id) has a molecular weight ranging from about 5 kDa to about 10 kDa, the sum of m, m1, _m2 , _m3a and _m3b is from about 40 to about 75, m1 is an integer from about 2 to about 35, m2 is an integer from about 2 to about 10, m _3a is an integer from 0 to about 4, m _3b is an integer from 1 to about 5, the sum of m _3a and m _3b is from 1 to about 5; and the ratio between PHF and the isolated NaPi2b-targeting antibody is an integer from 2 to about 8 (eg, from about 2 to about 6 or from about 2 to about 4).

In some embodiments, the ratio between auristatin F hydroxypropyl amide (AF HPA) and the isolated NaPi2b-targeting antibody may be approximately 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1 , 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11 :1, 10:1, 9:1, 8:1, 7:1 or 6:1.

In some embodiments, the ratio between the HPA AF and the isolated NaPi2b targeting antibody may be approximately 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1 , 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1 or 6:1.

- 9 043790

In other embodiments, the ratio between HPA AF and isolated NaPi2b targeting antibody may be approximately 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1 , 11:1, 10:1, 9:1, 8:1, 7:1 or 6:1.

In some embodiments, the ratio between the HPA AF and the isolated NaPi2b targeting antibody may be approximately 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, or 10:1.

In some embodiments, the ratio between the HPA AF and the isolated NaPi2b targeting antibody may be approximately 15:1, 14:1, 13:1, 12:1, or 11:1.

In some embodiments, the ratio between the HPA AF and the isolated NaPi2b targeting antibody may be approximately 15:1, 14:1, 13:1, or 12:1.

In some embodiments, the ratio between PHF and isolated NaPi2b targeting antibody may be approximately 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

In some embodiments, the ratio between PHF and isolated NaPi2b targeting antibody may be approximately 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

In other embodiments, the ratio between PHF and the isolated NaPi2b-targeting antibody may be approximately 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

In other embodiments, the ratio between PHF and the isolated NaPi2b-targeting antibody may be approximately 6:1, 5:1, 4:1, 3:1, or 2:1.

In other embodiments, the ratio between PHF and isolated NaPi2b-targeting antibody may be approximately 6:1, 5:1, 4:1, or 3:1.

In some embodiments, the ratio between PHF and isolated NaPi2b targeting antibody may be approximately 5:1, 4:1, or 3:1.

In some embodiments, the ratio between PHF and isolated NaPi2b targeting antibody may be approximately 4:1, 3:1, or 2:1.

Water-soluble maleimido blocking groups (e.g., X _a or X _b ) are groups that can be covalently attached to one of the two carbon atoms of the olefin after reaction of the maleimido group with a thiol-containing compound of formula (II):

R90-(CH2)d-SH (II) in which R ₉₀ is NHR91, OH, COOR ₉₃ , CH (NHR ₉₁ ) COOR ₉₃ or a substituted phenyl group;

R ₉₃ is hydrogen or C _1-4 alkyl;

R ₉₁ is hydrogen, CH ₃ or CH ₃ CO and d is an integer from 1 to 3.

In one embodiment, the water-soluble maleimido-blocking compound of formula (II) may be cysteine, N-acetyl cysteine, cysteine methyl ester, N-methyl cysteine, 2-mercaptoethanol, 3-mercaptopropanoic acid, 2-mercaptoacetic acid, mercaptomethanol (i.e. e. HOCH2SH), a benzyl thiol in which the phenyl is substituted with one or more hydrophilic substituents, or 3-aminopropane-1-thiol. One or more hydrophilic substituents on phenyl include OH, SH, methoxy, ethoxy, COOH, CHO, COC _1-4 alkyl, NH ₂ , F, cyano, SO ₃ H, PO ₃ H and the like.

In another aspect, the water-soluble blocking group for the maleimido group is -S-(CH2)d-R90, in which R ₉₀ is OH, COOH or CH (NHR ₉₁ ) COOR ₉₃ ;

R93 is hydrogen or CH3;

_R91 is hydrogen or _CH3CO and d is 1 or 2.

In another embodiment, the water-soluble blocking group for the maleimido group is -S-CH2-CH(NH2)COOH.

In some embodiments, the conjugate described herein includes one or more D-bearing PHFs, each independently having the formula (If), wherein the PHF has a molecular weight ranging from about 2 kDa to about 40 kDa:

l &

y % 2 (If) in which m is an integer from 1 to about 300, m1 is an integer from 1 to about 140, m ₂ is an integer from 1 to about 40, m _3a is an integer from 0 to about 17, m _3b is an integer from 1 to about 8;

- 10 043790 the sum of m _3a and m _3b varies from 1 to approximately 18;

the sum of m, m _b m2, m _3a and m _3b is from about 15 to about 300;

terminal indicates the attachment of one or more PHF polymer backbones to an isolated antibody that specifically binds to SLC34A2, wherein the isolated antibody that specifically binds to SLC34A2 is an isolated antibody comprising (i) a complementarity determining region heavy chain 1 (CDRH1) variable region comprising an amino acid sequence gytftgynih (seq id no: 5); the variable region of the complementarity determining region of the heavy chain 2 (CDRH2), including the amino acid sequence aiypgngdtsykqkfrg (seq id no: b); complementarity determining region heavy chain 3 (CDRH3) variable region comprising the amino acid sequence getaratfay (seq id NO: 7); complementarity determining region light chain 1 (CDRL1) variable region comprising the amino acid sequence sasqdignfln (seq id NO: 8); complementarity determining region light chain 2 (CDRL2) variable region comprising the amino acid sequence ytsslys (seq id no: 9); and complementarity determining region light chain 3 (CDRL3) variable region comprising the amino acid sequence QQYsklplt (seq id no: io), or (ii) CDRH1 comprising the amino acid sequence gfsfsdfams (seq id no: 18); CDRH2, including the amino acid sequence atigrvafhtyypdsmkg (seq id no: 19); CDRH3, including the amino acid sequence arhrgfdvghfdf (seq id no: 2 0); CDRL1, including the amino acid sequence RSSETLVHSSGNTYLE (seq id no: 21); CDRL2, including the amino acid sequence rvsnrfs (seq id no: 22); and CDRL3, including the amino acid sequence fqgsfnplt (seq id no: 23); and the ratio between PHF and antibody is 10 or less.

In some embodiments, the isolated NaPi2b-targeting antibody specifically binds to SLC34A2 and includes (i) CDRH1 comprising the amino acid sequence GYTFTGYNIH (SEQ ID NO: 5); CDRH2, comprising the amino acid sequence aiypgngdtsykqkfrg (seq id NO: 6); CDRH3 comprising the amino acid sequence GETARATFAY (SEQ ID NO: 7); CDRL1 comprising the amino acid sequence SASQDIGNFLN (SEQ ID NO: 8); CDRL2, including the amino acid sequence ytsslys (seq id NO: 9); and CDRL3, comprising the amino acid sequence qqysklplt (SEQ ID NO: 10).

In some embodiments, the isolated NaPi2b-targeting antibody specifically binds to SLC34A2 and includes CDRH1 comprising the amino acid sequence GFSFSDFAMS (SEQ ID NO: 18); CDRH2 comprising the amino acid sequence ATIGRVAFHTYYPDSMKG (SEQ ID NO: 19); CDRH3, including the amino acid sequence arhrgfdvghfdf (seq id no: 2 0); CDRL1, including the amino acid sequence rssetlvhssgntyle (seq id NO: 21); CDRL2 comprising the amino acid sequence RVSNRFS (SEQ ID NO: 22); _and CDRL3, comprising the amino acid sequence FQGSFNPLT (SEQ ID NO: 23).

The skeleton of the formula (If) may include one or more of the following features.

When the PHF in formula (If) has a molecular weight ranging from about 2 kDa to about 20 kDa, the sum of m, m _b m ₂ , m _3a and m _3b is from about 15 to about 150, m1 is an integer from 1 to about 70, m2 is an integer from 1 to about 20, m _3a is an integer from 0 to about 9, m _3b is an integer from 1 to about 8, the sum of m _3a and m _3b is from 1 to about 10, and the ratio between PHF and antibody is an integer from 2 to about 8.

When the PHF in formula (If) has a molecular weight ranging from about 3 kDa to about 15 kDa, the sum of m, m _b m ₂ , m _3a and m _3b is from about 20 to about 110, m ₁ is an integer from 2 to about 50, m2 is an integer from 2 to about 15, m _3a is an integer from 0 to about 7, m _3b is an integer from 1 to about 8, the sum of m _3a and m _3b is from 1 to about 8; and the ratio between PHF and antibody is an integer from 2 to about 8 (eg, from about 2 to about 6 or from about 2 to about 4).

When the PHF in formula (If) has a molecular weight ranging from about 5 kDa to about 10 kDa, the sum of m, m1, _m2 , _m3a and _m3b is from about 40 to about 75, m1 is an integer from about 2 to about 35, m2 is an integer from about 2 to about 10, m3a is an integer from 0 to about 4, m3b is an integer from 1 to about 5, the sum of m3a and m3b is from 1 to about 5; and the ratio between PHF and antibody is an integer from 2 to about 8 (eg, from about 2 to about 6 or from about 2 to about 4).

In some embodiments, the ratio between auristatin F hydroxypropyl amide (AF HPA) and antibody may be approximately 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1 , 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10 :1, 9:1, 8:1, 7:1 or 6:1.

- 11 043790

In some embodiments, the ratio between HPA AF and antibody may be approximately 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16 :1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1 or 6:1.

In other embodiments, the ratio between HPA AF and antibody may be approximately 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11 :1, 10:1, 9:1, 8:1, 7:1 or 6:1.

In some embodiments, the ratio between HPA AF and antibody may be approximately 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, or 10:1.

In some embodiments, the ratio between AF and antibody may be approximately 15:1, 14:1, 13:1, 12:1, or 11:1.

In some embodiments, the ratio between HPA AF and antibody may be approximately 15:1, 14:1, 13:1, or 12:1.

In some embodiments, the ratio between PHF and antibody may be approximately 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1: 1.

In some embodiments, the ratio between PHF and antibody may be approximately 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

In other embodiments, the ratio between PHF and antibody may be approximately 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

In other embodiments, the ratio between PHF and antibody may be approximately 6:1, 5:1, 4:1, 3:1, or 2:1.

In other embodiments, the ratio between PHF and antibody may be approximately 6:1, 5:1, 4:1, or 3:1.

In some embodiments, the ratio between PHF and antibody may be approximately 5:1, 4:1, or 3:1.

In some embodiments, the ratio between PHF and antibody may be approximately 4:1, 3:1, or 2:1.

Another aspect of the disclosure relates to the method of preparing the conjugate described herein. The method involves reacting an isolated antibody with a D-supporting polymer backbone of formula (Ia) such that a conjugate is formed:

in which L ^d1 is a carbonyl-containing group;

~—Q onRC- —sz-RC-D ~ each < in < independently denotes a first linker containing a biodegradable compound such that when the bond is broken, D is released in its active form for its intended therapeutic effect; and in c(-o)-l -|-d between L ^d1 and D denotes the direct or indirect attachment of D to L ^D1 ;

each independently denotes a second linker not yet associated with the isolated antibody, in which L ^P2 is a group containing a functional group that should form a covalent bond with the functional group of the isolated antibody, and I between L ^D1 and L ^P2 denotes direct or indirect attachment of L ^P2 to L ^D1 , and every second linker is different from every first linker;

m is an integer from 1 to about 300, m1 is an integer from 1 to about 140, m ₂ is an integer from 1 to about 40, m ₃ is an integer from 1 to about 18 and the sum of m, m1, _m2 and _m3 ranges from about 15 to about 300.

In the formulas for the polymer backbones disclosed herein, the gap or gap between the polyacetal units indicates that the units can be linked to each other in any order.

In other words, attachment groups containing, for example, D, L ^P2 and isolated antibody, can be randomly distributed along the polymer backbone.

The present disclosure also relates to methods of treating, preventing, delaying the progression of, or otherwise improving a symptom of one or more pathologies associated with aberrant NaPi2b expression, function, and/or activation, or alleviating a symptom associated with such pathologies, by administering a conjugate disclosed herein , the patient for whom such treatment or prophylaxis is desired. The patient receiving treatment is, for example, a human. The conjugates are administered in an amount sufficient to treat, prevent, or alleviate a symptom associated with a pathology.

- 12 043790

The present disclosure also relates to methods of treating, preventing, delaying the progression of, or otherwise improving a symptom of one or more pathologies associated with the expression, function and/or activation of NaPi2b, or alleviating a symptom associated with such pathologies, by administering a conjugate disclosed herein. the patient for whom such treatment or prophylaxis is desired. The patient receiving treatment is, for example, a human. The conjugate is administered in an amount sufficient to treat, prevent, or alleviate a symptom associated with the pathology.

Pathologies that are treated and/or prevented using the conjugates disclosed herein include, for example, cancer. In some embodiments, the conjugates disclosed herein may be used in the treatment, prevention, delay of progression, or other improvement of NaPi2b-expressing cancer, for example, the conjugates disclosed herein may be used in the treatment, prevention, delay of progression, or other improvement of in relation to a cancer symptom selected from the group consisting of ovarian cancer (such as epithelial ovarian cancer), thyroid cancer, colorectal cancer, lung cancer, non-small cell lung cancer (NSCLC) such as non-squamous NSCLC, breast cancer, cancer kidney and salivary duct cancer.

In some embodiments, the conjugates disclosed herein may be used in the treatment, prevention, delay of progression, or other improvement of a symptom of ovarian cancer. In some embodiments, the ovarian cancer is epithelial ovarian cancer.

In some embodiments, the conjugates disclosed herein may be used in the treatment, prevention, delay of progression, or other improvement of a symptom of NSCLC. In some embodiments, the NSCLC is non-squamous NSCLC.

In some embodiments, the conjugates disclosed herein may be used in the treatment, prevention, delay of progression, or other improvement of a symptom of ovarian cancer. In some embodiments, the ovarian cancer is epithelial ovarian cancer.

The disclosure also relates to kits and/or methods for identifying or otherwise specifying, e.g., stratifying, a population of patients suitable for therapeutic administration of the NaPi2b-targeting antibody-drug conjugate disclosed herein by identifying a patient's NaPi2b score prior to treatment with the NaPi2b-targeting antibody conjugate -medicine disclosed herein. In some embodiments, the patient is identified as having scores of 1+ or 2+, or 3+ for NaPi2b expression. In some embodiments, the test cell population is obtained from fresh, thawed tissue from a biopsy sample. In some embodiments, the test cell population is obtained from a primary or metastatic site. In some embodiments, the test cell population is obtained from frozen tissue from a biopsy or surgical specimen, or ascites fluid, or pleural fluid. In some embodiments, the test cell population is obtained from fixed tissue (eg, formalin fixation) from a biopsy or surgical specimen.

The IHC test measures the amount of NaPi2b receptor protein on the surface of cells in a cancer tissue sample, such as an ovarian cancer tissue sample or a lung cancer sample, and provides a score of the detected level of NaPi2b receptor on the cell surface as NaPi2b 0, 1+, 2+, or 3+.

In some embodiments, the patient is refractory to chemotherapy, including standard first-line chemotherapeutic agents.

In some embodiments, the patient has platinum-resistant ovarian cancer.

In some embodiments, the patient has platinum-sensitive ovarian cancer.

In some embodiments, the patient has platinum-refractory ovarian cancer.

In some embodiments, the patient has advanced ovarian cancer and has not received prior therapy to treat the cancer (eg, ovarian cancer). In some embodiments, the patient has advanced ovarian cancer and has not received prior chemotherapy to treat the cancer (eg, ovarian cancer).

The NaPi2b-targeting antibody-drug conjugates used in any of the embodiments of the methods and uses described herein can be administered at any stage of the disease, for example, such a NaPi2b-targeting antibody-drug conjugate can be administered to a patient suffering from cancer at any stage, from early to metastatic.

In some embodiments, the NaPi2b-targeting antibody-drug conjugates of the disclosure may be administered either alone or in combination with other compositions in therapy, for example, the conjugate of the disclosure may be used in conjunction with at least one additional therapeutic agent and/or adjuvant. In some embodiments, the additional therapeutic agent is a small molecule inhibitor, another antibody therapeutic agent, a polypeptide or peptide therapeutic agent, a nucleic acid therapeutic agent, and/or other biological agent. The additional therapeutic agent may be either the same as D used to form the conjugate or different.

In some embodiments, the additional therapeutic agent is a cytotoxic agent, a chemotherapeutic agent, a growth inhibitory agent, an angioinhibitor

- 13 043790 genesis, PARP inhibitor (poly(ADP)-ribose polymerase), alkylating agent, antimetabolite, antimicrotubule agent, topoisomerase inhibitor, cytotoxic antibiotic, any other nucleic acid degrading agent, or immune checkpoint inhibitor. In one embodiment, the therapeutic agent used in the treatment of cancer includes, but is not limited to, a platinum compound (eg, cisplatin or carboplatin); taxane (eg paclitaxel or docetaxel); a topoisomerase inhibitor (eg, irinotecan or topotecan); anthracycline (eg doxorubicin (ADRIAMYCIN®) or liposomal doxorubicin (DOXIL®)); antimetabolite (eg, gemcitabine, pemetrexed); cyclophosphamide; vinorelbine (NAVELBINE®); hexamethylmelamine; ifosfamide; etoposide; an angiogenesis inhibitor (eg, Bevacizumab (Avastin®), thalidomide, TNP-470, platelet factor 4, interferon, or endostatin); PARP inhibitor (eg, Olaparib (Lynparza™)); an immune checkpoint inhibitor, such as, for example, a monoclonal antibody targeting PD-1 or for PD-L ((for example, pembrolizumab (Keytruda®), atezolizumab (MPDL3280A) or nivolumab (Opdivo®)) or CTLA-4 (for example , ipilimumab (Yervoy®), kinase inhibitor (eg, sorafenib or erlotinib), ALK inhibitor (eg, crizotinib (Xalkori), ceritinib (Zykadia), alectinib (Alecensa), dalantercept (ACE 041), brigatinib (AP26113), entrectinib (NMS-E628), PF-06463922 TSR-011, CEP-37440 and X-396), proteasome inhibitor (eg, bortezomib or carfilzomib), immunomodulatory agent (eg, lenalidomide or IL-2), radiation agent and/or its biosimilar and/or combinations thereof Other suitable agents include an agent considered standard by those skilled in the art and/or a chemotherapeutic agent known to those skilled in the art.

In some embodiments, an immune checkpoint inhibitor suitable for the combinations and methods of the disclosure is a monoclonal antibody, a humanized antibody, a fully human antibody, a fusion protein, or a combination thereof.

In some embodiments, immune checkpoint inhibitors inhibit a checkpoint protein including CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligand, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD226, CD276, DR3, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell co- stimulator), LAIR1, LIGHT, MARCO (macrophage receptor with collagen structure), OX-40, SLAM, TIGHT, VTCN1 or a combination thereof.

In some embodiments, the immune checkpoint inhibitor interacts with a checkpoint protein ligand including CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligand, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD226, CD276, DR3, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulator), LAIR1, LIGHT, MARCO (macrophage receptor with collagen structure), OX-40, SLAM, TIGHT, VTCN1 or a combination thereof.

In some embodiments, the immune checkpoint inhibitor inhibits a checkpoint protein including CTLA-4, PDL1, PD1, or a combination thereof.

In some embodiments, the immune checkpoint inhibitor includes pembrolizumab (MK-3475), nivolumab (BMS-936558), pidilizumab (CT-011), AMP-224, MDX-1 105, durvalumab (MEDI4736), MPDL3280A, BMS-936559, IPH2101, TSR-042, TSR-022, ipilimumab, lirilumab, atezolizumab, avelumab, tremelimumab, or a combination thereof.

In some embodiments, the immune checkpoint inhibitor includes nivolumab (BMS-936558), ipilimumab, pembrolizumab, atezolizumab, tremelimumab, durvalumab, avelumab, or a combination thereof.

In some embodiments, the NaPi2b-targeting antibody-drug conjugate and the additional agent(s) are formulated into a single therapeutic composition, and the NaPi2b-targeting antibody-drug conjugate and the additional agent are administered simultaneously. Alternatively, the NaPi2b-targeting antibody-drug conjugate and the additional agent are kept separate from each other, for example, each formulated into a separate therapeutic composition, and the NaPi2b-targeting antibody-drug conjugate and the additional agent are administered simultaneously, or the NaPi2b-targeting antibody-drug conjugates are administered simultaneously, or the NaPi2b-targeting antibody-drug conjugates are administered simultaneously. the drug and the additional agent are administered at different times during the treatment regimen, for example, a NaPi2b-targeting antibody-drug conjugate is administered before administration of the additional agent, a NaPi2b-targeting antibody-drug conjugate is administered after administration of the additional agent, or a NaPi2b-targeting antibody-drug conjugate and the additional agent is administered alternately. As described herein, the NaPi2b-targeting antibody-drug conjugate and the additional agent are administered in single doses or multiple doses.

In some embodiments, the NaPi2b-targeting antibody-drug conjugate and the immune checkpoint inhibitor are formulated in the same formulation.

In some embodiments, the NaPi2b-targeting antibody-drug conjugate and the immune checkpoint inhibitor are formulated in separate formulations.

- 14 043790

In some embodiments, a combination comprising a NaPi2b-targeting antibody-drug conjugate and an immune checkpoint inhibitor disclosed herein can be used in the treatment, prevention, delay of progression, or other improvement of a symptom of ovarian cancer. In some embodiments, the ovarian cancer is epithelial ovarian cancer.

In some embodiments, a combination comprising a NaPi2b-targeting antibody-drug conjugate and an immune checkpoint inhibitor disclosed herein can be used in the treatment, prevention, delay of progression, or other improvement of a symptom of NSCLC. In some embodiments, the NSCLC is non-squamous NSCLC.

Also disclosed are kits comprising a NaPi2b-targeting antibody-drug conjugate and an immune checkpoint inhibitor. Kit components may be packaged together or divided into two or more containers. In some embodiments, the containers may be vials containing sterile lyophilized composition formulations suitable for reconstitution. The kit may also contain one or more buffers suitable for reconstitution and/or dilution of other reagents. Other containers that may be used include, but are not limited to, a bag, cuvette, box, test tube, and the like. The kit components can be packaged and stored in sterile containers. Another component that may be included is instructions for use of the kit by the consumer.

Pharmaceutical compositions according to the disclosure may include a NaPi2b-targeting antibody-drug conjugate disclosed herein and a suitable carrier. These pharmaceutical compositions may be included in kits, such as, for example, diagnostic kits.

One of ordinary skill in the art will appreciate that the antibodies disclosed herein have a variety of uses. for example, the proteins disclosed herein are used as therapeutic agents. The antibodies disclosed herein are also used as reagents in diagnostic kits or as diagnostic tools, or these antibodies can be used in competition tests to produce therapeutic reagents.

Unless otherwise specified, all technical and scientific terms used herein have the same meanings as commonly understood by one skilled in the art to which this disclosure pertains. In the description, the singular forms also include the plural unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, suitable methods and materials are described below. In the event of a conflict, this description, including definitions, will control. Moreover, the materials, methods, and examples are illustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

Brief description of the figures

Fig. 1 illustrates the antitumor efficacy of XMT-1535; example 3A, rituximab(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); example 4A ((1OH1.11.4B)-(MC-VC-PABA-MMAE)); example 2C ((10H1.11.4B)-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) and example IB, XMT 1535-(EG2-MI-(10 kDa PHF-BA- (AF-HPA-Ala)) measured in the OVCAR3 mouse tumor xenograft model.

Fig. 2 illustrates the antitumor efficacy of Example 3B, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); Example 4B ((10H1.11.4B)-(MC-VC-PABA-MMAE)); and Example 1C, XMT 1535(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), measured in the OVCAR3 mouse tumor xenograft model.

Fig. 3 illustrates the antitumor efficacy of Example 3B, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); Example 4B ((10H1.11.4B)-(MC-VC-PABA-MMAE)); and Example 1C, XMT 1535(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), measured in a patient-derived KRAS mutant non-small cell lung cancer xenograft model.

Fig. 4 illustrates the antitumor efficacy of Example 3B, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); example 4B ((10H1.11.4B)-(MC-VC-PABA-MMAE)); and Example 1C, XMT 1535(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), measured in a patient-derived xenograft model of non-small cell lung cancer.

Fig. 5 illustrates the antitumor efficacy of Example 1C, XMT 1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), measured in a patient-derived xenograft model of non-small cell lung cancer.

Fig. 6A and 6B, respectively, show plasma pharmacokinetics for the total antibody and total drug following administration of Example 1C at 1.25 mg/kg (1074 μg/m ² auristatin payload equivalent), 2.5 mg/kg (2147 μg/m m ² equivalent payload of auristatin) or 5 mg/kg (or 4294 μg/m ² equivalent payload of auristatin) as a single IV infusion in cynomolgus monkeys.

Fig. 7 illustrates the antitumor efficacy of Example 3B, rituximab-(EG2-MI-(10 kDa

- 15 043790

PHF-BA-(AF-HPA-Ala))); example 4B ((10H1.11.4B)-(MC-VC-PABA-MMAE)); and Example 1C, XMT 1535(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), measured in a patient-derived xenograft model of non-small cell lung cancer.

Fig. 8 illustrates the antitumor efficacy of Example 1C, XMT 1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), measured in a patient-derived xenograft model of non-small cell lung cancer.

Fig. 9 illustrates the antitumor efficacy of Example 1C, XMT 1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), measured in a patient-derived xenograft model of non-small cell lung cancer.

Fig. 10 illustrates the antitumor efficacy of Example 1C, XMT 1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))), measured in a patient-derived xenograft model of non-small cell lung cancer.

Detailed description

The present disclosure relates to a NaPi2b-targeting antibody-drug conjugate (such as a NaPi2b-targeting antibody-polymer-drug conjugate) that is biodegradable, biocompatible and exhibits high drug loading, and also specifically binds to the extracellular region of SLC34A2.

The NaPi2b-targeting antibody-drug conjugates (such as NaPi2b-targeting antibody-polymer-drug conjugates) of the invention include an antibody that specifically recognizes NaPi2b, also known as sodium-dependent phosphate transporter protein 2B. The NaPi2b antibodies used in the conjugates disclosed herein are capable of and useful for modulating, blocking, inhibiting, attenuating, counteracting, neutralizing, or otherwise interfering with at least one biological activity of NaPi2b. The antibodies disclosed herein also include antibodies that bind to soluble NaPi2b.

NaPi2b-targeting antibody-drug conjugates of the invention include antibodies that bind to a NaPi2b epitope with an equilibrium dissociation constant (Kd or Kd) of <1 μM, for example, <100 nM, preferably <10 nM, and more preferably <1 nM. For example, NaPi2b antibodies used in the antibody-drug conjugates disclosed herein exhibit a Kd ranging from about <1 nM to about 1 pM.

The NaPi2b-targeting antibody-drug conjugates of the invention may include antibodies that serve to modulate, block, inhibit, attenuate, counteract, neutralize, or otherwise interfere with the functional activity of NaPi2b. The functional activities of NaPi2b include, for example, participation in the absorption of transcellular inorganic phosphate (Pi), thereby helping to maintain phosphate homeostasis in the body. For example, NaPi2b antibodies completely or partially inhibit the functional activity of NaPi2b by partially or completely modulating, blocking, inhibiting, attenuating, counteracting, neutralizing, or otherwise interfering with transcellular inorganic phosphate absorption. Transcellular inorganic phosphate absorption activity is assessed using any method known in the art for detecting transcellular inorganic phosphate absorption activity, including, but not limited to, detecting levels of transcellular inorganic phosphate absorption in the presence and absence of the anti-NaPi2b antibody disclosed herein. .

The NaPi2b antibodies used in the antibody-drug conjugates disclosed herein may be antibodies that are believed to completely modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with the functional activity of NaPi2b when the level of functional activity of NaPi2b is in the presence of NaPi2b antibody is reduced by at least 95%, such as 96%, 97%, 98%, 99%, or 100%, compared to the level of functional activity of NaPi2b in the absence of binding by the NaPi2b antibody described herein. NaPi2b antibodies are considered to partially modulate, block, inhibit, reduce, antagonize, neutralize, or otherwise interfere with the functional activity of NaPi2b when the level of NaPi2b activity in the presence of the NaPi2b antibody is reduced by less than 95%, e.g., 10%, 20%, 25% , 30%, 40%, 50%, 60%, 75%, 80%, 85%, or 90% compared to the level of NaPi2b activity in the absence of binding by the NaPi2b antibody described herein.

Unless otherwise specified, scientific and technical terms used in connection with this disclosure shall have the meanings commonly understood by one skilled in the art. Further, unless the context otherwise requires, terms in the singular shall include the plural, and terms in the plural shall include the singular. In general, the nomenclatures and methods used in connection with cell and tissue culture, molecular biology and protein and oligo- or polynucleotide chemistry and hybridization described herein are known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (eg, electroporation, lipofection). Enzymatic

- 16 043790 These reactions and purification methods are carried out according to the manufacturer's specifications or as they are usually carried out in the art, or as described here. Known methods and procedures are generally carried out according to standard methods known in the art and as described in various general and more specific references cited and discussed throughout the present description, see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989)). The nomenclatures and laboratory procedures and methods used in connection with analytical chemistry, synthetic organic chemistry and medicinal and pharmaceutical chemistry described herein are known and commonly used in the art. Standard methods are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation and delivery, and patient treatment.

For purposes of this disclosure, the following terms, unless otherwise defined, have the following meanings:

For the purposes of the invention, the terms NaPi2b (also known as sodium-dependent phosphate transport protein 2B, SLC34A2, NaPiIIb, Npt2, Na(+)-dependent phosphate cotransporter 2B; sodium/phosphate cotransporter 2B; Na(+)/Pi cotransporter 2B; NaPi3b ; solute carrier family 34 member 2), when used herein, refers to human NaPi2b (e.g., GenBank accession no. O95436.3) and includes any variants, isoforms, and homologs of NaPi2b species naturally expressed by cells, including tumor cells, or expressed on cells transfected with the NaPi2b gene. These terms are synonymous and can be used interchangeably.

As used herein, the term NaPi2b antibody or anti-NaPi2b antibody refers to an antibody that specifically binds to the NaPi2b antigen.

When used in the context of two or more antibodies, the term competes with or cross-competes with indicates that the two or more antibodies compete for binding to NaPi2b, for example, compete for binding to NaPi2b in any assay known in the art. An antibody blocks or cross-blocks the binding of one or more other antibodies to NaPi2b if the antibody competes with one or more other antibodies by 25% or more, with 25%-74% representing a partial block and 75%-100% representing a complete block. which can be determined using any test known in the art. For some pairs of antibodies, competition or blocking in any test known in the art is observed only when one antibody is applied to the plate and the other is used to compete, but not vice versa. Unless otherwise indicated or inconsistent with context, the terms compete with, cross-compete with, block, or cross-block when used herein also include such antibody pairs.

Within the scope of the invention, the term antibody refers to immunoglobulin molecules and the immunologically active parts of the immunoglobulin (Ig) molecule, i.e. to molecules containing an antigen binding site that specifically binds (is immunoreactive towards it) to the antigen. Specific binds or is immunoreactive or directed to means that the antibody reacts with one or more antigenic determinants of the desired antigen and does not react with other polypeptides or binds with much lower affinity (Kd>10 ^-6 ). Antibodies include, but are not limited to, polyclonal, monoclonal and chimeric antibodies.

The basic structural unit of an antibody is known to include a tetramer. Each tetramer consists of two identical pairs of polypeptide chains, each of these pairs having one light chain (approximately 25 kDa) and one heavy chain (approximately 50-70 kDa). The amino-terminal portion of each chain includes a variable region of approximately 100-110 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function. In general, antibody molecules obtained from humans fall into one of the classes IgG, IgM, IgA, IgE and IgD, differing from each other by the nature of the heavy chain present in the molecule. Some classes also have subclasses, such as IgG1, IgG ₂ and others. Additionally, in humans, the light chain may be a kappa chain or a lambda chain.

The term monoclonal antibody (mAb) or monoclonal antibody composition, as used herein, refers to a population of antibody molecules containing only one molecular species of the antibody molecule, consisting of a unique light chain gene product and a unique heavy chain gene product. In particular, the complementarity determining regions (CDRs) of a monoclonal antibody are identical in all molecules of the population. Monoclonal antibodies (mAbs) contain an antigen-binding region capable of immunoreactivity with a specific epitope of an antigen, characterized by a unique binding affinity for it.

In general, antibody molecules obtained from humans belong to one of the classes IgG, IgM, IgA, IgE and IgD, differing from each other by the nature of the heavy chain present in the molecule. Some classes also have subclasses, such as IgG1, IgG ₂ and others. Additionally, in humans, the light chain may be a kappa chain or a lambda chain.

- 17 043790

The term antigen-binding site or binding moiety refers to the portion of an immunoglobulin molecule involved in binding an antigen. The antigen binding region is formed by amino acid residues of the variable N-terminal (V) regions of the heavy (H) and light (L) chains. Three highly divergent stretches in the variable regions of the heavy and light chains, called hypervariable regions, are inserted between more conserved flanking stretches known as reading frame regions, or FRs. Thus, the term FR refers to amino acid sequences naturally located between, and adjacent to, hypervariable regions in immunoglobulins. In an antibody molecule, three hypervariable regions of the light chain and three hypervariable regions of the heavy chain are located relative to each other in three-dimensional space, forming an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the bound antigen, and three hypervariable regions of each of the heavy and light chains are referred to as complementarity determining regions or CDRs. Amino acid assignment to each region is determined according to the definitions of Rabat Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987 and 1991)) or Chothia & Lesk J. Mol. Biol. 196:901-917 (1987), Chothia et al., Nature 342:878-883 (1989).

As used herein, the term epitope includes any protein determinant capable of specifically binding to an immunoglobulin or fragment thereof, or a T-cell receptor. The term epitope includes any protein determinant capable of specifically binding to an immunoglobulin or T-cell receptor. Epitope determinants typically consist of reactive surface groups of molecules such as amino acids or sugar side chains and typically have specific three-dimensional structural characteristics as well as specific charge characteristics. An antibody is said to bind specifically to an antigen when the dissociation constant is <1 μM; for example, <100 nM, preferably <10 nM, and more preferably <1 nM.

As used herein, the terms immunological binding and immunological binding properties refer to non-covalent interactions such as those occurring between an immunoglobulin molecule and an antigen for which the immunoglobulin is specific. The strength or affinity of immunological binding interactions can be expressed in terms of the dissociation constant ( _Kd ) of the interaction, where lower _Kd means greater affinity. The immunological binding properties of selected polypeptides can be quantified using methods known in the art. One such method involves measuring the rate of antigen-binding site/antigen complex formation and dissociation, and these rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both constant rate (K _on ) and non-constant rate (K _off ) can be determined by calculating concentrations and actual rates of association and dissociation (see Nature 361:186-87 (1993)). The ratio K _off /K _on ignores all non-affinity parameters and is equal to the dissociation constant K _d (see generally Davies et al. (1990) Annual Rev Biochem 59:439-473). An antibody of the present disclosure is said to specifically bind to NaPi2b when the equilibrium dissociation constant (K _d or K _D ) is <1 μM, preferably <100 nM, more preferably <10 nM, and most preferably <100 pM to about 1 pM , which can be measured by tests such as labeled ligand binding tests or similar tests known to one skilled in the art.

The term isolated polynucleotide, as used herein, means a polynucleotide of genomic, cDNA or synthetic origin, or a combination thereof, which, based on the origin of the isolated polynucleotide, (1) is not associated with all or part of the polynucleotide in which the isolated polynucleotide naturally occurs, (2) is operably linked to the polynucleotide , with which it is not associated in nature, or (3) does not occur in nature as part of a larger sequence. Polynucleotides according to the disclosure include nucleic acid molecules encoding heavy chain immunoglobulin molecules and parts thereof set forth in SEQ ID NOs: 1, 3, 14 and 16, and nucleic acid molecules encoding light chain immunoglobulin molecules and parts thereof set forth in SEQ ID NOs: 2, 4, 15 and 17.

The term isolated protein referred to herein means a protein derived from cDNA, recombinant RNA or synthetic origin, or a combination thereof, which, based on the origin or source of origin of the isolated protein (1) is not associated with naturally occurring proteins, (2) does not contain other proteins from the same source, (3) is expressed by cells of different species, or (4) does not occur in nature.

The term polypeptide is used herein as a generic term referring to the native protein, fragments or analogues of the polypeptide sequence. Therefore, fragments and analogues of the native protein are varieties of the polypeptide genus. Polypeptides according to the disclosure include immunoglobulin heavy chain molecules and portions thereof set forth in SEQ ID NOs: 1, 3,

- 18 043790 and 16, and light chain immunoglobulin molecules and parts thereof set forth in SEQ ID NOs: 2, 4, 15, and 17, as well as antibody molecules formed by combinations involving heavy chain immunoglobulin molecules with light chain immunoglobulin molecules, such as kappa light chain immunoglobulin molecules, and vice versa, as well as their fragments and analogues.

The term natural, as used herein, when applied to an object, refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence present in an organism (including viruses) that can be isolated from a source in nature and that has not been intentionally altered by humans in a laboratory or is otherwise naturally occurring.

The term operably linked within the scope of the invention refers to the positions of the components so described that are in a relationship that enables them to function in the intended manner. A control sequence operably linked to the coding sequence is ligated such that expression of the coding sequence is achieved under conditions compatible with the control sequences.

The term control sequence as used herein refers to polynucleotide sequences that are necessary to effect expression and processing of the coding sequences to which they are ligated. The nature of such control sequences varies among prokaryotes depending on the host organism, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence in eukaryotes, and typically such control sequences include promoters and a transcription termination sequence. The term control sequences includes, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is preferred, such as leader sequences and fusion partner sequences. The term polynucleotide as used herein means a polymer of nucleotides having a length of at least 10 bases, which may be either ribonucleotides or deoxyribonucleotides or a modified form of any type of nucleotide. This term includes single-stranded and double-stranded forms of DNA.

The term oligonucleotide as used herein includes natural and modified nucleotides linked by natural and non-natural oligonucleotide linkages. Oligonucleotides are a subpopulation of polynucleotides typically 200 bases or less in length. Preferably, the oligonucleotides are 10-60 bases in length, and most preferably 12, 13, 14, 15, 16, 17, 18, 19 or 20-40 bases in length. Oligonucleotides are typically single-stranded, for example in the case of probes, although oligonucleotides can be double-stranded, for example for use in constructing a gene mutant. The oligonucleotides disclosed herein are sense or antisense oligonucleotides.

The term natural nucleotides referred to herein includes deoxyribonucleotides and ribonucleotides. The term modified nucleotides referred to herein includes nucleotides with modified or substituted saccharide groups and the like. The term oligonucleotide linkages as used herein includes oligonucleotide linkages such as phosphorothioate, phosphorodithioate, phosphoroselerloate, phosphodiselenoate, phosphoroanilothioate, phosphoraniladate, phosphoroamidate and the like, see, for example, LaPlanche et al., Nucl. Acids Res. 14:9081 (1986); Stec et al., J. Am. Chem. Soc. 106:6077 (1984), Stein et al., Nucl. Acids Res. 16:3209 (1988), Zon et al., Anti Cancer Drug Design 6:539 (1991); Zon et al., Oligonucleotides and Analogues: A Practical Approach, pp. 87-108 (F. Eckstein, Ed., Oxford University Press, Oxford England (1991)); Stec et al., US Patent No. 5,151,510; Uhlmann and Peyman Chemical Reviews 90:543 (1990). The oligonucleotide may optionally include a detection label.

The following terms are used to describe sequence relationships between two or more polynucleotide or amino acid sequences: reference sequence, window of comparison, sequence identity, percent sequence identity, and substantive identity. A reference sequence is a specific sequence used as a basis for sequence comparison, a reference sequence may be a subpopulation of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence listed in a sequence listing, or may include a complete cDNA or gene sequence. Typically, the reference sequence is at least 18 nucleotides or 6 amino acids long, often at least 24 nucleotides or 8 amino acids long, and often at least 48 nucleotides or 16 amino acids long. Because two polynucleotide or amino acid sequences may each (1) include a sequence (i.e., part of the entire polynucleotide or amino acid sequence) that is similar between the two molecules, and (2) may further include a sequence that is different between the two polynucleotides or amino acid sequences, sequence comparisons between two (or more) molecules are typically made by comparing the sequences of the two molecules across a comparison window to identify and

- 19 043790 compare local regions of sequence similarity. A comparison window, as used herein, refers to a conceptual segment of at least 18 contiguous nucleotide positions or 6 amino acids, wherein the polynucleotide sequence or amino acid sequence can be compared to a reference sequence of at least 18 contiguous nucleotides or 6 amino acid sequences, and wherein the portion of the polynucleotide sequence in the comparison window may include insertions, deletions, substitutions, and the like (ie, gaps) that are 20 percent or less relative to the reference sequence (which does not include insertions or deletions) to optimally align the two sequences. Optimal sequence alignment to align the comparison window can be performed according to the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2:482 (1981), the Needleman and Wunsch J. Mol. homology alignment algorithm. Biol. 48:443 (1970), by searching for a method of similarity of Pearson and Lipman Proc. Natl. Acad. Sci (USA) 85:2444 (1988), computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin Genetics Software Package Release 7.0, (Genetics Computer Group, 575 Science Dr., Madison, Wis.), Geneworks , or MacVector software packages) or inspection, and the best alignment (i.e., resulting in the highest percentage of homology across the comparison window) generated by various methods can be selected.

The term sequence identity means that two polynucleotide or amino acid sequences are identical (ie, on a nucleotide or residue basis) across the window of comparison. The term percent sequence identity is calculated by comparing two optimally aligned sequences across a comparison window, determining the number of positions at which an identical nucleic acid base (e.g., A, T, C, G, U, or I) or residue is found in both sequences to obtain the number of matching positions , dividing the number of matching positions by the total number of positions in the comparison window (i.e., window size) and multiplying the result by 100 to obtain the percent sequence identity. The terms "substantial identity" as used herein means a characteristic of a polynucleotide or amino acid sequence, wherein the polynucleotide or amino acid comprises a sequence having at least 85% sequence identity, preferably at least 90% sequence identity, more typically at least 99% sequence identity, compared to with a reference sequence over a comparison window of at least 18 nucleotide (6 amino acid) positions, often over a window of at least 24-48 nucleotide (8-16 amino acid) positions, the percentage sequence identity being calculated by comparing the reference sequence with the sequence that may include deletions or insertions that are 20 percent or less of the reference sequence across the comparison window. The reference sequence may be a subset of a larger sequence.

Within the scope of the invention, the twenty standard amino acids and their abbreviations are used in a standard manner, see Immunology - A Synthesis (2nd Edition, E. S. Golub and D. R. Green, Eds., Sinauer Associates, Sunderland7 Mass (1991)). Stereoisomers (eg, D-amino acids) of the twenty standard amino acids, unnatural amino acids such as α,α-disubstituted amino acids, Nalkylamino acids, lactic acid and other non-conventional amino acids may also be suitable components for the polypeptides of the present disclosure. Examples of non-traditional amino acids include: 4-hydroxyproline, γ-carboxyglutamate, ε-N,N,N-trimethyllysine, ε-Nacetyl-lysine, O-phosphoserine, N-acetylserine, N-formylmethionine, 3-methylhistidine, 5-hydroxylysine, δN-methylarginine and other similar amino acids and imino acids (eg 4-hydroxyproline). In the polypeptide designations used herein, the left direction is the amino-terminal direction and the right direction is the carboxy-terminal direction, in accordance with standard usage and consensus.

Similarly, unless otherwise indicated, the left end of single-stranded polynucleotide sequences is the 5' end, the left direction of double-stranded polynucleotide sequences is referred to as the 5' direction. The direction of additions from 5' to 3' of synthesized RNA transcripts are referred to as transcription direction sequence regions on a DNA strand having the same sequence as RNA, and which represent the 5' to 5' ends of the RNA transcript, are referred to as upstream sequences, sequence regions on a DNA strand that has the same sequence as RNA and which are the 3' to 3' ends of the RNA transcript are referred to as downstream sequences.

As applied to polypeptides, the term identity essentially means that two peptide sequences, when optimally aligned, for example by the GAP or BESTFIT programs using default deletion penalties, have at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity, and most preferably at least 99 percent sequence identity.

Preferably, positions of residues that are not identical differ in conservative ways

- 20 043790 amino acid substitutions.

Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, the group of amino acids having aliphatic side chains is represented by glycine, alanine, valine, leucine and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is represented by serine and threonine; a group of amino acids having amide-containing side chains is represented by asparagine and glutamine; a group of amino acids having aromatic side chains is represented by phenylalanine, tyrosine and tryptophan; a group of amino acids having main side chains is represented by lysine, arginine and histidine; and a group of amino acids having sulfur-containing side chains is represented by cysteine and methionine. Preferred conservative groups of amino acid substitutions are as follows: valineleucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid and asparagine-glutamine.

As discussed herein, minor changes in the amino acid sequences of antibodies or immunoglobulin molecules are considered to be covered by the present disclosure if those changes in the amino acid sequence are supported by at least 75%, more preferably at least 80%, 90%, 95%, and most preferably 99 %. In particular, conservative amino acid substitutions are considered. Conservative substitutions are those that occur within a family of amino acids that are related in their side chains. Genetically encoded amino acids are usually divided into families: (1) acidic amino acids are aspartate, glutamate; (2) the main amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine and threonine. Hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other amino acid families include: (i) serine and threonine, which are the aliphatic hydroxy family; (ii) asparagine and glutamine, which are the amide-containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan and tyrosine, which are the aromatic family. For example, it is reasonable to expect that an isolated substitution of isoleucine or valine for leucine, glutamate for aspartate, serine for threonine, or a similar amino acid substitution for a structurally related amino acid would not have a significant effect on the binding or properties of the final molecule, especially if the substitution does not involve an amino acid in the reading frame. Whether an amino acid substitution can result in a functional peptide is easily determined by testing the specific activity of the derived polypeptide. The tests are described in detail here. Analogues of antibodies or immunoglobulin molecules can be easily obtained by a specialist. The preferred amino and carboxy termini of the analogues are located near the boundaries of the functional regions. Structural and functional regions can be identified by comparing nucleotide and/or amino acid sequence data with public or private sequence databases. Preferably, computerized comparison methods are used to identify sequence motifs or predicted protein conformational regions present in other proteins of known structure and/or function. There are known methods for identifying protein sequences that fold into a known spatial structure; Bowie et al., Science 253:164 (1991). Thus, the preceding examples demonstrate that one skilled in the art will be aware of sequence motifs and structural conformations that can be used to define structural and functional regions in accordance with the disclosure.

Preferred amino acid substitutions are those that: (1) reduce the propensity for proteolysis, (2) reduce the propensity for oxidation, (3) alter binding affinity for the formation of protein complexes, (4) alter binding affinity, and (4) impart or alter other physicochemical or functional properties of such analogues. Analogs may include different mutein sequences other than the natural sequence of the peptide. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) can be made in the natural sequence (preferably in a portion of the polypeptide outside the intermolecular junction forming region(s). A conservative amino acid substitution must not significantly alter the structural characteristics of the parent sequence (eg, the amino acid used for the substitution must not tend to break a helix in the parent sequence or disrupt other types of secondary structure characterizing the parent sequence). Examples of secondary and tertiary structures known in the polypeptide field are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W. H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, N.Y. (1991)) and Thornton et at. Nature 354:105 (1991).

The term agent is used here to refer to a chemical compound, a mixture of chemical compounds, a biological macromolecule, or an extract made from biological materials.

- 21 043790

As used herein, the terms labeled or labeled refer to the incorporation of a detectable marker, for example, the incorporation of a radiolabeled amino acid or the attachment of biotinyl groups to a polypeptide that can be detected by an avidin (e.g., streptavidin, containing a fluorescent marker or enzymatic activity that can be detected optical or calorimetric methods). In certain situations, the label or marker may also be therapeutic. Various methods for labeling polypeptides and glycoproteins are known in the art and can be used. Examples of labels for polypeptides include, but ^are not limited to, the following: radioisotopes or radionuclides (eg, ^3H , ^14C , ^15N , ^35S , 90Y, ^99Tc , ^n1In , ^125I , ^131I ), fluorescent labels ( e.g., FITC, rhodamine, lanthanide complex phosphor), enzymatic tags (e.g., horseradish peroxidase, p-galactosidase, luciferase, alkaline phosphatase), chemiluminescent, biotinyl groups, predefined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences , binding sites for secondary antibodies, metal-binding regions, epitope tags). In some embodiments, the tags are attached using spacers of varying lengths to alleviate potential steric hindrance. The term pharmaceutical or drug as used herein refers to a chemical compound or composition capable of inducing a desired therapeutic effect when properly administered to a patient.

Other chemical terms are used herein according to standard usage in the art, as illustrated in The McGraw-Hill Dictionary of Chemical Terms (Parker, S., Ed., McGraw-Hill, San Francisco (1985)).

For purposes of the invention, substantially pure means that the species is the predominant species (i.e., on a molar basis, it is more abundant than any other individual species in the composition), and preferably the substantially pure fraction is the composition in which the a species comprises at least about 50% (on a molar basis) of all macromolecular species present.

Typically, the substantially pure composition will include greater than about 80% of all macromolecular species present in the composition, more preferably greater than about 85%, 90%, 95%, and 99%. Most preferably, the species is purified to be substantially homogeneous (the species impurity cannot be detected in the composition by standard detection methods), with the composition consisting of substantially a single macromolecular species.

The use of the singular in the following description and in the claims is to be construed to include both the singular and the plural unless otherwise indicated or unless clearly inconsistent with the context. The terms including, having, being, as in having a chemical formula, and containing are to be construed as open-ended terms (ie, including, but not limited to), unless otherwise noted. For example, polymer backbones of a particular formula include all of the monomer units shown in the formula and may also include additional monomer units not shown in the formula. Additionally, whenever an inclusive or other open-ended term is used in an embodiment, it should be understood that the same embodiment can be stated more narrowly using the intermediate term consisting essentially of or the closed term consisting of.

The term about, about, or roughly, when used in connection with a numeric designation, means that a series or range of values is included. For example, approximately X includes a range of values that are ±20%, ±10%, ±5%, ±2%, ±1%, ±0.5%, ±0.2%, or ±0.1% of X. where X represents a numeric designation. In one embodiment, the term approximately refers to a range of values that are 5% greater or less than the specified value. In another embodiment, the term approximately refers to a range of values that are 2% greater or less than the specified value. In another embodiment, the term approximately refers to a range of values that are 1% greater or less than the specified value.

The indication of ranges of values serves merely as a shorthand way to individually refer to each individual value falling within that range unless otherwise noted, and each individual value is included in the description as if it were individually stated herein. The range used here, unless otherwise noted, includes two range boundaries. For example, the expression x is an integer from 1 to 6 and x is an integer 1-6 means x is 1, 2, 3, 4, 5 or 6, i.e. the terms X to y and the range X to Y include X and y and the integers in between.

All of the methods described herein can be performed in any suitable order unless otherwise noted or unless the context clearly dictates. The use of any and all examples or example language (such as, for example) is intended merely to better illustrate the disclosure and should not be construed as limiting the scope of the claims unless expressly stated otherwise. Nothing in the description should be construed to indicate that anything not stated

- 22 043790 element is important for what is stated.

Biologically compatible, as used herein, is intended to describe compounds that exhibit minimal destructive or organismal responses in contact with body fluids or living cells or tissues. Thus, a biocompatible group, for the purposes of the invention, refers to an aliphatic, cycloalkyl, heteroaliphatic, heterocycloalkyl, aryl or heteroaryl group falling within the definition of the word biocompatible as defined above. The term Biocompatibility, as used herein, also means that the compounds exhibit minimal interactions with recognition proteins, for example, natural antibodies, cellular proteins, cells and other components of biological systems, unless such interactions are specifically desired. Thus, substances and functional groups specifically designed to induce the above-mentioned minimal interactions, such as drugs and prodrugs, are considered biologically compatible. Preferably (except for compounds that are intended to be cytotoxic, such as, for example, antineoplastic agents), the compounds are biocompatible if their addition to normal cells in vitro at concentrations similar to the desired systemic concentrations in vivo results in less than or equal to 1% cell necrosis for a period of time equivalent to the in vivo half-life of the compound (e.g., the period of time required for removal/clearance of 50% of the compound administered in vivo), and their in vivo administration causes minimal and medically acceptable inflammation, foreign body reaction , immunotoxicity, chemical toxicity and/or other similar adverse effects. In the above sentence, the term normal cells refers to cells not intended to be destroyed or otherwise significantly affected by the test compound.

Biodegradable within the scope of the invention, biodegradable polymers are polymers that are bioprocessable in vivo. As used herein, biodegradable compounds or groups are those that, when taken up by cells, can be broken down by lysosomal or other chemical mechanisms or hydrolysis into components that the cells can either reuse or dispose of without significant toxic effect on the cells. The term biodegradable within the meaning of the invention has the same meaning as biodegradable. The degradation moieties preferably cause minimal or no organ or cellular overload or pathological processes associated with such overload or other adverse effects in vivo. Examples of biological degradation processes include enzymatic and non-enzymatic hydrolysis, oxidation and reduction.

Suitable conditions for non-enzymatic hydrolysis of biodegradable protein-polymer-drug conjugates (or components thereof, e.g., biodegradable polymer carrier and linkers between the carrier and the antibody or drug molecule) described herein, for example, include exposing the biodegradable conjugates to water at the temperature and pH of the lysosomal intracellular compartment. The biodegradation of some protein-polymer-drug conjugates (or components thereof, e.g., the biodegradable polymer carrier and linkers between the carrier and the antibody or drug molecule) may also be enhanced extracellularly, for example, in low pH regions of the animal body, such as areas of inflammation, in close proximity to activated macrophages or other cells that release factors that facilitate decomposition. In some preferred embodiments, the effective size of the polymer carrier at pH~7.5 does not change appreciably for 1 to 7 days and remains within 50% of the size of the original polymer for at least several weeks. At pH~5, on the other hand, the polymer carrier preferably degrades markedly within 1 to 5 days and is completely converted to low molecular weight fragments within a time interval of two weeks to several months. Polymer integrity in such tests can be measured, for example, using size exclusion HPLC. Although faster degradation may be preferable in some cases, in general it may be more desirable for the polymer to degrade in cells at a rate no greater than the rate of metabolism or excretion of polymer fragments by the cells. In preferred embodiments, the polymers and polymer biodegradation by-products are biocompatible.

A maleimido blocking compound as used herein refers to a compound that can react with maleimide to convert it to succinimide, and the maleimido protecting group refers to the chemical group attached to the succinimide after the conversion. In some embodiments, the maleimide blocking compound is a compound having a terminal thiol group for reaction with a maleimide. In one embodiment, the maleimide blocking compound is cysteine, N-acetyl cysteine, cysteine methyl ester, N-methyl cysteine, 2-mercaptoethanol, 3-mercaptopropanoic acid, 2-mercaptoacetic acid, mercaptomethanol (i.e. HOCH ₂ SH), benzylthiol , in which the phenyl is substituted with one or more hydrophilic substituents, or 3-aminopropane-1-thiol.

Hydrophilic: The term is hydrophilic as far as it refers to substituents, e.g.

- 23 043790 monomer units of a polymer macromolecule or on the protecting group of a maleimido group, making them hydrophilic or water-soluble, is not substantially different from the general meaning of this term in the art and denotes chemical groups containing ionizable, polar or polarizable atoms, or which can otherwise be solvated by water molecules. Thus, a hydrophilic group, for the purposes of the invention, refers to an aliphatic, cycloalkyl, heteroaliphatic, heterocycloalkyl, aryl or heteroaryl group falling within the definition of the word hydrophilic as defined above. Examples of particularly suitable hydrophilic organic groups include, but are not limited to, aliphatic or heteroaliphatic groups comprising a chain of atoms ranging from about one to twelve atoms, hydroxyl, hydroxyalkyl, amine, carboxyl, amide, carboxyl ester, thioester, aldehyde, nitrile, isonitrile, nitroso, hydroxylamine, mercaptoalkyl, heterocycle, carbamates, carboxylic acids and their salts, sulfonic acids and their salts, sulfonic acid esters, phosphoric acids and their salts, phosphate esters, polyglycol esters, polyamines, polycarboxylates, polyesters and polythioesters. In some embodiments, the hydrophilic substituents include a carboxyl group (COOH), an aldehyde group (CHO), a ketone group (COC ₁ - ₄ alkyl), methylol (CH2OH) or glycol (for example, CHOH-CH2OH or CH-( _CH2OH ) ₂ ), NH2, F, cyano, SO ₃ H, PO ₃ H, etc.

The term hydrophilic, as it relates to the polymers disclosed herein generally, does not differ from the use of this term in the art and refers to polymers including hydrophilic functional groups as defined above. In a preferred embodiment, the hydrophilic polymer is a water-soluble polymer. The hydrophilicity of a polymer can be directly measured by determining the energy of hydration or determined by interrogation between two liquid phases, or chromatography on solid phases of known hydrophobicity, such as, for example, C4 or C18.

Polymeric carrier: The term polymeric carrier, as used herein, refers to a polymer or modified polymer that is suitable for covalent attachment or can be covalently attached to one or more drug molecules with a specific linker and/or one or more PBRMs with a specific linker.

Physiological Conditions: The phrase physiological conditions, as used herein, refers to the range of chemical (eg pH, ionic strength) and biochemical (eg enzyme concentration) conditions likely to be encountered in the extracellular fluids of living tissues. For most normal tissues, the physiological pH is approximately 7.0 to 7.4. The circulation of blood plasma and normal interstitial fluid represents typical examples of normal physiological conditions.

Drug: As used herein, the term drug refers to a compound that is biologically active and provides a desired physiological effect upon administration to a patient (eg, an active pharmaceutical ingredient).

An antiangiogenesis agent or angiogenesis inhibitor refers to a small molecular weight substance, polynucleotide, polypeptide, isolated protein, recombinant protein, antibody, or conjugates or fusion proteins thereof that directly or indirectly inhibits angiogenesis, vasculogenesis, or unwanted vascular permeability. An angiogenesis inhibitor includes agents that bind to an angiogenic factor or its receptor and block their angiogenic activity. For example, the anti-angiogenic agent may be an antibody or other antagonist of the angiogenic agent, such as, but not limited to, anti-VEGF-A or anti-VEGF-A receptor (e.g., KDR receptor or Flt-1 receptor), VEGF-trap, anti-PDGFR inhibitors such as Gleevec™ (imatinib mesylate). Anti-angiogenesis agents also include native angiogenesis inhibitors, for example, angiostatin, endostatin, and the like.

Cytotoxic: As used herein, the term cytotoxic means a substance that is toxic to cells or a selected population of cells (eg, cancer cells). The toxic effect may lead to necrosis and/or cell lysis. In some cases, the toxic effect may be a sublethal destructive effect on the cell, such as slowing or stopping cell growth. To achieve a cytotoxic effect, the drug or prodrug may be selected from, among others, the group consisting of a DNA-disrupting agent, a microtubule-disrupting agent, or a cytotoxic protein or polypeptide.

Cytostatic: As used herein, the term cytostatic refers to a drug or other compound that inhibits or stops the growth and/or reproduction of cells.

Small Molecule: As used herein, the term small molecule refers to molecules, natural or artificially created (eg, by chemical synthesis), that have a relatively low molecular weight. Preferred small molecules are biologically active in that they have a local or systemic effect in animals, preferably mammals, more preferably humans. In some preferred embodiments, the small molecule is a drug, and the small molecule is referred to as a drug molecule or drug or tera

- 24 043790 pediatric remedy. In some embodiments, the drug molecule has an MW of less than or equal to about 5 kDa. In other embodiments, the drug molecule has an MW of less than or equal to about 1.5 kDa. In embodiments, the drug molecule may be selected from vinca alkaloids, auristatins, duocarmycins, tubulisins, unnatural camptothecin compounds, topoisomerase inhibitors, DNA-binding drugs, kinase inhibitors, MEK inhibitors, KSP inhibitors, calicheamicins, SN38, pyrrolobenzodiazepines, and analogs thereof. Preferably, although not necessarily, the drug is one that an appropriate government agency or agency, such as the FDA, already considers safe and effective for use. For example, drugs for human use listed by the FDA under 21 C.F.R. 330.5, 331-361, and 440-460; drugs for veterinary use listed by the FDA under 21 C.F.R. 500-589, which are incorporated herein by reference, are all considered suitable for use with the hydrophilic polymers of the invention.

A drug derivative or modified drug and the like, as used herein, refers to a compound comprising a drug molecule to be delivered by a conjugate disclosed herein and a functional group capable of attaching the drug molecule to a polymeric carrier.

Active form, as used herein, refers to a form of a compound that exhibits intended pharmaceutical efficacy in vivo or in vitro. In particular, when a drug molecule to be delivered by a conjugate disclosed herein is released from the conjugate, the active form may be the drug itself or derivatives thereof exhibiting the intended therapeutic properties.

Release of the drug from the conjugate can be achieved by cleavage of the biodegradable linker bond attaching the drug to the polymer carrier. Active drug derivatives may accordingly include a linker portion.

PHF refers to poly(1-hydroxymethylethylene hydroxymethyl formal).

As used herein, the terms polymer unit, macromolecule monomer unit, monomer, monomer unit, unit all refer to a repeating structural unit in a polymer.

For purposes of the invention, the molecular weight or MW of a polymer or polymer carrier/backbone or polymer conjugates refers to the average molecular weight of the unmodified polymer unless otherwise indicated.

An immune checkpoint inhibitor or an immune checkpoint inhibitory agent or an immune checkpoint blocking agent or an immune checkpoint modulator, as used herein, refers to an agent that binds to an immune checkpoint inhibitor protein and blocks its activity, thereby allowing immune system to recognize tumor cells and provide a prolonged immunotherapeutic response. Inhibition may be competitive or non-competitive inhibition, which may be steric or allosteric. In cases where the immune checkpoint protein is an immunostimulatory protein, the immune checkpoint inhibitor promotes the activity of the immune checkpoint protein, for example by binding and activating the immune checkpoint protein or by inhibiting by interfering with, for example by binding or deactivating, the immune checkpoint protein points. An example of an immune checkpoint inhibitor is an antibody to an immune checkpoint protein.

Immune checkpoints, as used herein, refer to inhibitory pathways of the immune system that are responsible for maintaining self-tolerance and modulating the duration and amplitude of physiological immune responses in peripheral tissues to reduce collateral tissue damage. Immune checkpoints are regulated by immune checkpoint proteins.

An immune checkpoint protein, as used herein, refers to a protein, typically a receptor (eg, CTLA4 or PD-1) or ligand (eg, PD-L1), that regulates or modulates the extent of the immune response. Immune checkpoint proteins can be inhibitory or stimulatory. In particular, immune checkpoint proteins inhibit the activation of the immune response. Thus, inhibition of immune checkpoint inhibitory protein stimulates or activates the immune response, such as T cell activation and proliferation.

The target of an immune checkpoint inhibitor, within the scope of the invention, is an immune checkpoint protein to which the immune checkpoint inhibitor or immune checkpoint inhibitory agent binds to block activity. Typically, an immune checkpoint inhibitor specifically binds to a target. For example, the target of an exemplary anti-CTLA4 antibody called ipilimumab is CTLA4.

Combination therapy as used herein refers to a treatment in which a patient is given two or more therapeutic agents, for example at least two or at least three therapeutic agents, to treat a single disease. For example, combination therapy includes therapy with a NaPi2b-targeting antibody-drug conjugate and an inhibitor thereof.

- 25 043790 moon control points.

Within the scope of the invention, co-administration or co-administered refers to the administration of at least two different therapeutic agents sufficiently close in time. Such administration can be carried out in any order, including simultaneous administration, as well as at time intervals ranging from several seconds to several days. Such administration may also include more than a single administration of one agent and/or independently of another agent. The agents can be administered by the same or different routes.

As used herein, an anti-CTLA4 antibody refers to any antibody that specifically binds to cytotoxic T lymphocyte associated protein 4 (CTLA4) or a soluble fragment thereof and blocks the binding of ligands to CTLA4, thereby resulting in competitive inhibition of CTLA4 and inhibition of CTLA4-mediated inhibition of T cell activation. Therefore, anti-CTLA4 antibodies are CTLA4 inhibitors. Reference to anti-CTLA4 antibodies herein includes the full-length antibody and derivatives thereof, such as antigen-binding fragments thereof that specifically bind CTLA4. Examples of anti-CTLA4 antibodies include, but are not limited to, ipilimumab or tremelimumab or derivatives or antigen binding fragment thereof.

As used herein, the antigen cytotoxic T lymphocyte associated protein 4 (CTLA4; also known as CD 152) refers to the inhibitory receptor of the immunoglobulin superfamily, bound by ligands such as CD80 (also called B7-1) and CD86 (also called B7-2). . CTLA4 includes human and non-human proteins. In particular, the CTLA4 antigen includes human CTLA4 having the amino acid sequence set forth in SEQ ID NO: 10 (see, for example, GenBank Accession No. AAL07473.1).

As used herein, an anti-PD-1 antibody refers to any antibody that specifically binds to programmed cell death protein 1 (PD-1) or a soluble fragment thereof and blocks the binding of ligands to PD-1, thereby resulting in competitive inhibition of PD-1 and inhibition of PD-1-mediated inhibition of T cell activation. Therefore, anti-PD-1 antibodies are PD-1 inhibitors. Reference to anti-PD-1 antibodies herein includes the full-length antibody and its derivatives, such as antigen-binding fragments that specifically bind to PD-1. Examples of anti-PD-1 antibodies include, but are not limited to, nivolumab, MK-3475, pidilizumab, or a derivative or antigen-binding fragment thereof.

As used herein, the programmed cell death protein 1 (PD-1) antigen refers to an inhibitory receptor which is a type 1 membrane protein and is bound by ligands such as PD-L1 and PD-L2, which are members of the B7 family. PD-1 includes human and non-human proteins. In particular, the PD-1 antigen includes human PD-1 having the amino acid sequence set forth in SEQ ID NO: 299 (see, for example, UniProt Accession No. Q15116.3). As used herein, an anti-PD-L1 antibody refers to an antibody that specifically binds to programmed death ligand 1 (PD-L1) or a soluble fragment thereof and blocks the binding of the ligand to PD-1, thereby resulting in competitive inhibition of PD-1 and inhibition PD-1-mediated inhibition of T cell activity. Therefore, anti-PD-LI antibodies are PD-1 inhibitors. Reference to anti-PD-L1 antibodies herein includes the full-length antibody and its derivatives, such as antigen-binding fragments that specifically bind to PD-L1. Examples of anti-PD-L1 antibodies include, but are not limited to, BMS-936559, MPDL3280A, MEDI4736 or a derivative or antigen binding fragment thereof.

As used herein, a dosage regimen or administration regimen refers to the amount of agent administered, eg, a composition containing a NaPi2b-targeting antibody-drug conjugate, and the frequency of administration. The mode of administration depends on the disease or condition being treated and thus may vary.

Within the scope of the invention, frequency of administration refers to the time between successive administrations for treatment. For example, the frequency may be days, weeks, or months. For example, the frequency may be several times per week, such as twice a week, three times a week, four times a week, five times a week, six times a week, or daily. The frequency can also be one, two, three or four weeks. The specific frequency depends on the specific disease or condition being treated. The usual frequency is several times a week, and usually twice a week.

As used herein, an administration cycle refers to a repeatable schedule of administration of an enzyme and/or a second agent repeated over successive administrations. For example, the administration cycle may be a 28-day cycle with administration twice a week for three weeks, followed by one week of cessation of administration.

For the purposes of the invention, when referring to dosage per mg/kg body weight of a patient, the average person is considered to have a body weight of approximately 70 kg-75 kg, for example 70 kg and a body surface area (BSA) of 1.73 m. within the scope of the invention, improvement in the symptoms of a particular disease or disorder as a result of treatment, such as the administration of a pharmaceutical composition or other

- 26 043790 of a therapeutic agent, refers to any reduction, permanent or temporary, lasting or transient, in the symptoms or adverse effects of a condition, such as, for example, a reduction in adverse effects associated with or that result from the administration of a NaPi2b-targeting antibody conjugate -medicine.

As used herein, treating or treating describes the management and care of a patient to combat a disease, condition or disorder and includes administering a conjugate of the disclosure or a pharmaceutical composition thereof to relieve symptoms or complications of a disease, condition or disorder or eliminate the disease, condition or disorder.

As used herein, prevention or prophylaxis refers to reducing the risk of developing a disease or condition, or reducing or eliminating the onset of symptoms or complications of a disease, condition or disorder.

The term effective amount or sufficient amount, as it relates to an active substance, refers to the amount necessary to induce the desired biological response. As used herein, a therapeutically effective amount or therapeutically effective dose refers to a dose or amount of an agent, compound, material or composition containing a compound that is at least sufficient to produce a detectable therapeutic effect. The effect can be detected by any method using a test known in the art. The exact effective amount for a patient depends on the patient's body weight, body size, and medical condition; the nature and extent of the condition; and the therapeutic agent selected for administration.

The patient includes mammals. The mammal can be, for example, any mammal, such as a human, primate, bird, mouse, rat, poultry, dog, cat, cow, horse, goat, camel, sheep or pig. Preferably, the mammal is human.

As used herein, unit dosage form or unit dosage form refers to physically discrete units suitable for human and animal administration and individually packaged as known in the art.

For the purposes of the invention, unit dose form refers to a composition formed as a single dose.

Within the scope of the invention, a kit refers to a combination of components, such as a combination of the compositions described herein and another object for the intended purpose, including, but not limited to, reconstitution, activation and tools/devices for delivery, administration, diagnosis and assessment of biological activity or property . Kits may include instructions for use.

The present disclosure includes all isotopes of atoms found in the compounds of the invention. Isotopes include atoms having the same atomic number but different mass numbers. By way of general example and without limitation, isotopes of hydrogen include tritium and deuterium. Isotopes of carbon include C-13 and C-14.

The present disclosure includes all isomers of a compound relating to and including optical isomers and tautomeric isomers, where the optical isomers include enantiomers and diastereomers, chiral isomers and non-chiral isomers, and the optical isomers include isolated optical isomers, as well as mixtures of optical isomers, including racemic and non-racemic mixtures ; wherein the isomer may be in isolated form or in mixture with one or more other isomers.

NaPi2b antibodies.

The antibody-drug conjugates disclosed herein include monoclonal antibodies that specifically recognize NaPi2b and have the ability to inhibit NaPi2b activity.

Examples of antibodies used in the antibody-drug conjugates disclosed herein include, for example, the antibodies referred to herein as antibody XMT 1535 and/or antibody 10H1.11.4B. These antibodies show specificity for human NaPi2b and have been shown to inhibit the functional activity of NaPi2b in vitro.

Each of the NaPi2b monoclonal antibodies described herein includes a heavy chain (HC), a heavy chain variable region (VH), a light chain (LC), and a light chain variable region (VL), as shown in the amino acid and corresponding nucleic acid sequences presented below . The heavy chain variable region and light chain variable region for each antibody are highlighted in color in the amino acid sequences shown below. The complementarity determining regions (CDRs) of the heavy chain and the light chain are highlighted in the amino acid sequences presented below. The amino acids encompassing the complementarity determining regions (CDRs) for the XMT 1535 antibody are as defined by E.A. Rabat et al. (See Rabat, E.A., et al., Sequences of Protein of immunological interest, Fifth Edition, US Department of Health and Human Services, US Government Printing Office (1991)), and are disclosed in US Patent 8,603,474, and amino acids covering the CDRs for antibody 10H1.11.4B are as defined in US Pat. No. 8,535,675.

Amino acid sequence of the heavy chain of XMT 1535 (heavy chain variable region

- 27 043790 (SEQ ID NO: 3)+IgG1 heavy chain constant region (SEQ ID NO: 11))

QVQLVQSGAEWKPGASVKMSCKASGYTFTGYNIHWVKQAPGQGLEWIGAIYPGNGDTS YKQKFRGRATLTADTSTSTVYMELSSLRSEDSAVYYCARGETARATFAYWGOGTLVTVSSGAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS WTVPSSSLGTQ TYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG* (SEQ ID NO: 1)

CDRH1: GYTFTGYNIH (SEQ ID NO: 5)

CDRH2: AIYPGNGDTSYKQKFRG (SEQ ID NO: 6)

CDRH3:GETARATFAY (SEQ ID NO: 7)

Amino acid sequence of XMT 1535 light chain (light chain variable region (SEQ ID NO: 4)+light chain constant region (SEQ ID NO: 12))

DIOMTOSPSSLSASVGDRVTITCSASODIGNFLNWYOOKPGKTVKVLIYYTSSLYSGVP SRFSGSGSGTDYTLTISSLQPEDFATYYCQQYSKLPLTFGOGTKLELKRRTVAAPSVFIFPPSD EQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADY EKH KVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 2)

CDRL1: SASQDIGNFLN (SEQ ID NO: 8)

CDRL2: YTSSLYS (SEQ ID NO: 9)

CDRL3: QQYSKLPLT (SEQ ID NO: 10)

Heavy chain amino acid sequence 10H1.11.4B (heavy chain variable region (SEQ ID NO: 16)+IgG1 heavy chain constant region (SEQ ID NO: 13))

EVOLVESGGGLVOPGGSLRLSCAASGFSFSDFAMSWVRQAPGKGLEWVATIGRVAFHTY YPDSMKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARHRGFDVGHFDFWGOGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSS WTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPK DTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD IAVEWESNGQPENNYKTT PPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS LSLSPG* (SEQ ID NO: 14) CDRH1: GFSFSDFAMS (SEQ ID NO: 18)

CDRH2: ATIGRVAFHTYYPDSMKG (SEQ ID NO: 19)

CDRH3: ARHRGFDVGHFDF (SEQ ID NO: 20)

Light chain amino acid sequence 10H1.11.4B (light chain variable region (SEQ ID NO: 17)+light chain constant region (SEQ ID NO: 12))

DIQMTQS PS S LSASVGDRVTITCRSSETLVHSSGNTYLEWYQQKPGKAPKLLIYRVSNR FSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCFQGSFNPLTFGQGTKVEIKRTVAAPSVFIF

PPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC* (SEQ ID NO: 15)

CDRL1: RSSETLVHSSGNTYLE (SEQ ID NO: 21)

CDRL2: RVSNRFS (SEQ ID NO: 22)

CDRL3: FQGSFNPLT (SEQ ID NO: 23)

Also included in the disclosure are antibodies that bind to the same epitope, or that cross-compete for binding to the same epitope, as the antibodies described herein. For example, the antibodies disclosed herein specifically bind to NaPi2b, wherein the antibody binds to an epitope comprising one or more amino acid residues on human NaPi2b (eg, GenBank accession no. 095436.3).

The antibodies disclosed herein specifically bind to an epitope on full-length human NaPi2b comprising the amino acid sequence:

MAPWPELGDA QPNPDKYLEG AAGQQPTAPD KSKETNKTDN TEAPVTKIEL

LPSYSTATLI DEPTEVDDPW NLPTLQDSGI KWSERDTKFK ILCFFQGIGR

101 LILLLGFLYF FVCSLDILSS AFQLVGGKMA GQFFSNSSIM SNPLLGLVIG

151 VLVTVLVQSS STSTSIWSM VSSSLLTVRA AIPIIMGANI GTSITNTIVA

201 LMQVGDRSEF RRAFAGATVH DFFNWLSVLV LLPVEVATHY LEIITQLIVE

251 SFHFKNGEDA PDLLKVITKP FTKLIVQLDK KVISQIAMND EKAKNKSLVK

301 IWCKTFTNKT QINVTVPSTA NCTSPSLCWT DGIQNWTMKN VTYKENIAKC

351 KIFVNFLP DLAVGTILI LSLLVLCGCL IMIVKILGSV LKFQVATVIK

401 KTINTDFPFP FAWLTGYLAI LVGAGMTFIV QSSSVFTSAL TPLIGIGVIT

451 IERAYPLTLG SNIGTTTTAI LAALASPGNA LRSSLQIALC HFFFNISGIL

501 LWYPIPFTRL PIRMAKFLGN ISAKYRWFAV FYLIIFFFLI PLTVFGLSLA

551 GWRVLVGVGV PWFIIILVL CLRLLQSRCP RVLPKKLQNW NFLPLWMRSL

601 KPWDAWSKF TGCFQMRCCC CCRVCCRACC LLCDCPKCCR CSKCCEDLEE

651 AQEGQDVPVK APETFDNITI SREAQGEVPA SDSKTECTAL (SEQ ID NO:

24)

The antibodies disclosed herein specifically bind to an epitope on the extracellular region (ECD) of human NaPi2b.

One skilled in the art will appreciate that it is possible to determine, without further experimentation, whether a monoclonal antibody has the same specificity as the monoclonal antibody disclosed herein (e.g., XMT 1535, 10H1.11.4B) by determining whether the former interferes with the binding of the latter to EC

- 28 043790 to a natural binding partner or other molecule known to bind NaPi2b. If a test monoclonal antibody competes with a monoclonal antibody disclosed herein, resulting in decreased binding by the monoclonal antibody disclosed herein, then the two monoclonal antibodies bind to the same or closely related epitope.

An alternative method for determining whether a monoclonal antibody has the specificity of the monoclonal antibody disclosed herein is to preincubate the monoclonal antibody disclosed herein with soluble NaPi2b (for which it is generally reactive) and then adding the test monoclonal antibody to determine whether it is inhibited the ability of the test monoclonal antibody to bind to NaPi2b. If the test monoclonal antibody is inhibited, then it is likely to have the same, or functionally equivalent, epitope specificity as the monoclonal antibody disclosed herein.

Screening for monoclonal antibodies disclosed herein can also be accomplished, for example, by measuring NaPi2b-mediated activity and determining whether the test monoclonal antibody is able to modulate, block, inhibit, reduce, counteract, neutralize, or otherwise interfere with NaPi2b activity.

NaPi2b antibodies are prepared, for example, using various procedures known in the art that can be used to produce monoclonal antibodies directed to NaPi2b or derivatives, fragments, analogs, homologues or orthologues thereof (see, for example, Antibodies: A Laboratory Manual, Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, incorporated herein by reference). Fully human antibodies are antibody molecules in which the entire light chain and heavy chain sequence, including the CDRs, is derived from human genes. Such antibodies are called human antibodies or fully human antibodies. Human monoclonal antibodies are prepared, for example, using the procedures described in the Examples below. Human monoclonal antibodies can also be produced using the triome method; the human B-cell hybridoma method (see Kozbor, et al., 1983 Immunol Today 4: 72); and the EBV hybridoma method for producing human monoclonal antibodies (see Cole, et al., 1985 In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Human monoclonal antibodies can be used and can be produced using human hybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80: 2026-2030) or by in vitro transformation of human B cells with Epstein-Barr virus ( see Cole, et al., 1985 In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).

Antibodies are purified by known methods such as affinity chromatography using protein A or protein G, which provide primarily the IgG fraction of the immune serum. Subsequently, or alternatively, the specific antigen that is the target of the immunoglobulin of interest, or its epitope, can be immobilized on a column to purify the immunospecific antibody by immunoaffinity chromatography. Purification of immunoglobulins is discussed, for example, by D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia PA, Vol. 14, No. 8 (April 17, 2000), pp. 25-28).

Monoclonal antibodies that modulate, block, inhibit, reduce, counteract, neutralize or otherwise interfere with NaPi2b activity are prepared, for example, by immunizing an animal with membrane-bound and/or soluble NaPi2b, such as, for example, murine, rat or human NaPi2b or an immunogenic fragment thereof , derivative or variant. The animal is also immunized with cells transfected with a vector containing a nucleic acid molecule encoding NaPi2b such that NaPi2b is expressed and binds to the surface of the transfected cells. Antibodies are also generated by screening a library containing antibody or antigen binding region sequences for binding to NaPi2b. This library is produced, for example, in a bacteriophage as a fusion protein or peptide with a bacteriophage coat protein expressed on the surface of assembled phage particles and DNA coding sequences contained in the phage particles (ie, a phage display library). Hybridomas resulting from myeloma/B-cell fusions are then tested for reactivity to NaPi2b. In addition, antibodies selected from and, if necessary, optimized in yeast antibody display libraries and yeast library display systems, as described in, for example:

- 29 043790

Blaise L, Wehnert A, Steukers MP, van den Beucken T, Hoogenboom HR, Hufton SE. Construction and diversification of yeast cell surface displayed libraries by yeast mating: application to the affinity maturation of Fab antibody fragments. Gene. 2004 Nov 2 4; 3 4 2 (2): 211 - 8; Boder ET, Wittrup KD. Yeast surface display for screening combinatorial polypeptide libraries. Nat Biotechnol. 1997 Jun;15(6):553-7; Kuroda K, Ueda M. Cell surface engineering of yeast for applications in white biotechnology. Biotechnol Lett. 2011 Jan;33(1):1-9. doi:10.1007/sl0529-010-0403-9. Review; Lauer TM, Agrawal NJ, Chennamsetty N, Egodage K, Helk B, Trout BL. Developability index: a rapid in silico tool for the screening of antibody aggregation propensity. J Pharm Sci. 2012 Jan;101(1):102-15; Orcutt K.D. and Wittrup K.D. (2010), 207-233 doi: 10.1007/978-3-642-01144-3_l5; Rakestraw JA, Aird D, Aha PM, Baynes BM, Lipovsek D. Secretion-and-capture cell-surface display for selection of target-binding proteins. Protein Eng Des Sei. 2011 Jun;24(6):525-30; US 8,258,082; US 6, 300, 064; US 6,696,248; US 6,165,718; US 6,500,644; US 6,291,158; US 6,291, 159; US 6,096,551; US 6, 368, 805; US 6, 500, 644.

Examples of yeast library presentation systems are described in, for example, WO2008118476; WO2009/036379; WO2010105256; and WO2012009568. In some embodiments, such yeast antibody display libraries or yeast library display systems are designed to mimic or reflect the diversity of characteristics of the human preimmune antibody spectrum. In some embodiments, such diversity of yeast antibody display libraries or diversity of yeast library display systems is generated in silico. In some embodiments, such yeast antibody display libraries or yeast library display systems include Saccharomyces yeast cells, such as Saccharomyces cerevisiae cells. In some embodiments, such yeast antibody display libraries or yeast library display systems include Pichia cells.

Monoclonal antibodies are produced, for example, using hybridoma methods such as those described by Kohler and Milstein, Nature, 256:495 (1975). In the hybridoma method, a mouse, hamster, or other suitable host is typically immunized with an immunizing agent to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunizing agent. Lymphocytes can also be immunized in vitro.

The immunizing agent typically includes a protein antigen, fragment or fusion protein thereof. Typically, either peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused to the immortalized cell line using a suitable fusion factor such as polyethylene glycol to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Immortalized cell lines are typically transformed mammalian cells, particularly myeloma cells derived from rodent, bovine and human origin. Typically, rat or mouse myeloma cell lines are used. Hybridoma cells can be cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if parenteral cells are deficient in the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), hybridoma culture media typically include hypoxanthine, aminopterin, and thymidine (HAT media), which prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that fuse efficiently, maintain stable high levels of antibody expression by the selected antibody-producing cells, and are sensitive to media such as HAT media. More preferred immortalized cell lines are murine myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, California and American Type Culture Collection, Manassas, Virginia. Human myeloma cell lines and mouse-human heteromyeloma cell lines have also been described for the production of monoclonal antibodies (see Kozbor, J. Immunol., 133:3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker , Inc., New York, (1987) pp. 51-63)).

The culture medium in which the hybridoma cells are cultured can then be tested for the presence of monoclonal antibodies directed to the antigen.

Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or an in vitro binding test such as a radioimmunoassay (RIA) or an enzyme-linked immunoabsorbent assay (ELISA). Such methods and tests are known in the art. The binding affinity of a monoclonal antibody can, for example, be determined by the assay of Munson and Pollard, Anal. Biochem., 107:220 (1980). In addition, in those

- 30 043790 therapeutic applications of monoclonal antibodies, it is important to identify antibodies that have a high degree of specificity and high binding affinity to the target antigen.

Once the desired hybridoma cells have been identified, clones can be subcloned by limiting dilution procedures and grown by standard methods (see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103). Suitable culture media include, for example, Dalbecco's modified Eagle's medium and RPMI-1640 medium. Hybridoma cells can also be grown in vivo as ascites in a mammal.

Monoclonal antibodies secreted by subclones can be isolated or purified from culture medium or ascites fluid by standard immunoglobulin purification procedures, such as, for example, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in US Pat. No. 4,816,567. DNA encoding the monoclonal antibodies disclosed herein can be readily isolated and sequenced using standard procedures (eg, using oligonucleotide probes that are capable of specifically binding to the genetic code of the heavy and light chains of murine antibodies). The hybridoma cells disclosed herein provide a preferred source of such DNA. Once isolated, the DNA can be placed into an expression vector, then transfected into host cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or otherwise non-immunoglobulin protein-producing myeloma cells, to enable the synthesis of monoclonal antibodies in recombinant host cells. The DNA may also be modified, for example, by substituting human sequences encoding the heavy and light chain constant regions for homologous murine sequences (see U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently joining a sequence encoding an immunoglobulin, all or part of a sequence encoding a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide can replace the constant regions of an antibody disclosed herein, or the variable regions of one antigen combining region of an antibody disclosed herein can be replaced to create a chimeric divalent antibody.

Monoclonal antibodies disclosed herein include fully human antibodies or humanized antibodies. These antibodies are suitable for administration to humans without causing an immune response in the individual against the administered immunoglobulin.

Humanized or fully human NaPi2b antibody is prepared, for example, using the procedures described in the Examples below.

In other alternative methods, the NaPi2b antibody is developed, for example, using bacteriophage display methods using antibodies containing only human sequences. Such approaches are known in the art, for example, as described in WO92/01047 and US Patent No. 6,521,404, which are incorporated herein by reference. In this approach, a combinatorial library of bacteriophage carrying random pairs of light and heavy chains is screened using a natural or recombinant source of NaPi2b. In another approach, a NaPi2b antibody can be produced by a method in which at least one process step involves immunizing a transgenic non-human animal with a human NaPi2b protein. In this approach, certain endogenous heavy and/or kappa light chain loci of this xenogeneic non-human animal were disabled and unable to rearrange required to generate genes encoding immunoglobulins in response to antigen. In addition, at least one human heavy chain locus and at least one human light chain locus were stably transfected into the animal. Thus, in response to an introduced antigen, human loci undergo rearrangement to form genes encoding human variable regions that are immunospecific for the antigen. Following immunization, therefore, XenoMouse produces B cells that secrete fully human immunoglobulins.

There are many methods known in the art to produce xenogeneic non-human animals. For example, see US Pat. No. 6,075,181 and US Pat. No. 6,150,584, which are incorporated herein by reference in their entirety. This general strategy was demonstrated in connection with the generation of the first XenoMouse™ strains, as published in 1994; see Green et al., Nature Genetics 7:13-21 (1994), which is incorporated herein by reference in its entirety, see also US Patent Nos. 6,162,963, 6,150,584, 6, 114,598, 6,075,181 and 5,939,598 and Japanese Patent Nos. 3,068,180 B2, 3 068 506 B2 and 3 068 507 B2 and European patent EP 0 4 63 151 B1, and International patent applications WO 94/02602, WO 96/34096, WO 98/24893, WO 00/76310, and members of these families .

In a complementary approach, others have used a minilocus approach, in which the exogenous Ig locus is mimicked by incorporating parts (individual genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a mu constant region, and a second constant region (preferably a gamma constant region) are formed into a construct for insertion into an animal, see, for example, US patents 5,545,806; 5,545,807; 5,591,669; 5,612,205; 5,625,825; 5,625,126; 5,633,425; 5,643,763; 5,661,016; 5,721,367; 5,770,429; 5,789,215;

- 31 043790

5,789,650; 5,814,318; 5.877; 397; 5,874,299; 6,023,010; and 6,255,458; and European Patent No. 0 54 6 073 B1; and International patent applications WO 92/03918, WO 92/22645, WO 92/22647, WO 92/22670, WO 93/12227, WO 94/00569, WO 94/25585, WO 96/14436, WO 97/13852 and WO 98/24884, and members of these families.

The generation of human antibodies from mice to which, by microfusion of cells, large portions of chromosomes or all chromosomes have been introduced has also been demonstrated; see European Patent Applications Nos. 773288 and 843961.

Human anti-mouse antibody (HAMA) responses have led industry to produce chimeric or otherwise humanized antibodies. While chimeric antibodies have a human constant region and an immune variable region, some human anti-chimeric antibody (HACA) responses are expected to be observed, especially with chronic or multi-dose use of the antibodies. Thus, it would be desirable to obtain fully human antibodies to NaPi2b to circumvent or mitigate the problems and/or effects of the HAMA or NASA responses.

The production of antibodies with reduced immunogenicity is also achieved through humanization, chimerization and display techniques using suitable libraries. It should be understood that murine antibodies or antibodies from other species can be humanized or primatized using methods known in the art, see, for example, Winter and Harris Immunol Today 14:43 46 (1993) and Wright et al. Crit, Reviews in Immunol. 12125-168 (1992). An antibody of interest can be produced by recombinant DNA techniques to replace the CH1, CH2, CH3, hinge and/or frame regions with the corresponding human sequence (see WO 92102190 and US Patents 5,530,101; 5,585,089; 5,693,761; 5,693,792; 5,714,350; and 5,777,085 ). In addition, the use of Ig cDNA to construct hybrid immunoglobulin genes is known in the art (Liu et al., P.N.A.S. 84:3439 (1987) and J. Immunol. 139:3521 (1987)). The mRNA is isolated from the hybridoma or other antibody-producing cell and used to make cDNA. The cDNA of interest can be amplified by polymerase chain reaction using specific primers (US Patent Nos. 4,683,195 and 4,683,202). The library is also prepared and screened to isolate the sequence of interest. The DNA sequence encoding the antibody variable region is then fused to human constant region sequences.

Sequences of human constant region genes can be found in Rabat et al. (1991) Sequences of Proteins of immunological Interest, N.I.H. Publication No. 91-3242. Human C region genes are readily available from known clones. The choice of isotype will be dictated by the desired effector functions, such as complement fixation or activity against antibody-dependent cellular cytotoxicity. The preferred isotypes are IgG1, IgG3 and IgG4. Any of the human light chain constant regions, kappa or lambda, can be used. The chimeric humanized antibody is then expressed using standard methods.

Further, human antibodies or antibodies from other species can be generated through display technologies, including, without limitation, bacteriophage display, retroviral display, ribosomal display, and other methods, using methods known in the art, and the resulting molecules can be subjected to additional maturation methods, such as affinity maturation, as known in the art. Wright et al. Crit, Reviews in Immunol. 12125-168 (1992), Hanes and Pluckthun PNAS USA 94:4937-4942 (1997) (ribosomal display), Parmley and Smith Gene 73:305-318 (1988) (phage display), Scott, TIBS, vol. 17:241-245 (1992), Cwirla et al. PNAS USA 87:6378-6382 (1990), Russel et al., Nucl. Acids Research 21:1081-1085 (1993), Hoganboom et al., Immunol. Reviews 130:43-68 (1992), Chiswell and McCafferty TIBTECH; 10:80-8A (1992), and US Patent No. 5,733,743. If display technologies are used to produce antibodies that are not human, such antibodies can be humanized as described above.

Using these methods, antibodies to NaPi2b-expressing cells, soluble forms of NaPi2b, epitopes or peptides thereof, and expression libraries thereof can be generated (see, for example, US Pat. No. 5,703,057), which can then be screened as described above. in relation to the activities described here.

The NaPi2b antibodies disclosed herein can be expressed by a vector containing a DNA segment encoding the single chain antibody described above.

These may include vectors, liposomes, naked DNA, adjuvanted DNA, gene gun, catheters, etc. Vectors include chemical conjugates, such as those described in WO93/64701, which have a targeting moiety (eg, a cell surface receptor ligand) and a nucleic acid binding moiety (eg, polylysine), a viral vector (eg, a DNA or RNA viral vector) fusion proteins, such as those described in PCT/US 95/02140 (WO95/22618), which is a fusion protein containing a targeting group (eg, target cell specific antibody) and a nucleic acid binding group (eg, protamine ), plasmids, phage, etc. Vectors can be chromosomal, non-chromosomal or synthetic.

Preferred vectors include viral vectors, fusion proteins and chemical conjugates. Retroviral vectors include Moloughney murine leukemia viruses. DNA-containing viral vectos

- 32 043790 ry are preferred. These vectors include poxvirus vectors such as orthopoxyl avipoxvirus vectors, herpes virus vectors such as herpes simplex virus I (HSV) vector (see Geller, A. I. et al., J. Neurochem, 64:487 (1995) ; Lim, F., et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed (Oxford Univ. Press, Oxford England) (1995); Geller, A. I. et al., Proc Natl. Acad. Sci.: U.S.A. 90:7603 (1993); Geller, A. I., et al., Proc Natl. Acad. Sci USA 87:1149 (1990), adenoviral vectors (see LeGal LaSalle et al., Science, 259:988 (1993); Davidson, et al., Nat. Genet 3:219 (1993); Yang, et al., J. Virol. 69:2004 (1995) and adeno-associated viral vectors (see Kaplitt, M. G. et al., Nat. Genet. 8:148 (1994).

Poxviral vectors introduce the gene into the cytoplasm of cells, while avipoxviral vectors lead only to short-term expression of the nucleic acid. Adenoviral vectors, adeno-associated viral vectors, and herpes simplex virus (HSV) vectors are preferred for introducing nucleic acid into nerve cells. The adenovirus vector results in shorter-term expression (approximately 2 months) than adeno-associated virus (approximately 4 months), which in turn is shorter than that of HSV vectors. The specific choice of vector will depend on the target cell and the condition being treated. Administration can be accomplished by standard methods, for example, infection, transfection, transduction or transformation. Examples of gene transfer methods include, for example, naked DNA, CaPO ₄ precipitation, DEAE dextran, electroporation, protoplast fusion, lipofection, cell microinjection, and viral vectors.

The vector can be used to target essentially any desired target cell. For example, stereotactic injection can be used to target vectors (eg, adenovirus, HSV) to the desired location. In addition, particles can be delivered by intracerebroventricular (icv) infusion using a minipump infusion system such as the SynchroMed Infusion System. A volume flow-based method called convection has also been effective in delivering large molecules to extended areas of the brain and can be used in delivering a vector to a target cell (see Bobo et al., Proc. Natl. Acad. Sci. USA 91 :2076-2080 (1994); Morrison et al., Am. J. Physiol. 266:292-305 (1994)). Other methods that may be used include catheters, intravenous, parenteral, intraperitoneal and subcutaneous injection and oral or other known routes of administration.

These vectors can be used to express large quantities of antibodies, which can be used in a variety of ways. For example, to detect the presence of NaPi2b in a sample. The antibody can also be used to attempt to bind to and disrupt the NaPi2b signaling pathway.

NaPi2b-targeting antibody conjugates.

The disclosure also relates to immunoconjugates comprising an antibody conjugated to a cytotoxic agent, such as a toxin (eg, an enzymatically active toxin of bacterial, fungal, plant or animal origin or fragments thereof) or a radioactive isotope (ie, a radioconjugate).

Enzymatically active toxins and fragments thereof that may be used include diphtheria A chain, non-binding active diphtheria toxin fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modecin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacca americana proteins (PAPI, PAPII and PAPS), Momordica charantia inhibitor, curcin, crotin, Saponaria officinalis inhibitor, gelonin, mitogellin, restrictocin, fenomycin, enomycin and trichothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re.

Antibody-cytotoxic agent conjugates are prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional imiddoester derivatives (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)-hexanediamine), bisdiazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as tolylene-2,6diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, ricin antitoxin can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for radionucleotide-antibody conjugation (see WO94/11026).

One of ordinary skill in the art will appreciate that a wide variety of possible moieties can be coupled to the resulting antibodies disclosed herein (see, for example, Conjugate Vaccines, Contributions to Microbiology and Immunology, J. M. Cruse and R. E. Lewis, Jr (eds), Carger Press, New York, (1989), the entire contents of which are incorporated herein by reference).

The coupling can be accomplished by any chemical reaction that links two molecules while the antibody and the other group retain their respective activities. This binding may involve a variety of chemical mechanisms, such as covalent binding, affinity binding, intercalation, coordinate binding and complexation. Preferred connections

- 33 043790 However, covalent bonding is important. Covalent bonding can be accomplished either by direct condensation of existing side chains or by the incorporation of external molecules that provide bridging. A variety of di- or multivalent coupling agents can be used in linking protein molecules, such as antibodies of the present disclosure, with other molecules. For example, representative coupling agents may include organic compounds such as thioesters, cyanamides, succinimide esters, diisocyanates, glutaraldehyde, diazobenzenes, and hexamethylenediamines. This list is not an exhaustive list of the various classes of binding agents known in the art, but is an example of more conventional binding agents (see Killen and Lindstrom, Jour. Immun. 133:1335-2549 (1984); Jansen et al., Immunological Reviews 62:185-216 (1982); and Vitetta et al., Science 238:1098 (1987).

Preferred linkers are described in the literature (see, for example, Ramakrishnan, S. et al., Cancer Res. 44:201-208 (1984) which describes the use of MBS (M-maleimidobenzoyl-Nhydroxysuccinimide ester). See also US Pat. No. 5,030,719, which describes the use of a halogenated acetyl hydrazide derivative linked to an antibody via an oligopeptide linker. Particularly preferred linkers include: (i) EDC (1-ethyl-3-(3-dimethylaminopropyl)cyanamide hydrochloride; (ii) SMPT (4-succinimidyloxycarbonyloxy- alpha-methyl-alpha-(2pyridyldithio)-toluene (Pierce Chem. Co., Cat (21558G); (iii) SPDP (succinimidyl-6[3-(2pyridyldithio)propionamido]hexanoate (Pierce Chem. Co., Cat .# 21651G); (iv) sulfo-LC-SPDP (sulfosuccinimidyl-6[3-(2-pyridyldithio)-propionamide]hexanoate (Pierce Chem. Co. Cat. # 2165-G); and (v) sulfo-NHS (N-Hydroxysulfo-succinimide: Pierce Chem. Co., Cat.# 24510) conjugated with EDC.

The linkers described above contain components having different characteristics, thus leading to conjugates with different physicochemical properties. For example, sulfo-NHS esters of alkyl carboxylates are more stable than sulfo-NHS esters of aromatic carboxylates. NHS ester-containing linkers are less soluble than sulfo-NHS esters. Further, the SMPT linker contains a hindered disulfide bond and can form conjugates with increased stability. Disulfide bonds are generally less stable than other bonds because the disulfide bond is cleaved in vitro, resulting in a less accessible conjugate. Sulfo-NHS in particular can improve the stability of carbodiimide bonds. Carbodiimide linkages (such as EDC), when used in combination with sulfo-NHS, form esters that are more resistant to hydrolysis than the carbodiimide coupling reaction alone.

In one aspect, the conjugate described herein includes an isolated NaPi2b-targeting antibody that specifically binds to the extracellular region of SLC34A2 linked directly or indirectly to one or more D-bearing polymer backbones independently comprising poly(1hydroxymethylethylene hydroxymethyl formal) (PHF), having a molecular weight ranging from about 2 kDa to about 40 kDa, each one or more D-supporting polymer backbones independently having formula (Ic):

(1c) wherein each D is independently a therapeutic or diagnostic agent;

L ^D1 is a carbonyl-containing group;

each ^{ct 0)1} in ^C < ^=O > ^L 4-d independently represents a first linker containing a biodegradable bond such that when the bond is broken, D is released in an active form for its intended therapeutic effect; and in ^C <= ^O > ^L between L ^D1 and D denotes the direct or indirect attachment of D to L ^D1 ;

each <''' independently represents a second linker not yet associated with the isolated antibody NaPi2b, in which LP ² is a group containing a functional group that should form a covalent bond with the functional group of the isolated antibody, and I between L ^D1 and L ^P2 denotes direct or indirect attachment of L ^P2 to L ^D1 , and every second linker is different from every first linker;

each < ? independently represents a third linker connecting each D-bearing polymer backbone to the isolated antibody, in which the terminal ^_ ^ attached to L ^P2 denotes direct or indirect attachment of L ^P2 to the isolated antibody after the formation of a covalent bond between the functional group of L ^P2 and the functional group of the isolated antibody ; and every third linker is different from every first linker;

m is an integer from 1 to approximately 300, m1 is an integer from 1 to approximately

- 34 043790 140, m2 is an integer from 1 to about 40, _m3 is an integer from 0 to about 18, m4 is an integer from 1 to about 10; the number of m, m1, m2, _m3 and m4 is from about 15 to about 300; and the total number of L ^P 2 attached to the isolated antibody is 10 or less.

The conjugate may include one or more of the following features.

For example, in formula (Ic), m1 is an integer from 1 to about 120 (eg, about 1-90), and/or m ₃ is an integer from 1 to about 10 (eg, about 1-8).

For example, when the PHF in formula (Ic) has a molecular weight ranging from about 6 kDa to about 20 kDa (i.e., the number of m, m1, _m2 , _m3 , and _m4 is in the range from about 45 to about 150 ), m2 is an integer from 2 to about 20, m3 is an integer from 0 to about 9, m4 is an integer from 1 to about 10, and/or m1 is an integer from 1 to about 75 (for example, m1 is about 4-45).

For example, when the PHF in formula (Ic) has a molecular weight ranging from about 8 kDa to about 15 kDa (i.e., the number of m, m1, _m2 , _m3 , and _m4 is in the range from about 60 to about 110 ), m ₂ is an integer from 2 to about 15, m ₃ is an integer from 0 to about 7, m ₄ is an integer from 1 to about 10, and/or m1 is an integer from 1 to about 55 (for example, m1 is approximately 4-30).

For example, when the PHF in formula (Ic) has a molecular weight ranging from about 2 kDa to about 20 kDa (i.e., the number of m, m1, _m2 , _m3 , and _m4 is in the range from about 15 to about 150 ), m2 is an integer from 1 to about 20, _m3 is an integer from 0 to about 10 (for example, m3 is in the range from 0 to about 9), m4 is an integer from 1 to about 8, and/or m1 is an integer from 1 to about 70, and the total number of L ^P2 bound to the isolated antibody is from about 2 to about 8 (eg, about 2, 3, 4, 5, 6, 7 or 8).

For example, when the PHF in formula (Ic) has a molecular weight ranging from about 3 kDa to about 15 kDa (i.e., the number of m, m1, m2, _m3 , and m4 is in the range from about 20 to about 110), m ₂ is an integer from 2 to about 15, m ₃ is an integer from 0 to about 8 (for example, m ₃ is in the range from 0 to about 7), m ₄ is an integer from 1 to about 8, and/or m1 is an integer from 2 to about 50, and the total number of L ^P2 associated with the isolated antibody is from about 2 to about 8 (eg, about 2, 3, 4, 5, 6, 7 or 8).

For example, when the PHF in formula (Ic) has a molecular weight ranging from about 5 kDa to about 10 kDa, (ie, the number of m, m1, _m2 , _m3 and m4 is from about 40 to about 75), m2 is an integer from about 2 to about 10 (for example, m2 is about 3-10), _m3 is an integer from 0 to about 5 (for example, _m3 is in the range from 0 to about 4), m4 is an integer from 1 to about 8 (for example, m ₄ is in the range from 1 to about 5), and/or m1 is an integer from about 2 to about 35 (for example, m 1 is about 5-35), and the total number of L ^P2 associated with isolated antibody, is from about 2 to about 8 (for example, about 2, 3, 4, 5, 6, 7 or 8).

For example, when PHF has a molecular weight ranging from 2 kDa to 40 kDa, (for example, about 6-20 kDa or about 8-15 kDa, about 2-20 kDa or about 3-15 kDa, or about 5-10 kDa) The number of drugs per PHF (eg, m2) is an integer from 1 to about 40 (eg, about 1-20 or about 2-15, or about 3-10, or about 2-10). These backbones can be used, for example, to conjugate an isolated antibody having a molecular weight of 40 kDa or more (e.g., 60 kDa or more; 80 kDa or more; 100 kDa or more; 120 kDa or more; 140 kDa or more; 160 kDa or more more than, 180 kDa or more, or 200 kDa or more, or approximately 40-200 kDa, 40-180 kDa, 40-140 kDa, 60-200 kDa, 60-180 kDa, 60-140 kDa, 80-200 kDa, 80-180 kDa, 80-140 kDa, 100-200 kDa, 100-180 kDa, 100-140 kDa or 140-150 kDa). In this embodiment, the ratio of isolated anti-PHF antibody is from about 1:1 to about 1:10, from about 1:1 to about 1:9, from about 1:1 to about 1:8, from about 1:1 to about 1:7, from about 1:1 to about 1:6, from about 1:1 to about 1:5, from about 1:1 to about 1:4, from about 1:1 to about 1:3, from about 1:1 to about 1:2, about 1:2 to about 1:4, about 1:2 to about 1:3, about 1:3 to about 1:4, or about 1:3 to about 1:5.

For example, when PHF has a molecular weight ranging from 2 kDa to 40 kDa, (for example, about 6-20 kDa or about 8-15 kDa, about 2-20 kDa or about 3-15 kDa, or about 5-10 kDa) The number of drugs per PHF (eg, m2) is an integer from 1 to about 40 (eg, about 1-20 or about 2-15, or about 3-10, or about 2-10). These scaffolds can be used, for example, for conjugation

- 35 043790 tion of an isolated antibody having a molecular weight from 140 kDa to 180 kDa or from 140 kDa to 150 kDa. In this embodiment, the ratio of isolated anti-PHF antibody is from about 1:1 to about 1:10, from about 1:1 to about 1:9, from about 1:1 to about 1:8, from about 1:1 to about 1:7, from about 1:1 to about 1:6, from about 1:1 to about 1:5, from about 1:1 to about 1:4, from about 1:1 to about 1:3, from about 1:1 to about 1:2, about 1:2 to about 1:4, about 1:2 to about 1:3, about 1:3 to about 1:4, or about 1:3 to about 1:5.

NaPi-2b antibodies in this molecular weight range include, but are not limited to, for example, complete antibodies such as IgG, IgM.

For example, when PHF has a molecular weight ranging from 2 kDa to 40 kDa, the number of drugs per PHF (eg, m ₂ ) is an integer from 1 to about 40 (eg, about 1-20 or about 2-15, or about 3-10, or approximately 2-10). These backbones can be used, for example, to conjugate an isolated antibody having a molecular weight of 60 kDa to 120 kDa. In this embodiment, the ratio of isolated anti-PHF antibody is from about 1:1 to about 1:10, from about 1:1 to about 1:9, from about 1:1 to about 1:8, from about 1:1 to about 1:7, from about 1:1 to about 1:6, from about 1:1 to about 1:5, from about 1:1 to about 1:4, from about 1:1 to about 1:3, from about 1:1 to about 1:2, about 1:2 to about 1:4, about 1:2 to about 1:3, about 1:3 to about 1:4, or about 1:3 to about 1:5.

In some embodiments, D is a therapeutic agent. In some embodiments, the therapeutic agent is a small molecule having a molecular weight of ^about 5 kDa, ^about 4 kDa, ^about 3 kDa, ^about 1.5 kDa, or < about 1 kDa.

In some embodiments, the therapeutic agent has an IC ₅₀ of less than approximately 1 nM.

In another embodiment, the therapeutic agent has an IC ₅₀ of greater than about 1 nM, for example, the therapeutic agent has an IC ₅₀ of from about 1 to 50 nM.

Some therapeutics having an IC ₅₀ greater than about 1 nM (eg, less potent drugs) are unsuitable for conjugation to an antibody using conjugation techniques known in the art. Without being bound by theory, such therapeutics have a potential that is not sufficient for use in targeted antibody-drug conjugates using standard methods because a sufficient number of copies of the drug (i.e., greater than 8) cannot be conjugated using methods known in the art. techniques of methods without deteriorating the pharmacokinetic and physicochemical properties of the conjugate. However, sufficiently high loadings of these less potent drugs can be achieved using the conjugation strategies described here, thereby resulting in high therapeutic loadings while maintaining the desired pharmacokinetic and physicochemical properties. Thus, the disclosure also relates to an antibody-polymer-drug conjugate that includes an isolated antibody, PHF, and at least eight therapeutic agent groups, wherein D is auristatin, dolastatin (e.g., dolastatin 10 or dolastatin 15), monomethyl auristatin E (MMAE) , monomethyl auristatin F (MMAF), auristatin F, AF HPA, monomethyl AF HPA, phenylenediamine (AFP).

For example, duocarmycin or analogues thereof are duocarmycin A, duocarmycin B1, duocarmycin B2, duocarmycin C1, duocarmycin C2, duocarmycin D, duocarmycin SA, CC-1065, adozelesin, bizelesin or carzelesin.

Other examples of D include those described, for example, in US Pat. No. 8,815,226; and US Patent Application Publication No. 2015-0104407; the contents of each of which are incorporated herein in their entirety.

In some embodiments, the number of D-bearing polymer backbones that can be conjugated to an antibody is limited by the number of free cysteine residues. In some embodiments, free cysteine residues are introduced into the amino acid sequence of the antibody by methods described herein. Examples of conjugates disclosed herein may include antibodies having 1, 2, 3 or 4 cysteine amino acids (Lyon, R. et al. (2012) Methods in Enzym. 502:123-138). In some embodiments, one or more free cysteine residues are already present in the antibody, without the use of engineering, where existing free cysteine residues can be used to conjugate the antibody to the D-supporting polymer backbone. In some embodiments, the antibody is subjected to reducing conditions prior to antibody conjugation to generate one or more free cysteine residues.

- 36 043790

In some embodiments, the functional group L ^P2 that is to form a covalent bond with the functional group of the isolated antibody (such as a functional group or reactive group on an amino acid residue of the antibody, e.g., a functional group on a cysteine residue or a lysine residue of the antibody) is selected from -SR ^p , -SS-LG, ^Xt b and halogen, wherein LG is a leaving group, R ^p is H or a sulfur protecting group, and one of Xa and Xb is H and the other is a water-soluble maleimido protecting group, or Xa and Xb, together with the carbon atoms to which they are attached, form a carbon-carbon double bond. For example, the functional group L ^P2 that is to form a covalent bond is a functional group that does not react with the functional group of the isolated antibody, for example, ° as the functional group L ^P2 in which one of Xa and X _b denotes H and the other denotes a water-soluble protective group for the maleimido group, or X _a and X _b . In some embodiments, in the conjugate described herein, the D-supporting polymer backbone of formula (Ic) has formula (Ie):

wherein, PHF has a molecular weight ranging from about 2 kDa to about 40 kDa;

each D is independently a therapeutic agent having a molecular weight of <5 kDa, and “I between D and a carbonyl group represents direct or indirect attachment of D to a carbonyl group, X represents CH ₂ , O or NH;

one of X _a and X _b is H and the other is a water-soluble protecting group for the maleimido group, or X _a and X _b , together with the carbon atoms to which they are attached, form a carbon-carbon double bond, m1 is an integer from 1 to about 140, m ₇ is an integer from 1 to about 40, and the number of m1 and m ₇ is from about 2 to about 180, m is an integer from 1 to about 300, m _3a is an integer from 0 to about 17, m _3b is an integer from 1 to about 8, and the number of m _3a and m _3b is from 1 to 18, and the number of m, m1, m ₇ , m _3a and m _3b is from about 15 to about 300.

In some embodiments, in the conjugate described herein, the D-supporting polymer backbone of formula (Ie) has formula (Id):

wherein one of Xa and Xb is H and the other is a water-soluble protecting group for a maleimido group, or _Xa and _Xb , together with the carbon atoms to which they are attached, form a carbon-carbon double bond;

m _3a is an integer from 0 to about 17, m _3b is an integer from 1 to about 8, and the number of m _3a and m _3b is from 1 to 18, and the number of m, m1, m ₂ , m _3a and m _3b is from about 15 to about 300.

For example, the ratio between m ₂ and m _3b is greater than 1:1 and less than or equal to 10:1.

For example, the ratio between m ₂ and m _3b is approximately 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1 or 2:1.

For example, the ratio between m ₂ and m _3b is from 2:1 to 8:1.

For example, the ratio between m ₂ and m _3b is approximately 8:1, 7:1, 6:1, 5:1, 4:1, 3:1 or 2:1.

For example, the ratio between m ₂ and m _3b is from 2:1 to 4:1.

For example, the ratio between m ₂ and m _3b is approximately 4:1, 3:1 or 2:1.

For example, the ratio between m ₂ and m _3b is approximately 3:1 and 5:1.

For example, the ratio between m ₂ and m _3b is approximately 3:1, 4:1 or 5:1.

For example, when the PHF in formula (Id) has a molecular weight ranging from about 2 kDa to about 20 kDa, the number of m, m1, _m2 , _m3a and _m3b is from about 15 to about 150, m1 is an integer from 1 to approximately 70, m ₂ is an integer from 1 to

- 37 043790 about 20, m _3a is an integer from 0 to about 9, m _3b is an integer from 1 to about 8, and the ratio between PHF and the isolated NaPi2b-targeting antibody is an integer from 2 to about 8.

For example, when the PHF in formula (Id) has a molecular weight ranging from about 3 kDa to about 15 kDa, the number of m, m1, _m2 , _m3a and _m3b is from about 20 to about 110, m1 is an integer from 2 to about 50, m2 is an integer from 2 to about 15, _m3a is an integer from 0 to about 7, _m3b is an integer from 1 to about 8, and the ratio between PHF and the isolated NaPi2b-targeting antibody is an integer from 2 to about 8 (eg, an integer from 2 to about 6 or an integer from 2 to about 4).

For example, when the PHF in formula (Id) has a molecular weight ranging from about 5 kDa to about 10 kDa, the number of m, m _b m ₂ , m _3a and m _3b is from about 40 to about 75, m1 is an integer from about 2 to about 35, m ₂ is an integer from about 2 to about 10, m3a is an integer from 0 to about 4, m _3b is an integer from 1 to about 5, and the ratio between PHF and the isolated NaPi2b targeting antibody is an integer from 2 to about 8 (eg, an integer from 2 to about 6 or an integer from 2 to about 4).

For example, water-soluble maleimido protecting groups are groups that can be covalently attached to one of the two carbon atoms of the olefin after reaction of the maleimido group with a thiol-containing compound of formula (II):

R90-(CH2)d-SH (II) in which R90 is NHR91, OH, COOR93, CH(NHR ₉₁ )COOR ₉₃ or a substituted phenyl group;

R ₉₃ is hydrogen or C1.4 alkyl;

R91 represents hydrogen, _CH3 or _CH3CO and d is an integer from 1 to 3.

For example, the water-soluble protecting group for the maleimido compound of formula (II) may be cysteine, N-acetyl cysteine, cysteine methyl ester, N-methyl cysteine, 2-mercaptoethanol, 3-mercaptopropanoic acid, 2-mercaptoacetic acid, mercaptomethanol (i.e., HOCH ₂ SH), a benzyl thiol in which the phenyl is substituted with one or more hydrophilic substituents, or 3-aminopropane-1-thiol. One or more hydrophilic substituents on phenyl include OH, SH, methoxy, ethoxy, COOH, CHO, COC1.4 alkyl, NH ₂ , F, cyano, SO ₃ H, PO ₃ H, and the like.

For example, a water-soluble protecting group for maleimido is -S-(CH ₂ )dR ₉₀ in which R ₉₀ is OH, COOH or CH(NHR ₉₁ )COOR ₉₃ ;

R ₉₃ is hydrogen or CH ₃ ;

R91 stands for hydrogen or _CH3CO and d is 1 or 2.

For example, a water-soluble protecting group for the maleimido group is -S-CH ₂ -CH(NH ₂ )COOH.

For example, when the PHF has a molecular weight ranging from 2 kDa to 40 kDa, (for example, about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa), the number of drugs per PHF (for example, m ₂ ) is an integer from 1 to about 40 (for example, about 1-20 or about 2-15, or about 3-10, or about 2-10). These backbones can be used, for example, to conjugate an isolated antibody having a molecular weight of 40 kDa or more (e.g., 60 kDa or more; 80 kDa or more; or 100 kDa or more; 120 kDa or more; 140 kDa or more; 160 kDa or more, 180 kDa or more, or 200 kDa or more, or about 40-200 kDa, 40-180 kDa, 40-140 kDa, 60-200 kDa, 60-180 kDa, 60-140 kDa, 80-200 kDa , 80180 kDa, 80-140 kDa, 100-200 kDa, 100-180 kDa, 100-140 kDa or 140-150 kDa). In this embodiment, the ratio of isolated anti-PHF antibody is from about 1:1 to about 1:10, from about 1:1 to about 1:9, from about 1:1 to about 1:8, from about 1:1 to about 1:7, from about 1:1 to about 1:6, from about 1:1 to about 1:5, from about 1:1 to about 1:4, from about 1:1 to about 1:3, from about 1:1 to about 1:2, about 1:2 to about 1:8, about 1:2 to about 1:6, about 1:2 to about 1:5, about 1:2 to about 1 :4, from about 1:2 to about 1:3, from about 1:3 to about 1:4, or from about 1:3 to about 1:5.

For example, when the PHF has a molecular weight ranging from 2 kDa to 40 kDa, (for example, about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa), the number of drugs per PHF (for example, m2) is an integer from 1 to about 40 (for example, about 1-20 or about 2-15, or about 3-10, or about 2-10). These backbones can be used, for example, to conjugate an isolated antibody having a molecular weight of 140 kDa to 180 kDa or 140 kDa to 150 kDa. In this embodiment, the relation

- 38 043790 isolated anti-PHF antibody is from about 1:1 to about 1:10, from about 1:1 to about 1:9, from about 1:1 to about 1:8, from about 1:1 to about 1: 7, from about 1:1 to about 1:6, from about 1:1 to about 1:5, from about 1:1 to about 1:4, from about 1:1 to about 1:3, from about 1: 1 to about 1:2, about 1:2 to about 1:8, about 1:2 to about 1:6, about 1:2 to about 1:5, about 1:2 to about 1:4 , from about 1:2 to about 1:3, from about 1:3 to about 1:4, or from about 1:3 to about 1:5.

Isolated antibodies in this molecular weight range include, but are not limited to, for example, complete antibodies such as IgG, IgM.

For example, when PHF has a molecular weight ranging from 2 kDa to 40 kDa (for example, about 2-20 kDa or about 3-15 kDa, or about 5-10 kDa), the number of drugs per PHF (for example, m ₂ ) is an integer from 1 to about 40, (for example, about 1-20 or about 2-15, or about 3-10 or 2-10). These backbones can be used, for example, to conjugate an isolated antibody having a molecular weight of 60 kDa to 120 kDa. In this embodiment, the ratio of isolated anti-PHF antibody is from about 1:1 to about 1:10, from about 1:1 to about 1:9, from about 1:1 to about 1:8, from about 1:1 to about 1:7, from about 1:1 to about 1:6, from about 1:1 to about 1:5, from about 1:1 to about 1:4, from about 1:1 to about 1:3, from about 1:1 to about 1:2, about 1:2 to about 1:8, about 1:2 to about 1:6, about 1:2 to about 1:5, about 1:2 to about 1 :4, from about 1:2 to about 1:3, from about 1:3 to about 1:4, or from about 1:3 to about 1:5.

For example, when the PHF has a molecular weight ranging from 2 kDa to 40 kDa, (for example, about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa), the number of drugs per PHF (for example, m ₂ ) is an integer from 1 to about 40 (for example, about 1-20 or about 2-15, or about 3-10 or 2-10). These backbones can be used, for example, to conjugate an isolated antibody having a molecular weight of 40 kDa to 80 kDa. In this embodiment, the ratio of isolated anti-PHF antibody is from about 1:1 to about 1:10, from about 1:1 to about 1:9, from about 1:1 to about 1:8, from about 1:1 to about 1:7, from about 1:1 to about 1:6, from about 1:1 to about 1:5, from about 1:1 to about 1:4, from about 1:1 to about 1:3, from about 1:1 to about 1:2, about 1:2 to about 1:8, about 1:2 to about 1:6, about 1:2 to about 1:5, about 1:2 to about 1 :4, from about 1:2 to about 1:3, from about 1:3 to about 1:4, or from about 1:3 to about 1:5.

In some embodiments, in the conjugate described herein, the D-supporting polymer backbone of formula (Ie) has formula (If), wherein the polymer is PHF having a molecular weight ranging from about 2 kDa to about 40 kDa:

wherein m is an integer from 1 to about 300, m1 is an integer from 1 to about 140, m ₂ is an integer from 1 to about 40, m _3a is an integer from 0 to about 17, m _3b is an integer from 1 to approximately 8;

the number of m _3a and m _3b varies from 1 to about 18;

the number of m, m1, _m2 , _m3a and _m3b is from about 15 to about 300;

terminal denotes the attachment of one or more polymer backbones to an isolated antibody that specifically binds to the extracellular region of SLC34A2 comprising (i) a complementarity determining region heavy chain 1 (CDRH1) variable region comprising the amino acid sequence gytftgynih (seq id no: 5); the variable region of the complementarity determining region of the heavy chain 2 (CDRH2), including the amino acid sequence

- 39 043790 activity aiypgngdtsykqkfrg (seq id no: b); complementarity determining region heavy chain 3 (CDRH3) variable region comprising the amino acid sequence getaratfay (seq id no: 7); complementarity determining region light chain 1 (CDRL1) variable region comprising the amino acid sequence sasqdignfln (seq id no: 8); complementarity determining region light chain 2 (CDRL2) variable region comprising the amino acid sequence YTSSLYS (SEQ ID NO: 9); and complementarity determining region light chain 3 (CDRL3) variable region comprising the amino acid sequence QQYSKLPLT (seq ID NO: 10) or (ii) CDRH1 comprising the amino acid sequence GFSFSDFAMS (SEQ ID NO: 18); CDRH2 comprising the amino acid sequence ATIGRVAFHTYYPDSMKr (SEQ ID NO: 19); CDRH3, comprising the amino acid sequence arhrgfdvghfdf (SEQ id NO: 2 0); CDRL1 comprising the amino acid sequence RSSETLVHSSGNTYLE (SEQ ID NO: 21); CDRL2 comprising the amino acid sequence RVSNRFS (SEQ ID NO: 22); and CDRL3, including the amino acid sequence fqgsfnplt (seq id no: 2 3); and the ratio between PHF and antibody is 10 or less.

In some embodiments, the isolated NaPi2b-targeting antibody specifically binds to SLC34A2 and includes (i) CDRH1 comprising the amino acid GYTFTGYNIH (SEQ ID NO: 5); CDRH2, including the amino acid AIYPGNGDTSYKQKFRG (SEQ ID NO: 6); CDRH3 comprising the amino acid GETARATFAY (SEQ ID NO: 7); CDRL1, including amino acid

SASQDIGNFLN(SEQ ID NO: 8); CDRL2, including amino acid ytsslys (seq id NO: 9); and CDRL3, which includes the amino acid

QQYSKLPLT (seq id no: io) .

sequence sequence sequence sequence sequence sequence

In some embodiments, the isolated NaPi2b-targeting antibody specifically binds to SLC34A2 and includes CDRH1 comprising the amino acid sequence GFSFSDFAMS (SEQ ID NO: 18); CDRH2 comprising the amino acid sequence ATIGRVAFHTYYPDSMKr (SEQ ID NO: 19); CDRH3, comprising the amino acid sequence arhrgfdvghfdf (seq id NO: 2 0); CDRL1, including the amino acid sequence rssetlvhssgntyle (seq id NO: 21); CDRL2, including the amino acid sequence RVSNRFS (seq id NO: 22); and CDRL3, including the amino acid sequence

FQGSFNPLT(SEQ ID NO: 23).

Formula skeletons (If) may include one or more of the following features.

When the PHF in formula (If) has a molecular weight ranging from about 2 kDa to about 20 kDa, the number of m, m1, _m2 , _m3a and _m3b is from about 15 to about 150, m1 is an integer from 1 to about 70, m2 is an integer from 1 to about 20, m _3a is an integer from 0 to about 9, m _3b is an integer from 1 to about 8, the sum of m _3a and m _3b is from 1 to about 10, and the ratio between PHF and antibody is an integer from 2 to about 8.

When the PHF in formula (If) has a molecular weight ranging from about 3 kDa to about 15 kDa, the number of m, m1, _m2 , _m3a and _m3b is from about 20 to about 110, m1 is an integer from 2 to about 50, m2 is an integer from 2 to about 15, m _3a is an integer from 0 to about 7, m _3b is an integer from 1 to about 8, the sum of m _3a and m _3b is from 1 to about 8; and the ratio between PHF and antibody is an integer from 2 to about 8 (eg, from about 2 to about 6 or from about 2 to about 4).

When the PHF in formula (If) has a molecular weight ranging from about 5 kDa to about 10 kDa, the number of m, m1, _m2 , _m3a and _m3b is from about 40 to about 75, m1 is an integer from about 2 to about 35, m2 is an integer from about 2 to about 10, m _3a is an integer from 0 to about 4, m _3b is an integer from 1 to about 5, the sum of m _3a and m _3b is from 1 to about 5; and the ratio between PHF and antibody is an integer from 2 to about 8 (eg, from about 2 to about 6 or from about 2 to about 4).

In some embodiments, the ratio between auristatin F hydroxypropyl amide (AF HPA) and antibody may be approximately 30:1, 29:1, 28:1, 27:1, 26:1, 25:1, 24:1, 23:1 , 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10 :1, 9:1, 8:1, 7:1 or 6:1.

In some embodiments, the ratio between HPA AF and antibody may be approximately 25:1, 24:1, 23:1, 22:1, 21:1, 20:1, 19:1, 18:1, 17:1, 16 :1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1, 8:1, 7:1 or 6:1.

In other embodiments, the ratio between HPA AF and antibody may be approximately 20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11 :1, 10:1, 9:1, 8:1, 7:1 or 6:1.

In some embodiments, the ratio between HPA AF and antibody may be approximately 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, or 10:1.

In some embodiments, the ratio between AF and antibody may be at

- 40 043790 approximately 15:1, 14:1, 13:1, 12:1 or 11:1.

In some embodiments, the ratio between HPA AF and antibody may be approximately 15:1, 14:1, 13:1, or 12:1.

In some embodiments, the ratio between PHF and antibody may be approximately 10:1, 9:1, 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, 2:1, or 1: 1.

In some embodiments, the ratio between PHF and antibody may be approximately 8:1, 7:1, 6:1, 5:1, 4:1, 3:1, or 2:1.

In other embodiments, the ratio between PHF and antibody may be approximately 6:1, 5:1, 4:1, 3:1, 2:1, or 1:1.

In other embodiments, the ratio between PHF and antibody may be approximately 6:1, 5:1, 4:1, 3:1, or 2:1.

In other embodiments, the ratio between PHF and antibody may be approximately 6:1, 5:1, 4:1, or 3:1.

In some embodiments, the ratio between PHF and antibody may be approximately 5:1, 4:1, or 3:1.

In some embodiments, the ratio between PHF and antibody may be approximately 4:1, 3:1, or 2:1.

Other embodiments of antibody-polymer-drug conjugates are those described, for example, in US Pat. No. 8,815,226; and US Patent Application Publication No. 2015-0104407; the contents of each of which are incorporated herein in their entirety.

This disclosure also relates to a drug derivative modified so that it can be directly conjugated to an antibody in the absence of a polymeric carrier, and to corresponding antibody-drug conjugates.

In some embodiments, the NaPi2b-targeting antibody-drug conjugates include an antibody conjugated, i.e. covalently attached to a drug group. In some embodiments, the antibody is covalently attached to the drug moiety via a linker, such as a non-polymeric linker.

D (eg, drug moiety) of NaPi2b-targeted antibody-drug conjugates (ADC) may include any compound, moiety, or moiety having a cytotoxic or cytostatic effect as defined herein. In some embodiments, the NaPi2b-targeting antibody-drug conjugate (ADC) includes a NaPi2b-targeting antibody (Ab) targeting a tumor cell, a D (e.g., a drug moiety), and a linker group (L) that attaches the Ab to the D. In some embodiments, the antibody is attached to a linker group (L) via one or more amino acid residues, such as lysine and/or cysteine.

In some embodiments, the ADC has the formula (Ig):

Ab- (L-D)p (Ig) where p=1 to approximately 20.

In some embodiments, the number of drug moieties that can be conjugated to the antibody is limited by the number of free cysteine residues. In some embodiments, free cysteine residues are introduced into the amino acid sequence of the antibody by methods described herein. Examples of formula Ig ADCs include, but are not limited to, antibodies having 1, 2, 3 or 4 cysteine amino acids (Lyon, R. et al. (2012) Methods in Enzym. 502:123-138). In some embodiments, one or more free cysteine residues are already present in the antibody, without the use of engineering, where the existing free cysteine residues can be used to conjugate the antibody to the drug. In some embodiments, the antibody is subjected to reducing conditions prior to antibody conjugation to generate one or more free cysteine residues.

In some embodiments, the Linker (L) is a bifunctional or multifunctional group that can be used to connect one or more D (eg, drug groups) to an antibody (Ab) to form an antibody-drug conjugate (ADC) of formula Ig. In some embodiments, antibody-drug conjugates (ADCs) can be prepared using a linker having reactive functional groups for covalent attachment to the drug and antibody. For example, in some embodiments, a cysteine thiol of an antibody (Ab) may form a bond with a reactive functional group of a linker or drug-linker intermediate to create an ADC.

In one aspect, the linker has a functional group that is capable of reacting with free cysteine present on the antibody to create a covalent bond. Non-limiting examples of such reactive functional groups include maleimide, haloacetamides, αhaloacetyl, activated esters such as succinimide esters, 4-nitrophenyl esters, pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides,

- 41 043790 acid chlorides, sulfonyl chlorides, isocyanates and isothiocyanates. See, for example, the conjugation method on page 766 of Klussman, et al. (2004), Bioconjugate Chemistry 15 (4):765-773, and Examples therein.

In some embodiments, the linker has a functional group that is capable of reacting with an electrophilic group present on the antibody. Examples of such electrophilic groups include, but are not limited to, aldehyde and ketone carbonyl groups. In some embodiments, the heteroatom of the reactive functional group of the linker can react with an electrophilic group on the antibody and form a covalent bond with the antibody unit. Non-limiting examples of such reactive functional groups include, but are not limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone, hydrazine carboxylate and arylhydrazide.

The linker may include one or more linker components. Examples of linker components include 6-maleimidocaproyl (MC), maleimidopropanoyl (MP), valine-citrulline (val-cit or vc), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyloxy (PAB), N-succinimidyl-4(2 -pyridylthio)pentanoate (SPP) and 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (MCC). Various linker components are known in the art, some of which are described below.

The linker may be a cleavable linker to facilitate drug release. Non-limiting examples of cleavable linkers include acid-labile linkers (eg, including hydrazone), protease-sensitive (eg, peptidase-sensitive) linkers, photolabile linkers, or disulfide-containing linkers (Chari et al., Cancer Research 52:127-131 (1992 ; U.S. Pat. No. 5,208,020).

In some embodiments, the linker has the following formula (IIg):

^- A _a -W _w -Yy (IIg) in which A is an extension unit and a is an integer from 0 to 1;

W is the unit of amino acid, and w is an integer from 0 to 12;

Y is a spacer unit, and y is an integer of 0, 1 or 2. An ADC comprising a linker of formula (IIg) has the formula I(A): Ab-(Aa-Ww-Yy-D)p, in which Ab, D and p have the meanings defined above for formula (Ig). Examples of such linkers are described in US Pat. No. 7,498,298, which is incorporated herein by reference in its entirety.

In some embodiments, the linker component includes an extension unit (A) that links the antibody to another linker component or to a drug moiety. Non-limiting examples of extension units are shown below (in which the wavy line indicates the sites of covalent attachment to the antibody, drug, or additional linker components):

In some embodiments, the linker component includes an amino acid (W) unit. In some such embodiments, the amino acid unit allows cleavage of the linker by a protease, thereby facilitating release of the drug from the immunoconjugate after exposure to intracellular proteases such as lysosomal enzymes (Doronina et al. (2003) Nat. Biotechnol. 21:778-784). Examples of amino acid units include, but are not limited to, dipeptides, tripeptides, tetrapeptides and pentapeptides. Examples of dipeptides include, but are not limited to, valine-citrulline (vc or val-cit), alanine-phenylalanine (AF or ala-phe); phenylalanine-lysine (fk or phe-lys); phenylalanine-homolysine (phe-homolys); and N-methyl-valine-citrulline (Me-val-cit). Examples of tripeptides include, but are not limited to, glycine-valine-citrulline (gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). The amino acid unit may include naturally occurring amino acid residues and/or minor amino acids, and/or unnatural amino acid analogs such as citrulline. Amino acid units can be designed and optimized for enzymatic degradation by a specific enzyme, for example, tumor-associated protease, cathepsin B, C and D or plasmin protease.

Typically, peptide-type linkers can be obtained by forming a peptide bond between two or more amino acids and/or peptide fragments. Such peptide bonds can be prepared, for example, according to a liquid phase synthesis method (eg E. Schrider and K. Lubke (1965) The Peptides, volume 1, pp. 76-136, Academic Press).

- 42 043790

In some embodiments, the linker component includes a spacer unit that links the antibody to a drug moiety, either directly or through an extension unit and/or an amino acid unit. The spacer unit may be self-cleaving or non-self-cleaving. A non-self-cleaving spacer unit is one in which part or all of the spacer unit remains associated with the drug group after cleavage by the ADC. Examples of non-self-cleaving spacer units include, but are not limited to, a glycine spacer unit and a glycine-glycine spacer unit. In some embodiments, enzymatic cleavage of an ADC containing a glycine-glycine spacer unit by a tumor cell-associated protease results in the release of the glycine-glycine drug moiety from the ADC moiety. In some such embodiments, the glycine-glycine-drug group is subjected to a hydrolysis step in the tumor cell, thereby cleaving the glycine-glycine spacer unit from the drug group.

The self-cleaving spacer unit allows the release of the drug group. In some embodiments, the linker spacer unit includes a paminobenzyl unit. In some such embodiments, p-aminobenzyl alcohol is attached to an amino acid unit through an amide bond, and a carbamate, methyl carbamate, or carbonate is formed between the benzyl alcohol and the drug (Hamann et al. (2005) Expert Opin. Ther. Patents (2005) 15:1087 -1103). In some embodiments, the spacer unit includes paminobenzyloxycarbonyloxy (PAB). In some embodiments, the ADC including the self-cleaving linker has the structure:

wherein Q is -C1-C8 alkyl, -Q-(C1- _C8 alkyl), halogen, nitro or cyano;

n ₆ is an integer from 0 to 4;

_Xa may be one or more additional spacer units or may be absent, and p is an integer from 1 to about 20.

In some embodiments, p is an integer from 1 to 10, 1 to 7, 1 to 5, or 1 to 4. Non-limiting examples of the spacer unit X _a include:

^R 102

-wherein R ₁₀₁ and R ₁₀₂ are independently selected from H and C1-C6 alkyl. In some embodiments, R ₁₀₁ and R ₁₀₂ are each -CH ₃ .

Other examples of self-cleaving spacers include, but are not limited to, aromatic compounds that are electronically similar to the PAB group, such as 2-aminoimidazole-5-methanol derivatives (U.S. Patent No. 7,375,078; Hay et al. (1999) Bioorg. Med. Chem. Lett 9:2237) and ortho- or para-aminobenzylacetals. In some embodiments, spacers that undergo cyclization after hydrolysis of the amide bond may be used, such as substituted and unsubstituted 4-aminobutyric acid amides (Rodrigues et al. (1995) Chemistry Biology 2:223), suitably substituted with bicyclo[2.2.1] and bicyclo[2.2.2]ring systems (Storm et al. (1972) J. Mer. Chem. Soc. 94:5815) and 2-aminophenylpropionic acid amides (Amsberry, et al. (1990) J. Org. Chem. 55:5867). Linking a drug to the α-carbon of a glycine residue is another example of a self-cleaving spacer that can be used in an ADC (Kingsbury et al. (1984) J. Med. Chem. 27:1447).

In some embodiments, linker L may be a dendritic type linker for covalently attaching more than one drug moiety to an antibody via branching, a multifunctional linker moiety (Sun et al. (2002) Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al. (2003) Bioorganic & Medicinal Chemistry 11:1761-1768). Dendritic linkers can increase the molar ratio of drug to antibody, i.e. loading, which refers to the ADC potential. Thus, when an antibody has only one reactive cysteine thiol group, multiple drug groups can be attached via a dendritic linker.

Non-limiting examples of linkers are shown below for ADCs of formula (Ig):

- 43 043790

where R ₁₀₁ and R1o2 are independently selected from H and C1-C6 alkyl;

n ₅ is an integer from 0 to 12.

In some embodiments, n is an integer from 2 to 10. In some embodiments, n is an integer from 4 to 8.

In some embodiments, R ₁₀₁ and R ₁₀₂ are each -CH ₃ .

Other non-limiting examples of ADCs include the structures:

Y stands for:

) :-N—(CH ₂ ) _n7 — in which X _a means:

or

-N-(CH ₂ ) _n7 or ;

each R ₁₀₃ is independently H or C1-C6 alkyl; and n7 is an integer from 1 to 12.

In some embodiments, the linker is replaced with groups that modulate solubility and/or reactivity. As a non-limiting example, a charged substituent such as sulfonate (-SO3-) or ammonium may increase the water solubility of the linker reagent and facilitate the coupling reaction of the linker reagent to the antibody and/or drug moiety, or facilitate the Ab-L coupling reaction (antibody intermediate -linker) with D or D-L (intermediate

- 44 043790 drug-linker compound) with Ab, depending on the synthesis route used to obtain the ADC. In some embodiments, a linker portion is coupled to an antibody, and a linker portion is coupled to a drug, and then Ab-(linker portion) ^a is coupled to drug-(linker portion) ^b to form an ADC of formula Ig.

Compounds expressly disclosed herein include, but are not limited to, ADC prepared with the following linker reagents: bis-maleimido-trioxyethylene glycol (BMPEO), N-(βmaleimidopropyloxy)-N-hydroxy succinimide ester (BMPS), N ester -(smaleimidocaproyloxy)succinimide (EMCS), N-[γ-maleimidobutyryloxy]succinimide ester (GMBS), 1,6-hexane-bis-vinylsulfone (HBVS), succinimidyl 4-(N-maleimidomethyl)cyclohexane-1carboxy-(6 -amidocaproate) (LC-SMCC), m-maleimidobenzoyl-K-hydroxysuccinimide ester (MBS), 4-(4-K-maleimidophenyl)butyric acid hydrazide (MPBH), succinimidyl 3(bromoacetamido)propionate (SBAP), succinimidyl iodoietate (SIA) )cyclohexane-1carboxylate (SMCC), succinimidyl 4-(p-maleimidophenyl) butyrate (SMPB), succinimidyl 6-[(βmaleimidopropionamido)hexanoate] (SMPH), iminothiolane (IT), sulfo-EMCS, sulfo-GMBS, sulfoKMUS, sulfo -MBS, sulfo-SIAB, sulfo-SMCC and sulfo-SMPB and succinimidyl-(4vinylsulfone)benzoate (SVSB), and including bis-maleimide reagents: dithiobismaleimidoethane (DTME), 1,4-bismaleimidobutane (BMB), 1,4- bismaleimidyl-2,3-dihydroxybutane (BMDB), bismaleimidohexane (BMH), bismaleimidoethane (BMOE), BM(PEG) ₂ (shown below) and BM(PEG) ₃ (shown below); bifunctional imidoester derivatives (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such such as bis(pdiazoniumbenzoyl)ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate), and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). In some embodiments, bis-maleimide reagents couple a cysteine thiol group in an antibody to a drug thiol group, linker, or linker-drug intermediate. Other functional groups that are reactive with thiol groups include, but are not limited to, iodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyl disulfide, isocyanate and isothiocyanate.

VM(PEG) 2

Some useful linker reagents can be obtained from various commercial sources, such as Pierce Biotechnology, Inc (Rockford, IL), Molecular Biosciences Inc (Boulder, CO), or synthesized according to procedures described in the art; for example, in Toki et al. (2002) J. Org. Chem. 67:1866-1872; Dubowchik, et al. (1997) Tetrahedron Letters, 38:5257-60; Walker, M. A (1995) J. Org. Chem. 60:5352-5355; Frisch et al. (1996) Bioconjugate Chem. 7:180186; U.S. Pat. No. 6,214,345; WO 02/088172; US 2003130189; US2003096743; WO 03/026577; WO 03/043583 and WO 04/032828.

Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an example of a chelating agent for radionucleotide-antibody conjugation. See, for example, WO 94/11026.

Methods for preparing polymer-NaPi2b-targeting antibody conjugates.

In some embodiments, the conjugates are formed in multiple steps. These steps include (1) modifying the polymer so that it contains a functional group that can react with a functional group of the drug or its derivative; (2) reacting the modified polymer with a drug or derivative thereof such that the drug binds to the polymer; (3) modifying the polymer-drug conjugate such that the polymer contains a functional group that can react with the functional group of the isolated NaPi2b-targeting antibody or derivative thereof; and (4) reacting the modified polymer-drug conjugate with a NaPi2b-targeting antibody to form the conjugate disclosed herein. Step (3) may be omitted if the modified polymer obtained in step (1) contains a functional group that can react with a functional group of the antibody.

In another embodiment, the conjugates are formed in several steps: (1) modifying the polymer so that it contains a functional group that can react with the functional group

- 45 043790 group of the first drug or its derivative; (2) reacting the modified polymer with a first drug or derivative thereof such that the first drug binds to the polymer; (3) modifying the polymer-drug conjugate so that it contains a different functional group that can react with the functional group of the second drug or derivative thereof (4) reacting the modified polymer-drug conjugate with the second drug or derivative thereof so that the second drug was bound to the polymer-drug conjugate; (5) modifying a polymer-drug conjugate containing 2 different drugs so that the polymer contains a functional group that can react with the functional group of the NaPi2b-targeting antibody; and (6) reacting the modified polymer-drug conjugate from step (5) with an isolated NaPi2b-targeting antibody or derivative thereof to form the conjugate disclosed herein. Steps (5) and (6) can be repeated if 2 different isolated NaPi2b-targeting antibodies or derivatives thereof are to be conjugated to form a polymer-drug conjugate comprising two different drugs and two different antibodies.

In yet another embodiment, the conjugates are formed in several steps. These steps include (1) modifying the polymer so that it contains a functional group that can react with the functional group of the drug or its derivative; (2) further modifying the polymer so that it also contains a functional group that can react with the functional group of the NaPi2b-targeting antibody; (3) reacting the modified polymer with a drug or derivative thereof such that the drug binds to the polymer; and (4) reacting the modified polymer-drug conjugate with a NaPi2b-targeting antibody to form the conjugate disclosed herein. The sequence of stages (1) and (2) or stages (3) and (4) may be reversed. Further, one of steps (1) or (2) may be omitted if the modified polymer contains a functional group that can react with both the functional group of the drug or derivative thereof and the functional group of the NaPi2b-targeted antibody.

In another embodiment, the conjugates are formed in several steps: (1) modifying the polymer so that it contains a functional group that can react with the functional group of the first drug or derivative thereof; (2) further modifying the polymer so that it contains a functional group that can react with the functional group of the NaPi2b-targeting antibody; (3) reacting the modified polymer with a first drug or derivative thereof such that the first drug binds to the polymer; (4) modifying the polymer-drug conjugate so that it contains a different functional group that can react with the functional group of the second drug or derivative thereof (5) reacting the modified polymer-drug conjugate with the second drug or derivative thereof so that the second drug was bound to the polymer-drug conjugate; (6) reacting a modified polymer-drug conjugate containing 2 different drugs so that the polymer is isolated with a NaPi2b-targeting antibody or derivative thereof to form the conjugate disclosed herein. Step (6) can be repeated if 2 different isolated antibodies or derivatives thereof are to be conjugated to form a polymer-drug conjugate comprising two different drugs and two different antibodies. Step (4) can be carried out after step (1) so that the modified polymer contains two different functional groups that can react with two different drugs or derivatives thereof. In this embodiment, the modified polymer containing two different functional groups that can react with two different drugs or derivatives thereof so that it contains a functional group that can react with the functional group of the antibody can be further modified; before reacting the modified polymer with either two different drugs (step (3) and step (5) or with an antibody (step (6).

In some embodiments, the conjugates disclosed herein have applications in biomedical fields such as drug delivery and tissue engineering, and the polymeric carrier is biocompatible and biodegradable. In some embodiments, the carrier is a soluble polymer, nanoparticle, gel, liposome, micelle, suture, implant, etc. In some embodiments, the term soluble polymer includes a biodegradable, biocompatible polymer such as a polyal (eg, a hydrophilic polyacetal or polyketal). In some other embodiments, the carrier is a fully synthetic, semi-synthetic, or natural polymer. In some other embodiments, the carrier is hydrophilic. Examples of suitable polymeric carriers for preparing the conjugates disclosed herein are described in US Pat. No. 8,815,226, the contents of which are incorporated herein by reference in their entirety.

- 46 043790

In one embodiment, the polymeric carrier includes units of formula (IV):

OH X' (IV), in which X' represents a substituent for the hydroxyl group of the polymer backbone. As shown in formula (IV) and other formulas described herein, each polyacetal unit has a single hydroxyl group attached to the glycerol group of that unit and an X' group attached to the glycol aldehyde group of that unit. For convenience only, it should be understood that a polymer having units of formula (IV) and other formulas described herein may contain a random distribution of units having X' groups (or other substituent such as a linker including a maleimide terminus) attached to a glycol aldehyde group these units, and those having a single X' group (or other substituent, such as a linker including a maleimide terminus) attached to the glycerol group of these units, as well as units having two X' groups (or other substituents, such as a linker , including a maleimide termination), one of which is attached to the glycol-aldehyde group, and the other is attached to the glycerol group of these units.

In one embodiment, biodegradable biocompatible polyals suitable for practicing the present disclosure have a molecular weight of about 2 kDa to about 40 kDa, about 6 kDa to about 20 kDa, or about 8 kDa to about 15 kDa). For example, the biodegradable biocompatible polyal used for the polymer backbones or conjugates disclosed herein is PHF having a molecular weight of about 2 to about 40 kDa (e.g., about 2-20 kDa, 3-15 kDa, or 5-10 kDa. )

Methods for preparing polymeric carriers (eg, biocompatible, biodegradable polymeric carriers) suitable for conjugation with modifiers are known in the art. For example, guidance on the synthesis can be found in US Patents 5,811,510; 5,863,990; 5,958,398; 7,838,619; 7,790,150; and 8,685,383. One skilled in the art will know how to adapt these methods to create polymeric carriers for use in the practice of the present disclosure.

In one embodiment, a method for forming biodegradable biocompatible polyaldehyde conjugates according to the present disclosure includes a method in which a suitable polysaccharide is combined with an effective amount of a glycol-specific oxidizing agent to form an aldehyde intermediate. The aldehyde intermediate, which is itself a polyal, can then be reduced to the corresponding polyol, succinylated, and coupled with one or more suitable modifiers to form a biodegradable, biocompatible polyal conjugate comprising succinamide-containing linkages.

In another preferred embodiment, fully synthetic biodegradable biocompatible polyals for use in the present disclosure can be prepared by reacting a suitable initiator with a suitable precursor compound.

For example, fully synthetic polyals can be prepared by condensation of vinyl esters with protected substituted diols. Other methods, such as ring opening polymerization, can be used, and the effectiveness of the method may depend on the degree of substitution and the size of the protecting groups.

One skilled in the art will appreciate that solvent systems, catalysts, and other factors can be optimized to produce high molecular weight products.

In some embodiments, the carrier is PHF.

In embodiments, the polymeric carrier is PHF having a polydispersity index (PDI) of <1.5, such as <1.4, <1.3, <1.2, or <1.1.

For example, for the conjugation of an isolated NaPi2b-targeting antibody having a molecular weight of 40 kDa to 200 kDa, the polymeric backbone support is a polyacetal, e.g., PHF, having a molecular weight (i.e., MW of unmodified PHF) ranging from about 2 kDa to about 40 kDa (eg, about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa).

For example, for the conjugation of an antibody having a molecular weight of 40 kDa to 80 kDa, the polymeric support of the backbones disclosed herein is a polyacetal, e.g., PHF, having a molecular weight (i.e., MW of unmodified PHF) ranging from about 2 kDa to about

- 47 043790 specifically 40 kDa (for example, approximately 2-20 kDa, or approximately 3-15 kDa, or approximately 510 kDa). For example, PHF has a molecular weight of approximately 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, or 15 kDa.

For example, for the conjugation of an antibody having a molecular weight of 60 kDa to 120 kDa, the polymeric support of the backbones disclosed herein is a polyacetal, e.g., PHF, having a molecular weight (i.e., MW of unmodified PHF) ranging from about 2 kDa to about 40 kDa (eg, about 2-20 kDa, or about 3-15 kDa, or about 510 kDa). For example, PHF has a molecular weight of approximately 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, or 15 kDa.

For example, for the conjugation of an antibody having a molecular weight of 140 kDa to 180 kDa or 140 kDa to 150 kDa, the polymeric support of the backbones disclosed herein is a polyacetal, e.g., PHF, having a molecular weight (i.e., MW of unmodified PHF) of ranging from about 2 kDa to about 40 kDa (eg, about 2-20 kDa, or about 3-15 kDa, or about 5-10 kDa). For example, PHF has a molecular weight of approximately 5 kDa, 6 kDa, 7 kDa, 8 kDa, 9 kDa, 10 kDa, 11 kDa, 12 kDa, 13 kDa, 14 kDa, or 15 kDa.

Antibodies in this molecular weight range include, but are not limited to, for example, full length antibodies such as IgG, IgM.

The biodegradable biocompatible conjugates disclosed herein can be formulated to meet desired biodegradability and hydrophilicity requirements. For example, under physiological conditions, a balance between biodegradability and stability can be achieved. For example, it is known that molecules with molecular weights outside a certain threshold (usually above 40-100 kDa, depending on the physical form of the molecule) are not excreted through the kidneys like small molecules and can only be eliminated from the body through cellular uptake and degradation in the body. intracellular compartments, primarily lysosomes. This observation illustrates how functionally stable yet biodegradable materials can be developed by adjusting their stability under normal physiological conditions (pH 7.5 ± 0.5) and at lysosomal pH (pH about 5). For example, it is known that the hydrolysis of acetal and ketal groups is catalyzed by acids, so polyals will generally be less stable in the acidic lysosomal environment than, for example, in blood plasma. An assay can be designed to compare the degradation profile of polymers at, for example, pH 5 and pH 7.5 at 37°C in aqueous media and thereby determine the expected balance of polymer stability in a normal physiological environment and in the digestive lysosomal compartment after cellular uptake. The integrity of the polymer in such tests can be measured, for example, by size exclusion HPLC. One skilled in the art may select other suitable methods for studying the various fragments of the degradable conjugates disclosed herein.

In many cases, it will be preferable that at pH 7.5 the effective size of the polymer does not change in a detectable manner for 1 to 7 days and remains within 50% of the original size for at least several weeks. At pH 5, on the other hand, the polymer should preferably degrade in a detectable manner within 1 to 5 days and be completely converted to low molecular weight fragments within two weeks to several months. Although faster degradation may be preferable in some cases, in general it may be more desirable for the polymer to degrade in cells at a rate no greater than the rate of metabolism or excretion of polymer fragments by the cells. Accordingly, in some embodiments, the conjugates of the present disclosure are expected to be biodegradable, particularly after uptake by cells, and relatively inert to biological systems. The decomposition products of the carrier are preferably uncharged and do not significantly shift the pH of the medium. It is proposed that the abundance of alcohol groups can ensure a low percentage of recognition of the polymer by cellular receptors, in particular, phagocytes.

Polymer scaffolds according to the present disclosure typically contain few, if any, antigenic determinants (characteristic of, for example, some polysaccharides and polypeptides) and typically do not include rigid structures capable of engaging in key-lock interactions in vivo unless desired. . Thus, the soluble, cross-linked and solid conjugates disclosed herein have predicted low toxicity and bioadhesiveness making them suitable for several biomedical applications.

In some embodiments of the present disclosure, the biodegradable, biocompatible conjugates may form linear or branched structures. For example, the biodegradable, biocompatible polyal conjugates of the present disclosure may be chiral (optically active). If necessary, the biodegradable biocompatible polyal conjugates according to the present disclosure may be scalemic.

In some embodiments, the conjugates disclosed herein are water soluble. In some embodiments, the conjugates disclosed herein are water insoluble. In some embodiments, the conjugate of the invention is in solid form. In some embodiments, the conjugates disclosed herein are colloids. In some variants

- 48 043790 embodiments, the conjugates disclosed herein are in particulate form. In some embodiments, the conjugates disclosed herein are in the form of a gel.

Scheme 1 below demonstrates a synthetic design for the polymeric drug scaffolds disclosed herein. In one embodiment, the conjugates are formed in several steps: (1) the polymer, PHF, is modified to contain a COOH group (eg, -C(O)-X-(CH ₂ ) ₂ COOH); (2) the polymer is then further modified to contain a maleimido group (eg, EG2-MI) that can react with the PBRM functionality; (3) a modified polymer containing two different functional groups is reacted with a functional group of a drug or a derivative thereof (eg, AF-HPA-Ala) to form a polymer-drug conjugate; (4) PBRM disulfide bonds are reduced; (5) the reduced PBRM is then reacted with a polymer-drug conjugate to form a protein-polymer-drug conjugate; and (6) the remaining maleimido groups are optionally reacted with a maleimido-blocking compound (eg, cysteine).

In another embodiment, the order of steps (2) and (3) may be reversed, as depicted on the right side of Scheme 1 below.

Scheme 1

In yet another embodiment, steps (2) and (3) above are performed simultaneously, as depicted in Scheme 2 below.

Scheme 2

- 49 043790

1. Reconstituted NaPi2b antibody

2. Cysteine

Use of NaPi2b-targeted antibody-drug conjugates.

It should be understood that administration of the therapeutic agents according to the disclosure is carried out with suitable carriers, excipients and other agents included in the formulations to provide improved transport, delivery, tolerability and the like. A variety of suitable formulations can be found in a formulary known to all those skilled in the art of pharmaceutical chemistry: Remington's Pharmaceutical Sciences ( ^15th ed., Mack Publishing Company, Easton, Pennsylvania (1975)), especially Chapter 87, Blaug, Seymour. These formulations include, for example, powders, pastes, ointments, jellies, waxes, oils, lipids, lipid- (cationic or anionic) containing vesicles (such as Lipofectin™), DNA conjugates, anhydrous absorbent pastes, oil-in-water emulsions and emulsions such as water in oil, carbowax emulsions (polyethylene glycols with different molecular weights), semi-solid gels and semi-solid mixtures containing carbowax. Any of the above-described mixtures may be suitable for the treatments and methods of treatment in accordance with the present disclosure, provided that the active ingredient in the formulation is not inactivated by the formulation and the formulation is physiologically compatible and tolerated by its route of administration. See also Baldrick P. Pharmaceutical excipient development: the need for preclinical guidance. Regul. Toxicol Pharmacol. 32(2):210-8 (2000), Wang W. Lyophilization and development of solid protein Pharmaceuticals. Int. J. Pharm. 203 (1-2):1-60 (2000), Charman WN Lipids, lipophilic drugs, and oral drug delivery—some emerging concepts. J Pharm Sci. 89(8):967-78 (2000), Powell et al. Compendium of excipients for parenteral formulations PDA J Pharm Sci Technol. 52:238-311 (1998) and the citations therein for additional information regarding compositions, excipients and carriers known to those skilled in the art of pharmaceutical chemistry.

In one embodiment, the NaPi2b antibody conjugates disclosed herein can be used as therapeutic agents. Such agents are typically used to diagnose, prognosis, monitor, treat, alleviate, prevent and/or delay the progression of disease or pathology associated with, for example, aberrant NaPi2b activity and/or expression in a patient. The therapeutic regimen is carried out by identifying a patient, for example a human patient, suffering from (or at risk of developing) a disease or disorder associated with aberrant NaPi2b activity and/or expression, such as cancer, using standard methods. A NaPi2b antibody conjugate preparation, preferably having high specificity and high affinity for its target antigen, is administered to a patient and generally has an effect due to its binding to the target. Administration of a conjugate may suppress or inhibit or interfere with the signaling function of the target. Administration of a conjugate may suppress or inhibit or prevent the target from binding to the endogenous ligand to which it binds naturally. For example, the conjugate can bind to a target and modulate, block, inhibit, reduce, counteract, neutralize, or otherwise interfere with the activity and/or expression of NaPi2b.

Diseases or disorders associated with aberrant NaPi2b activity and/or expression include, but are not limited to, cancer. The target cancer may be ovarian cancer such as epithelial ovarian cancer, thyroid cancer, colorectal cancer, lung cancer, non-small cell lung cancer (NSCLC) such as non-squamous NSCLC, breast cancer, kidney cancer and salivary duct cancer.

In another aspect, the diseases or disorders are cancers selected from the group consisting of non-small cell lung cancer (NSCLC), such as non-squamous NSCLC, and ovarian cancer, such as epithelial ovarian cancer. Typically, alleviating or treating a disease or disorder involves reducing one or more symptoms or medical problems associated with the disease or disorder. For example, in the case of cancer, the therapeutically effective amount of the conjugate or pharmaceutical composition thereof may be one of or a combination of the following: a reduction in the number of cancer cells; reduction in tumor size; inhibiting (ie reducing to some extent and/or stopping) the infiltration of cancer cells into peripheral organs; inhibition of tumor metastasis; inhibition, to some extent, of tumor growth; and/or alleviation, to some extent, of one or more symptoms associated with cancer. In some embodiments, a composition disclosed herein can be used to prevent the onset or recurrence of a disease or disorder in a patient.

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A therapeutically effective amount of the NaPi2b antibody conjugate disclosed herein generally refers to the amount necessary to achieve the therapeutic goal. As noted above, this may be a binding interaction between the antibody and its target antigen, in some cases preventing the target from functioning. The amount required to administer further depends on the binding affinity of the antibody to its specific antigen and also depends on the rate at which the administered antibody is cleared from the free volume of the patient to whom it is administered. The mode of administration in which the conjugates disclosed herein are used is also selected in accordance with a variety of other factors, including the type, species, age, body weight, sex and disease of the patient; the severity of the condition being treated; route of administration; the patient's renal and hepatic function; and the specific conjugate used. Typically, a physician or veterinarian can easily determine and prescribe the effective amount of conjugate needed to prevent, counter, or stop the progression of the condition. General ranges for therapeutically effective administration of the NaPi2b antibody conjugate disclosed herein may be, by way of non-limiting example, from 0.1 mg/kg body weight to about 50 mg/kg body weight, from 0.1 mg/kg body weight to about 100 mg/kg body weight or from 0.1 mg/kg body weight to approximately 150 mg/kg body weight. The ranges disclosed herein are expressed as amounts administered based on the patient's body weight, and can be easily converted by one skilled in the art to amounts administered per patient body surface area. For example, 1 mg/kg body weight for an adult is equivalent to approximately 37 mg/m ² and 1 mg/kg body weight for a child is equivalent to approximately 25 mg/m ² .

Typical frequency of administration may be, for example, twice a day to once a month (eg, once a day, once a week; once every two weeks; once every 3 weeks or once a month). For example, the 1535 XMT or 10H1.11.4B conjugates disclosed herein can be administered (eg, as a single dose once a week, every 2 weeks, every 3 weeks, or once a month) in an amount of from about 0.1 mg/day. kg to approximately 20 mg/kg (e.g., 0.2 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.67 mg/kg, 0.8 mg/kg, 1 mg/kg, 1.2 5 mg/kg, 1.67 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/ kg, 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/ kg, 18 mg/kg, 19 mg/kg or 20 mg/kg). For example, the 1535 XMT or 10H1.11.4B conjugates disclosed herein can be administered (eg, as a single dose once a week, every 2 weeks, every 3 weeks, or once a month) in an amount of from about 0.1 mg/day. kg to approximately 20 mg/kg (e.g., 0.2 mg/kg, 0.4 mg/kg, 0.5 mg/kg, 0.67 mg/kg, 0.8 mg/kg, 1 mg/kg, 1.25 mg/kg, 1.67 mg/kg, 2 mg/kg, 2.5 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg , 8 mg/kg, 9 mg/kg, 10 mg/kg, 11 mg/kg, 12 mg/kg, 13 mg/kg, 14 mg/kg, 15 mg/kg, 16 mg/kg, 17 mg/kg , 18 mg/kg, 19 mg/kg, or 20 mg/kg) for the treatment of NaPi2b-expressing ovarian cancer or NaPi2b-expressing NSCLC cancer.

The effectiveness of treatment is determined in association with any known method of diagnosing or treating a particular NaPi2b-related disorder. Relief of one or more symptoms of a NaPi2b-related disorder indicates that the antibody provides clinical benefit.

Methods for screening antibodies having the desired specificity include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and other immunologically mediated methods known in the art.

Therapeutic administration and formulations of NaPi2b-targeted antibody-drug conjugates.

The conjugates disclosed herein (also referred to herein as active compounds) may be included in pharmaceutical compositions suitable for administration. The principles and considerations involved in preparing such compositions, as well as guidance in the selection of components, are given, for example, in Remington's Pharmaceutical Sciences: The Science And Practice Of Pharmacy 19 ^th ed (Alfonso R. Gennaro, et al., editors) Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts, Possibilities, Limitations, And Trends, Harwood Academic Publishers, Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances In Parenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

Such compositions typically include an antibody and/or conjugates thereof and a pharmaceutically acceptable carrier.

As used herein, the phrase pharmaceutically acceptable refers to compounds, materials, compositions, carriers and/or dosage forms that are, within the bounds of sound medical judgment, suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reaction or other problems or complications commensurate with a reasonable benefit/risk ratio.

As used herein, the term pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Suitable carriers are described in the new edition of Remington's Pharmaceutical Sciences, the standard reference in the field, which is incorporated herein by reference. Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous carriers such as

- 51 043790 non-volatile oils, can also be used. The use of such media and agents for pharmaceutically active substances is known in the art. In addition, unless any standard vehicles or agents are incompatible with the active compound, their use in the compositions is also contemplated.

Formulations that are used for in vivo administration must be sterile. This is easily achieved by filtration through sterilizing filtration membranes.

The pharmaceutical composition disclosed herein is formulated so that it is compatible with its intended route of administration. Example routes of administration include parenteral, eg, intravenous, dermal, subcutaneous, oral (eg, inhalation), transdermal (ie, topical), transmucosal, and rectal. Solutions or suspensions used for parenteral, dermal or subcutaneous administration may include the following components: a sterile diluent such as water for injection, saline, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates; and agents to adjust tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. The parenteral drug may be in ampoules, single-use syringes, or multi-dose ampoules made of glass or plastic.

In one embodiment, the pharmaceutical composition is in bulk or unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, an IV bag, a tablet, a single pump on an aerosol inhaler, or an ampoule. The amount of active ingredient (eg, a conjugate disclosed herein) in a single dose of the composition is an effective amount and will vary depending on the particular treatment. One skilled in the art will appreciate that it is sometimes necessary to make routine dosage changes depending on the age and condition of the patient. The dosage also depends on the route of administration.

Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (if the substance is water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable vehicles include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and must be fluid to the extent that it can be easily administered by syringe. It must be stable under the conditions of manufacture and storage and must be preserved against contamination by microorganisms such as bacteria and fungi. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (eg, glycerin, propylene glycol and liquid polyethylene glycol, etc.) and suitable mixtures thereof. Proper fluidity can be maintained, for example, by using a coating agent such as lecithin, maintaining the desired particle size in the case of a dispersion, and by using surfactants. Prevention of the action of microorganisms can be achieved with various antibacterial and antifungal substances, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc. In many cases, it is preferable to include isotonic substances in the composition, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride. Prolonged absorption of injectable compositions can be achieved by including an absorption delaying agent in the composition, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in a suitable solvent with one or a combination of the ingredients listed above, if desired, followed by sterilizing filtration. Typically, the dispersion is prepared by incorporating the active compound into a sterile vehicle containing the basic dispersion medium and the other required ingredients listed above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation methods are vacuum drying and freeze drying, yielding a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution.

Oral compositions typically include an inert diluent or edible carrier. They may be in gelatin capsules or compressed into tablets. For purposes of oral therapeutic administration, the active compound may be included with excipients and used in the form of tablets, lozenges or capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash, wherein the compound in the liquid carrier is administered orally and rinsed and spit or swallowed. Pharmaceutically compatible vehicles and/or adjuvants may be included as part of the composition. Tablets, pills, capsules, lozenges, etc. may contain any of the following ingredients or compounds of a similar nature: a binder such as microcrystalline cellulose, tragacanth or gelatin; excipient such as starch or lactose, disintegrant such as alginic acid, Primogel or corn

- 52 043790 starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as fumed silica; a sweetener such as sucrose or saccharin; or a flavoring such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are supplied in the form of an aerosol spray from a sealed container or dispenser containing a suitable propellant, for example a gas such as carbon dioxide, or a nebulizer.

Systemic administration may also be accomplished by transmucosal or transdermal means. For transmucosal or percutaneous administration, the composition uses penetrants that are adequate for the barrier through which it is required to penetrate. Such penetrants are known in the art and include, for example, for transmucosal administration, detergents, bile salts and fusidic acid derivatives. Transmucosal administration can be accomplished using nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, balms, gels or creams, as is known in the art.

The compounds may also be formulated as suppositories (eg, with standard suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are formulated with carriers that protect the compound from rapid elimination from the body, such as sustained/controlled release formulations, including implants and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyortho-esters and polylactic acid can be used. Methods for preparing such compositions are known to those skilled in the art.

For example, the active ingredients can be enclosed in microcapsules obtained, for example, by coacervation or surface polymerization methods, for example, hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposome, protein microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions.

Sustained release preparations can be prepared. Suitable examples of sustained release formulations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which are in the form of molded articles, for example, films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (eg, poly(2-hydroxyethyl methacrylate), or polyvinyl alcohol), polylactides (US Patent No. 3,773,919), copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradable ethylene vinyl acetate, degradable glycolic acid-lactic acid copolymers such as DEPOT LUPRON™ (injectable microspheres formulated from glycolic acid-lactic acid copolymer and leuprolide acetate), and poly-O-(-)-3-hydroxybutyric acid. Although polymers such as ethylene vinyl acetate and lactic acid-glycolic acid release molecules over 100 days, some hydrogels release proteins over shorter time intervals.

Materials may also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeting infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. They can be prepared according to methods known to one skilled in the art, for example, as described in US Pat. No. 4,522,811.

It is particularly preferred to formulate oral or parenteral compositions in unit dosage form for ease of administration and uniformity of dosage. Unit dosage form as used herein refers to physically discrete units suitable for use as unit doses for a patient being treated; each form containing a predetermined amount of the active compound calculated to produce the desired therapeutic effect, in combination with the desired pharmaceutical carrier. The specification for the unit dosage forms disclosed herein is dictated by and directly depends on the unique characteristics of the active compound and the particular therapeutic effect that will be achieved and the limitations inherent in formulating such an active compound for human treatment.

The pharmaceutical compositions may be included in a container, pouch or dispenser along with instructions for administration.

The formulation may also contain more than one active compound as needed for the particular indication being treated, preferably with additional activities that do not adversely affect each other. Also or in addition, the composition may include an agent that improves its function, such as, for example, a cytotoxic agent, cytokine, chemotherapeutic agent or growth inhibitory agent. Such molecules are suitably present in combination in amounts that are effective for the intended purpose.

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In one embodiment, the active compounds are administered as part of a combination therapy, i.e. combined with other agents, for example therapeutic agents, which are useful for treating pathological conditions or disorders, such as various forms of cancer, autoimmune disorders and inflammatory diseases. The term in combination in this context means that the agents are given at substantially the same time, either simultaneously or sequentially. If given sequentially, at the start of administration of the second compound, the first of the two compounds is preferably still detectable in effective concentrations at the treatment site.

For example, a combination therapy may include one or more of the conjugates disclosed herein, formulated in conjunction with and/or used in conjunction with one or more additional antibodies, e.g., NaPi2b antibody, which may be the same as the antibody used to form the conjugate, or another antibody.

For example, combination therapy may include one or more therapeutic agents and/or adjuvants. In some embodiments, the additional therapeutic agent is a small molecule inhibitor, another antibody therapeutic, a polypeptide or peptide therapeutic, a nucleic acid therapeutic, and/or other biologic.

In some embodiments, the additional therapeutic agent is a cytotoxic agent, a chemotherapeutic agent, a growth inhibitory agent, an angiogenesis inhibitor, a PARP inhibitor, an alkylating agent, an antimetabolite, an antimicrotubule agent, a topoisomerase inhibitor, a cytotoxic antibiotic , any other nucleic acid-disrupting agent or immune checkpoint inhibitor. In one embodiment, the therapeutic agent used in the treatment of cancer includes, but is not limited to, a platinum compound (eg, cisplatin or carboplatin); taxane (eg paclitaxel or docetaxel); a topoisomerase inhibitor (eg, irinotecan or topotecan); anthracycline (eg doxorubicin (ADRIAMYCIN®) or liposomal doxorubicin (DOXIL®)); antimetabolite (eg, gemcitabine, pemetrexed); cyclophosphamide; vinorelbine (NAVELBINE®); hexamethylmelamine; ifosfamide; etoposide; an angiogenesis inhibitor (eg, Bevacizumab (Avastin®), thalidomide, TNP-470, platelet factor 4, interferon, or endostatin); PARP inhibitor (eg, Olaparib (Lynparza™)); an immune checkpoint inhibitor, such as, for example, a monoclonal antibody targeting either PD-1 or PD-L ((Pembrolizumab (Keytruda®), atezolizumab (MPDL3280A) or Nivolumab (Opdivo®)) or CTA-4 (Ipilimumab (Yervoy®), kinase inhibitor (eg, sorafenib or erlotinib), proteasome inhibitor (eg, bortezomib or carfilzomib), immunomodulatory agent (eg, lenalidomide or IL-2), radiation agent, ALK inhibitor (eg, crizotinib (Xalkori), ceritinib (Zykadia), alectinib (Alecensa), dalantercept (ACE-041), brigatinib (AP26113), entrectinib (NMS-E628), PF-06463922 TSR-011, CEP-37440 and X-396) and/or their biosimilar and /or combinations thereof Other suitable agents include an agent that is considered a medical standard and/or a chemotherapeutic agent known in the art.

In some embodiments, the immune checkpoint inhibitor is a CTLA-4 inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-CTLA-4 monoclonal antibody. In other embodiments, the immune checkpoint inhibitor is a human or humanized anti-CTLA-4 antibody. In one embodiment, an anti-CTLA-4 antibody blocks the binding of CTLA-4 to CD80 (B7-1) and/or CD86 (B7-2) expressed on antigen presenting cells. Examples of anti-CTLA-4 antibodies include, but are not limited to, Bristol Meyers Squibb anti-CTLA-4 antibody ipilimumab (also known as Yervoy®, MDX-010, BMS-734016 and MDX-101); anti-CTLA4 antibody, clone 9H10 from Millipore; Pfizer tremelimumab (CP-675.206, ticilimumab); and anti-CTLA4 antibody clone BNI3 from Abeam.

In some embodiments, the anti-CTLA-4 antibody is an anti-CTLA-4 antibody disclosed in any of the following patent publications (incorporated herein by reference): WO 2001014424; WO 2004035607; US2005/0201994; EP 1212422 B1; WO2003086459; WO2012120125; WO2000037504; WO2009100140; WO200609649; WO2005092380; WO2007123737; WO2006029219; WO20100979597; WO200612168; and WO1997020574. Additional CTLA-4 antibodies are described in US Patent Nos. 5,811,097, 5,855,887, 6,051,227, and 6,984,720; in PCT publications WO 01/14424 and WO 00/37504; and US Publications No. 2002/0039581 and 2002/086014; and/or US Patent Nos. 5,977,318, 6,682,736, 7,109,003 and 7,132,281, incorporated herein by reference). In some embodiments, the anti-CTLA-4 antibody is, for example, those disclosed in: WO 98/42752; US Patent Nos. 6,682,736 and 6,207,156; Hurwitz et al., Proc. Natl. Acad. Sci. USA, 95(17): 1006710071 (1998); Camacho et al., J. Clin. Oncol., 22(145): Abstract no. 2505 (2004) (antibody CP-675206); Mokyr et al., Cancer Res., 58:5301-5304 (1998) (incorporated herein by reference).

In some embodiments, the CTLA-4 inhibitor is a CTLA-4 ligand, as disclosed in WO1996040915.

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In some embodiments, the CTLA-4 inhibitor is an inhibitor of a CTLA-4 expressing nucleic acid. For example, anti-CTLA4 PHKi molecules may take the form of molecules described by Mello and Fire in PCT publications WO 1999/032 619 and WO 2001/029058; US publications No. 2003/0051263, 2003/0055020, 2003/0056235, 2004/265839, 2005/0100913, 2006/0024798, 2008/0050342, 2008/0081373, 2008/0 248576 and 2008/055443; and/or US Patent Nos. 6,506,559, 7,282,564, 7,538,095, and 7,560,438 (incorporated herein by reference). In some cases, the anti-CTLA4 PHKi molecules take the form of double-stranded PHKi molecules described by Tuschl in European patent EP 1309726 (incorporated herein by reference). In some cases, anti-CTLA4 PHKi molecules take the form of double-stranded PHKi molecules described by Tuschl in US Pat. Nos. 7,056,704 and 7,078,196 (incorporated herein by reference). In some embodiments, the CTLA4 inhibitor is an aptamer described in PCT Publication No. WO2004081021.

In addition, the anti-CTLA4 PHKi molecules of the present disclosure may take the form of RNA molecules described by Crooke in US Patent Nos. 5,898,031, 6,107,094, 7,432,249 and 7,432,250, and European Patent EP 0928290 (incorporated herein by reference).

In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-PD-L1 monoclonal antibody. In other or additional embodiments, the immune checkpoint inhibitor is a human or humanized anti-PD-L1 antibody. In one embodiment, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as PD-L1. In another embodiment, an immune checkpoint inhibitor reduces the interaction between PD-1 and PD-L1. Examples of immune checkpoint inhibitors include antibodies (eg, anti-PD-L1 antibody), PHKi molecules (eg, anti-PD-L1 PHKi), antisense molecules (eg, anti-PD-L1 antisense RNA), dominant negative proteins ( for example, dominant negative protein PD-L1) and small molecule inhibitors. Antibodies include monoclonal antibodies, humanized antibodies, deimmunized antibodies, and Ig fusion proteins. An example of an anti-PD-L1 antibody includes clone EH12. Examples of anti-PD-L1 antibodies include: Genentech MPDL3280A (RG7446); anti-mouse PD-L1 antibody clone 10F.9G2 (Cat #BE0101) from BioXcell; anti-PD-L1 monoclonal antibody MDX-1105 (BMS-936559) and BMS-935559 from BristolMeyer's Squibb; MSB0010718C; mouse anti-PD-L1 clone 29E.2A3; and MEDI4736 AstraZeneca. In some embodiments, the anti-PD-L1 antibody is an anti-PD-L1 antibody disclosed in any of the following patent publications (incorporated herein by reference): WO2013079174; CN101104640; WO2010036959; W02013056716; WO2007005874; WO2010089411; WO2010077634; WO2004004771; WO2006133396; WO201309906; US 20140294898; WO2013181634 or WO2012145493.

In some embodiments, the PD-L1 inhibitor is an inhibitor of a nucleic acid that expresses PD-L1. In some embodiments, a PD-L1 inhibitor is disclosed in one of the following patent publications (incorporated herein by reference): WO2011127180 or WO2011000841. In some embodiments, the PD-L1 inhibitor is rapamycin.

In some embodiments, the immune checkpoint inhibitor is a PD-L2 inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 monoclonal antibody. In other or additional embodiments, the immune checkpoint inhibitor is a human or humanized anti-PD-L2 antibody. In some embodiments, the immune checkpoint inhibitor reduces the expression or activity of one or more immune checkpoint proteins, such as PD-L2. In other embodiments, an immune checkpoint inhibitor reduces the interaction between PD-1 and PD-L2. Examples of immune checkpoint inhibitors include antibodies (eg, anti-PD-L2 antibody), PHKi molecules (eg, anti-PD-L2 PHKi), antisense molecules (eg, anti-PD-L2 antisense RNA), dominant negative proteins ( for example, dominant negative protein PD-L2) and small molecule inhibitors. Antibodies include monoclonal antibodies, humanized antibodies, deimmunized antibodies, and Ig fusion proteins.

In some embodiments, the PD-L2 inhibitor is GlaxoSmithKline AMP-224 (Amplimmune). In some embodiments, the PD-L2 inhibitor is rHIgM12B7.

In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-PD-1 monoclonal antibody. In other embodiments, the immune checkpoint inhibitor is a human or humanized anti-PD-1 antibody. For example, inhibi

- 55 043790 tors of biological activity of PD-1 (or its ligands) disclosed in US patents 7.02 9.67 4; 6,808,710; or US Patent Applications 20050250106 and 20050159351 may be used in the combinations of the invention. Examples of anti-PD-1 antibodies include: anti-mouse PD-1 antibody clone J43 (Cat #BE0033-2) from BioXcell; anti-mouse PD-1 antibody clone RMP1-14 (Cat #BE0146) from BioXcell; mouse anti-PD-1 antibody clone EH12; Merck anti-mouse PD-1 antibody (Keytruda®, pembrolizumab, lambrolizumab, H409A1 1); and AnaptysBio's anti-PD-1 antibody known as ANB011; antibody MDX-1106 (ONO4538); human IgG4 monoclonal antibody Bristol-Myers Squibb nivolumab (Opdivo®, BMS936558, MDX1106); AstraZeneca AMP 514 and AMP 224 and Pidilizumab (CT-011 or hBAT-1), CureTech Ltd.

Additional examples of anti-PD-1 antibodies are described in Goldberg et al., Blood 1 10(1): 186-192 (2007), Thompson et al., Clin. Cancer Res. 13(6): 1757-1761 (2007), and Korman et al., International Application No. PCT/JP2006/309606 (publication WO 2006/121168 A1, each of which is expressly incorporated herein by reference. In some embodiments, an anti-PD-1 antibody is an anti-PD-1 antibody disclosed in any of the following patent publications (incorporated herein by reference): WO014557; WO2011110604; WO2008156712; US2012023752; WO2011110621; WO2004072286; WO2004056875; W O20100036959;WO2010029434;W0201213548; WO2002078731; WO2012145493; WO2010089411; WO2001014557; WO2013022091; WO2013019906; WO2003011911; US20140294898; and WO2010001617.

In some embodiments, the PD-1 inhibitor is a PD-1 binding protein as disclosed in WO200914335 (incorporated herein by reference).

In some embodiments, the PD-1 inhibitor is a PD-1 peptidomimetic inhibitor, as disclosed in WO2013132317 (incorporated herein by reference).

In some embodiments, the PD-1 inhibitor is an anti-mouse PD-1 mAb: clone J43, BioXCell (West Lebanon, N.H.).

In some embodiments, the PD-1 inhibitor is PD-L1 protein, PD-L2 protein or fragments, and the antibody MDX-1106 (ONO-4538) tested in clinical studies for the treatment of certain cancers (Brahmer et al., J Clin Oncol 2010 28(19): 3167-75, Epub 2010 Jun 1). Other blocking antibodies can be readily identified and prepared by one skilled in the art based on the known region of interaction between PD-1 and PD-L1/PD-L2, as discussed above. For example, a peptide corresponding to the IgV PD-1 or PD-L1/PD-L2 region (or part of this region) can be used as an antigen to produce blocking antibodies using methods known in the art.

In some embodiments, the immune checkpoint inhibitor is an IDO1 inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-IDO1 small molecule. Examples of small molecules against IDO1 include: INCB024360 Incyte, NSC 721782 (also known as 1-methyl-O-tryptophan) and F001287 Bristol Meyers Squibb.

In some embodiments, the immune checkpoint inhibitor is a LAG3 (CD223) inhibitor. In some embodiments, the immune checkpoint inhibitor is an anti-LAG3 antibody. In some embodiments, the immune checkpoint inhibitor is an anti-LAG3 monoclonal antibody. In other or additional embodiments, the immune checkpoint inhibitor is a human or humanized anti-LAG3 antibody. In additional embodiments, an anti-LAG3 antibody blocks the interaction of LAG3 with major histocompatibility complex (MHC) class II molecules. Examples of anti-LAG3 antibodies include: anti-Lag-3 antibody clone eBioC9B7W (C9B7W) from eBioscience; anti-Lag3 antibody LS-B2237 from LifeSpan Biosciences; IMP321 (ImmuFact) from Immutep; anti-Lag3 antibody BMS-986016; and chimeric antibody LAG 3 A9H12. In some embodiments, the anti-LAG3 antibody is an anti-LAG3 antibody disclosed in any of the following patent publications (incorporated herein by reference): WO2010019570; WO2008132601; or WO2004078928.

In some embodiments, the immune checkpoint inhibitor is an anti-TIM3 (also known as HAVCR2) antibody. In some embodiments, the immune checkpoint inhibitor is an anti-TIM3 monoclonal antibody. In other or additional embodiments, the immune checkpoint inhibitor is a human or humanized anti-TIM3 antibody. In additional embodiments, an anti-TIM3 antibody blocks the interaction of TIM3 with galectin-9 (Gal9). In some embodiments, the anti-TIM3 antibody is an anti-TIM3 antibody disclosed in any of the following patent publications (incorporated herein by reference): WO2013006490; WO201155607; WO2011159877; or WO200117057. In another embodiment, the TIM3 inhibitor is the TIM3 inhibitor disclosed in WO200905262 3.

In some embodiments, the immune checkpoint inhibitor is an anti-B7-H3 antibody. In one embodiment, the immune checkpoint inhibitor is MGA271.

- 56 043790

In some embodiments, the immune checkpoint inhibitor is an anti-MR antibody. In one embodiment, the immune checkpoint inhibitor is Lirilumab (IPH2101). In some embodiments, the anti-MR antibody blocks the interaction of KIR with HLA.

In some embodiments, the immune checkpoint inhibitor is an anti-CD137 antibody (also known as 4-1BB or TNFRSF9). In one embodiment, the immune checkpoint inhibitor is urelumab (BMS-663513, Bristol-Myers Squibb), PF-05082566 (anti-4-1BB, PF-2566, Pfizer), or XmAb-5592 (Xencor). In one embodiment, the anti-CD137 antibody is the antibody disclosed in US patent application US2005/0095244; the antibody disclosed in issued US Pat. No. 7,288,638 (such as 20H4.9-IgG4 [1007 or BMS-663513] or 20H4.9-IgG1 [BMS-663031]); the antibody disclosed in issued US Pat. No. 6,887,673 [4E9 or BMS-554271]; the antibody disclosed in issued US Pat. No. 7,214,493; the antibody disclosed in issued US Pat. No. 6,303,121; the antibody disclosed in issued US Pat. No. 6,569,997; the antibody disclosed in issued US Pat. No. 6,905,685; the antibody disclosed in issued US Pat. No. 6,355,476; the antibody disclosed in issued US Patent No. 6,362,325 [1D8 or BMS-469492; 3Н3 or BMS-469497; or 3E1]; the antibody disclosed in issued US Pat. No. 6,974,863 (such as 53A2); or an antibody disclosed in issued US Pat. No. 6,210,669 (such as 1D8, 3B8 or 3E1). In another embodiment, the immune checkpoint inhibitor is as disclosed in WO 2014036412. In another embodiment, the anti-CD137 antibody blocks the interaction of CD137 with CD137L.

In some embodiments, the immune checkpoint inhibitor is an anti-PS antibody. In one embodiment, the immune checkpoint inhibitor is bavituximab.

In some embodiments, the immune checkpoint inhibitor is an anti-CD52 antibody. In one embodiment, the immune checkpoint inhibitor is alemtuzumab.

In some embodiments, the immune checkpoint inhibitor is an anti-CD30 antibody. In one embodiment, the immune checkpoint inhibitor is brentuximab vedotin. In another embodiment, an anti-CD30 antibody blocks the interaction of CD30 with CD30L.

In some embodiments, the immune checkpoint inhibitor is an anti-CD33 antibody. In one embodiment, the immune checkpoint inhibitor is gemtuzumab ozogamicin.

In some embodiments, the immune checkpoint inhibitor is an anti-CD20 antibody. In one embodiment, the immune checkpoint inhibitor is ibritumomab tiuxetan. In another embodiment, the immune checkpoint inhibitor is ofatumumab. In another embodiment, the immune checkpoint inhibitor is rituximab. In another embodiment, the immune checkpoint inhibitor is tositumomab.

In some embodiments, the immune checkpoint inhibitor is an anti-CD27 (also known as TNFRSF7) antibody. In one embodiment, the immune checkpoint inhibitor is CDX-1127 (Celldex Therapeutics). In another embodiment, an anti-CD27 antibody blocks the interaction of CD27 with CD70.

In some embodiments, the immune checkpoint inhibitor is an anti-OX40 (also known as TNFRSF4 or CD134) antibody. In one embodiment, the immune checkpoint inhibitor is a mouse anti-OX40 IgG. In another embodiment, an anti-OX40 antibody blocks the interaction of OX40 with OX40L.

In some embodiments, the immune checkpoint inhibitor is an anti-glucocorticoid-inducible tumor necrosis factor receptor (GITR) antibody. In one embodiment, the immune checkpoint inhibitor is TRX518 (GITR, Inc.). In another embodiment, an anti-GITR antibody blocks the interaction of GITR with GITRL.

In some embodiments, the immune checkpoint inhibitor is an anti-inducible T-cell costimulator (ICOS, also known as CD278) antibody. In one embodiment, the immune checkpoint inhibitor is MEDI570 (Medlmmune, LLC) or AMG557 (Amgen). In another embodiment, an anti-ICOS antibody blocks the interaction of ICOS with ICOSL and/or B7-H2.

In some embodiments, the immune checkpoint inhibitor is an inhibitor against BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2, or SLAM. As described elsewhere herein, an immune checkpoint inhibitor can be one or more of binding proteins, antibodies (or fragments or variants thereof) that bind to immune checkpoint molecules, nucleic acids that reduce the expression of immune checkpoint molecules, or any other molecules that bind to immune control molecules

- 57 043790 points (i.e. small organic molecules, peptidomimetics, aptamers, etc.). In some cases, the BTLA (CD272) inhibitor is HVEM. In some cases, the CD160 inhibitor is HVEM. In some cases, the 2B4 inhibitor is CD48. In some cases, the LAIR1 inhibitor is collagen. In some cases, the TIGHT inhibitor is CD112, CD113 or CD155. In some cases, the CD28 inhibitor is CD80 or CD86. In some cases, the LIGHT inhibitor is HVEM. In some cases, the DR3 inhibitor is TL1A. In some cases, the CD226 inhibitor is CD155 or CD112. In some cases, the CD2 inhibitor is CD48 or CD58. In some cases, SLAM is self-inhibiting, and the inhibitor of SLAM is SLAM.

In some embodiments, the immune checkpoint inhibitor inhibits a checkpoint protein that includes, but is not limited to, CTLA4 (cytotoxic T lymphocyte antigen 4, also known as CD152), PD-L1 (programmed cell death ligand 1, also known as CD274), programmed cell death protein PDL2 2), PD-1 (programmed cell death protein 1, also known as CD279), B-7 family of ligands (B7-H1, B7-I3, B7-H4) BTLA (B and T lymphocytes, also known as CD272), HVEM, TIM3 (T cell membrane protein 3), GAL9, LAG 3 (lymphocyte activation gene 3; CD223), VISTA, KIR (killer immunoglobulin receptor), 2B4 (also known as CD244), CD160, CGEN-15049, CHK1 (checkpoint kinase 1), CHK2 (checkpoint kinase 2), A2aR (adenosine A2a receptor), CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD226, CD276 , DR3, GITR, HAVCR2, HVEM, IDO1 (indoleamine 2,3-dioxygenase 1), IDO2 (indoleamine 2,3-dioxygenase 2), ICOS (inducible T cell costimulator), LAIR1, LIGHT (also known as TNFSF14, member TNF family), MARCO (macrophage receptor with collagen structure), OX40 (also known as tumor necrosis factor receptor superfamily member 4, TNFRSF4 and CD134) and its ligand OX40L (CD252), SLAM, TIGHT, VTCN1 or a combination thereof.

In some embodiments, the immune checkpoint inhibitor interacts with a checkpoint protein ligand including CTLA-4, PDL1, PDL2, PD1, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1, CHK2, A2aR, B-7 family ligand, CD2, CD27, CD28, CD30, CD40, CD70, CD80, CD86, CD137, CD226, CD276, DR3, GITR, HAVCR2, HVEM, IDO1, IDO2, ICOS (inducible T cell costimulator), LAIR1, LIGHT, MARCO (macrophage receptor with collagen structure), OX40, SLAM, TIGHT, VTCN1 or a combination thereof.

In some embodiments, the immune checkpoint inhibitor inhibits a checkpoint protein including CTLA-4, PDL1, PD1, or a combination thereof.

In some embodiments, the immune checkpoint inhibitor inhibits a checkpoint protein including CTLA-4 and PD1 or a combination thereof.

In some embodiments, the immune checkpoint inhibitor includes pembrolizumab (MK 3475), nivolumab (BMS-936558), pidilizumab (CT 011), AMP 224, MDX-1 105, durvalumab (MEDI4736), MPDL3280A, BMS-936559, IPH2101, TSR -042, TSR-022, ipilimumab, lirilumab, atezolizumab, avelumab, tremelimumab, or a combination thereof.

In some embodiments, the immune checkpoint inhibitor is nivolumab (BMS-936558), ipilimumab, pembrolizumab, atezolizumab, tremelimumab, durvalumab, avelumab, or a combination thereof.

In some embodiments, the immune checkpoint inhibitor is pembrolizumab.

Diagnostic and prophylactic compositions.

The NaPi2b antibody conjugates disclosed herein are used in diagnostic and prophylactic formulations. In one embodiment, the NaPi2b antibody conjugate disclosed herein is administered to patients at risk of developing one or more of the aforementioned diseases, such as, for example, but not limited to, cancer. The susceptibility of a patient or organ to one or more of the above indications can be determined using genotypic, serological or biochemical markers.

In another embodiment of the disclosure, the NaPi2b antibody conjugate disclosed herein is administered to humans diagnosed with a clinical indication associated with one or more of the above diseases, such as, for example, but not limited to, cancer. Following diagnosis, the NaPi2b antibody conjugate disclosed herein is administered to mitigate or reverse the effects of a clinical indication associated with one or more of the above-mentioned diseases.

In another embodiment of the disclosure, a method of identifying a patient with ovarian cancer amenable to treatment with the conjugates disclosed herein includes measuring the status of certain characteristics in a tumor sample obtained from the patient, and identifying the patient for treatment based on the status of certain characteristics in the tumor sample.

In another embodiment of the disclosure, a method of identifying a patient with NSCLC amenable to treatment with the conjugates disclosed herein includes measuring the status of certain characteristics in a tumor sample obtained from the patient, and identifying the patient for treatment based on the status of certain characteristics in the tumor sample.

- 58 043790

The antibodies disclosed herein can also be used in the detection of NaPi2b in patient samples and accordingly can be used as diagnostic agents. For example, the NaPi2b antibodies disclosed herein are used in in vitro tests, such as ELISA, to detect levels of NaPi2b in a patient sample.

In one embodiment, the NaPi2b antibody disclosed herein is immobilized on a solid support (eg, well(s) of a microtiter plate). The immobilized antibody serves as an immobilized antibody for any NaPi2b that may be present in the test sample. Before contacting the immobilized antibody with the patient sample, the solid support is rinsed and treated with a blocking agent, such as milk protein or albumin, to prevent nonspecific adsorption of the analyte.

The well is then treated with a test sample suspected of containing the antigen or a solution containing a standard amount of antigen. Such a sample, for example a serum sample from a patient, is suspected of having levels of circulating antigen that are considered diagnostic for the pathology. After rinsing the test sample or standard, the solid support is treated with a detectably labeled second antibody. The labeled second antibody serves as a detection antibody. The level of detectable label is measured and the concentration of NaPi2b antigen in the test sample is determined for comparison with a standard curve constructed from standard samples.

It should be understood that based on the results obtained using the NaPi2b antibodies disclosed herein, in an in vitro diagnostic test, it is possible to determine the stage of a patient's disease based on NaPi2b antigen expression levels. For a given disease, blood samples are collected from patients who are diagnosed at various stages of disease progression and/or who are at various points in the therapeutic treatment of the disease. Using a population of samples that provide statistically significant results for each stage of progression or therapy, a range of antigen concentrations is determined that can be considered representative of each stage.

All publications and patent documents cited herein are incorporated herein by reference as if each such publication or document were specifically and individually designated as being incorporated herein by reference. Citation of publications and patent documents is not intended as an admission that any or all of them constitute relevant prior art, nor does it constitute an admission as to content or date. Although the present disclosure has been described by written description, one skilled in the art will appreciate that the disclosure may be practiced in a variety of embodiments and that the foregoing description and examples below are presented for purposes of illustration only and are not intended to limit the following claims.

Examples

The following worked examples illustrate linkers, drug molecules and PBRMs and methods for their preparation. They are not intended to be limiting, and one skilled in the art will appreciate that other reagents or methods may be used.

Abbreviations.

The following abbreviations are used in the following reaction schemes and synthetic examples. This list is not an exhaustive list of abbreviations used in the application, since other standard abbreviations understood by one skilled in the art of organic synthesis may also be used in the synthesis schemes and examples.

AF-HPA - Auristatin F-hydroxypropylamide,

FBS - fetal bovine serum,

MMAE - monomethyl auristatin E,

NaPi2b - sodium phosphate cotransporter type II,

NSCLC - non-small cell lung cancer.

General information.

CDRs were identified according to the Kabat numbering scheme.

Tumor growth inhibition (%TGI) was defined as the percentage difference in mean tumor volumes (MTVs) between treatment and control groups.

The effectiveness of treatment was determined by the frequency and amplitude of tumor size regression observed during the study. Treatment can cause partial regression (PR) or complete regression (CR) of the tumor in the animal. In the PR response, tumor volume was 50% or less of its volume on day 1 for three periodic measurements during the study and was equal to or greater than 13.5 mm ³ for one or more of these three measurements. In the CR response, tumor volume was less than 13.5 mm ³ for three periodic measurements throughout the study. An animal with a CR response at the end of the study was further classified as a tumor-free survivor (TFS). Animals were tested for regression reactions.

AF-HPA was prepared in a manner similar to that described in US Pat. No. 8,685,383, Example 48.

Anti-NaPi2b antibody (XMT-1535) was prepared using SEQ ID NO: 38 and SeQ ID NO: 39 from US Patent 8,603,474B2.

- 59 043790

Antibody aHTu-NaPi2b (10H1.11.4B) was prepared using SEQ ID NO: 80 and SEQ ID NO: 81 from US Patent 8,535,675B2.

HPLC purification was carried out on a semipreparative Phenomenex Gemini 5 µm 110 A, 250x10 mm, 5 µm column.

SEC was performed on a Tosoh Biosciences TSK gel G4000 column (7.8 mm x 30 cm, 10 μm) or Superose 12 (GE Healthcare).

WCX was performed on a ProPac WCX-10 column (94 mm x 250 mm) (ThermoFisher).

Whenever possible, the drug content of the conjugates was determined spectrophotometrically, otherwise LC/MS or 1H-NMR was performed to quantify the drug content.

The protein content of the protein-polymer-drug conjugate was determined spectrophotometrically at 280 nm or by ELISA.

The molecular weights of the polymer conjugates (reported as apparent weight average molecular weights or peak molecular weights) were determined by SEC with either polysaccharide or protein molecular weight standards. More specifically, for polymer or polymer-drug conjugates, a polysaccharide molecular weight standard was used, and for protein-polymer-drug conjugates, protein standards are used. Unless specifically indicated, the stated molecular weight of the polymer carrier is the weight average molecular weight of PHF; and for the polymer-drug conjugate and the polymer-protein-drug conjugate, the molecular weight is the peak molecular weight. NaPi2b-targeting antibody-polymer-drug conjugates have peak molecular weights ranging from approximately 170 kDa to approximately 250 kDa. Synthesized/measured polymers and polymer conjugates typically have a polydispersity of <1.5.

NaPi2b-targeting antibody-polymer-drug conjugates were separated from the remaining unreacted polymer-drug conjugates (preparative-WCX HPLC). If necessary, additional purification by size exclusion chromatography was performed to remove any aggregated NaPi2b-targeting antibody-polymer-drug conjugates. In general, NaPi2b polymer-drug-antibody conjugates typically contain <5% (w/w) aggregate fraction as defined by SEC; <0.5% (w/w, e.g. <0.1% w/w) free (unconjugated) drug as determined by RP HPLC or LC-MS/MS; <1% (w/w) free polymer-drug conjugate as determined by RP HPLC and <2% (w/w, e.g., <1% w/w) unconjugated NaPi2b as determined by HIC HPLC and /or WCX HPLC. Reduced or partially reduced antibodies were obtained using procedures described in the literature, see, for example, Francisco et al., Blood 102 (4): 1458-1465 (2003). All drug concentrations (conjugated and unconjugated) were determined by LC-MS/MS.

To determine the pharmacokinetics of NaPi2b-targeting antibody-polymer drug conjugates, assays were developed to measure the plasma concentration of NaPi2b antibody-PHF-AF-HPA conjugate (i.e., AF-HPA conjugate) and the concentration of released unconjugated AF-HPA and AF (free drugs ). To determine plasma concentrations of free drugs, an oxidized plasma sample was treated with acetonitrile to precipitate plasma proteins, and the antibody-drug conjugate and acetonitrile-containing supernatant were analyzed for free drugs by LCMS/MS. To determine the concentration of the AF-HPA conjugate, the oxidized plasma sample was subjected to hydrolysis in an alkaline medium, followed by oxidation and precipitation of the protein with acetonitrile. The acetonitrile-containing supernatant containing released AF-HPA and AF was analyzed by LC-MS/MS. Standard curves for free drugs and AF-HPA conjugate in plasma were linear across the concentration series 1–3000 ng/mL and 10–20,000 ng/mL, respectively. Total NaPi2b concentration was determined by ELISA.

General procedures.

General Procedure A. Conjugation of polymer to linker or drug.

In general, the conjugation of a polymer (PHF-BA or PHF-GA) with an amine-containing linker, such as, for example, EG2-maleimide, or an amine-containing drug linker, such as, for example, AF-HPA-Ala, HPA-Ala, is carried out in water or a mixture of organic solvent/water 10-90% in the presence of an activating agent, such as, for example, EDC.HCl. Typical organic solvents include, but are not limited to, water-miscible solvents such as, for example, DMSO, DMF, DMA, NMP, propylene glycol and ACN. A coactivator such as NHS is added to speed up the coupling. The polymer is first mixed with an amine compound, followed by the addition of a coactivator (NHS) and then the addition of an activator (EDC.HCl). The reaction is carried out at 0-10°C, pH from 4.5 to 7.5 for 1 hour to 24 hours at ambient temperature. The resulting polymer-conjugated product is purified by diafiltration or SEC. The product is concentrated to 2-50 mg/ml, the pH is adjusted to a value from 4.5 to 6.5 to maintain drug stability

- 60 043790 polymer linker, and the conjugate is stored frozen at -20 to -80°C until further use.

Conjugation of the polymer with the amine-containing linker or drug can be carried out sequentially in any order or simultaneously.

General procedure B. Partial selective protein reduction (NaPi2b antibody).

Partial selective reduction of intrachain disulfide groups or unpaired disulfide in the corresponding NaPi2b antibody prior to conjugation with the polymer-drug conjugate is achieved using a reducing agent such as, for example, TCEP, DTT or βmercaptoethanol. When reduction is carried out with an excess of reducing agent, the reducing agent is removed prior to conjugation by diafiltration or SEC. The extent to which NaPi2b disulfide groups are converted to reactive sulfhydryl groups depends on the stoichiometry of NaPi2b, the reducing agent, pH, temperature and/or reaction time. When some, but not all, of the disulfide groups in PBRM are reduced, the reduced PBRM is partially reduced NaPi2b.

General Procedure C. Conjugation of Partially Reduced NaPi2b-Targeting Antibody to a Polymer-Drug Conjugate.

Conjugation of the partially reduced NaPi2b-targeting antibody to the polymer-drug conjugate is carried out under neutral or weakly basic conditions (pH 6.5-8.5) at antibody concentrations of 1-10 mg/ml and polymer-drug conjugate concentrations of 0.5-10 mg/mL. ml. The polymer-drug conjugate is typically used in a 1- to 5-fold excess relative to the desired protein-polymer-drug conjugate stoichiometry. When an antibody is conjugated to the maleimido group of a polymer-drug conjugate, the conjugation is completed, if necessary, by adding a water-soluble maleimido-blocking compound such as, for example, N-acetyl cysteine, cysteine methyl ester, N-methyl cysteine, 2-mercaptoethanol, 3-mercaptopropane acid, 2-mercaptoacetic acid, mercaptomethanol (i.e. HOCH2SH), benzyl thiol, etc.

The resulting NaPi2b-targeting antibody-polymer-drug conjugate is typically purified by diafiltration to remove any unconjugated polymer-drug conjugate, unconjugated drug, and small molecule impurities. Alternatively or additionally, suitable chromatographic separation procedures such as, for example, size exclusion chromatography, hydrophobic interaction chromatography, such as ion chromatography, for example, WCX chromatography; Reverse phase chromatography, hydroxyapatite chromatography, affinity chromatography, or a combination thereof, can be used to purify the NaPi2b targeting antibody-polymer-drug conjugate. The resulting purified NaPi2b polymer-drug conjugate is typically formulated in a buffer at pH 5.0-6.5.

Example 1. Synthesis of XMT-1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala)))

XMT-1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates were prepared using the procedure described in US patent application publication US-2015-0104407-A1. In table I details of antibody-polymer-drug conjugates are given.

Table I

Example No. DAR (drug:antibody ratio) 1Ά From approximately 8:1 to approximately 12:1 1B From approximately 10:1 to approximately 14:1 1C From approximately 11:1 to approximately 15:1

The XMT-1535-(EG2-MI-(1O kDa PHF-BA-(AF-HPA-Ala))) conjugates have peak molecular weights ranging from approximately 180 kDa to approximately 250 kDa. Synthesized/measured polymer and polymer conjugates typically have a polydispersity of <1.5. The polymer-drug conjugates (i.e., a drug-carrying polymer chain attached to an antibody) contained from about 25 mol.% to about 35 mol.% beta-alanine, from about 7.0 mol.% to

- 61 043790 about 10 mol.% AF-HPA-Ala and from about 1.5 mol.% to about 4 mol.% EG2MI.

Example 2. Synthesis of (1OH1.11.4B)-(EG2-MI-(1O kDa PHF-BA-(AF-HPA-Ala)))

(10:11.1.4B)-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) (non-binding control) conjugates were prepared using the procedure described in US Patent Application Publication US-20150104407 -A1. In table II details of antibody-polymer-drug conjugates are given.

_____________________________________Table II

Example No. DAR (drug:antibody ratio) 2A From approximately 10:1 to approximately 14:1 2B From approximately 14:1 to approximately 19:1 20 From approximately 9:1 to approximately 13:1

The (1OH1.11.4B)-(EG2-MI-(1O kDa PHF-BA-(AF-HPA-Ala))) conjugates had peak molecular weights ranging from approximately 170 kDa to approximately 230 kDa. Synthesized/measured polymer and polymer conjugates typically have a polydispersity of <1.5. The polymer-drug conjugates (ie, a drug-carrying polymer chain attached to an antibody) contained from about 25 mol.% to about 35 mol.% beta-alanine, from about 7 mol.% to about 10 mol.% AF-HPA-Ala and from about 1.5 mol.% to about 4 mol.% EG2MI.

Example 3. Synthesis of rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala)))

Rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates (non-binding control) were prepared using the procedure described in US patent application publication US-20150104407-A1. In table III gives details of antibody-polymer-drug conjugates.

________________________________________Table III

Example No. DAR (drug:antibody ratio) BEHIND From approximately 15:1 to approximately 21:1 ZV From approximately 14:1 to approximately 19:1

Rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates had peak molecular weights ranging from approximately 170 kDa to approximately 230 kDa. Synthesized/measured polymer and polymer conjugates typically have a polydispersity of <1.5. The polymer-drug conjugates (ie, a drug-carrying polymer chain attached to an antibody) contained from about 25 mol.% to about 35 mol.% beta-alanine, from about 7 mol.% to about 10 mol.% AF-HPA-Ala and from about 1.5% to about 4 mol.% EG2-MI.

- 62 043790

Example 4 Synthesis of (10H1.11.4B)-(MC-VC-PABA-MMAE)

(10H1.11.4B)-(maleimide-VC-PABA-MMAE) conjugates were prepared using the procedure described in Doronina et al., Nature biotechnology, 21: 778-784 (2003). In table IV details of antibody-drug conjugates are given.

Table IV

Example No. DAR (drug:antibody ratio) 4Ά 4:1 4B 3:1

Example 5. Testing of NaPi2b-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates for cytotoxicity.

Example 1A, XMT-1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); example 2C, (10H1.11.4B)-(EG2MI-(10 kDa PHF-BA-(AF-HPA-Ala))); example 3A, Rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) and AF-HPA or example 1C, XMT-1535-(EG2-MI-(1O kDa PHF-BA- (AF-HPA-Ala))); example 2C, (10H1.11.4B)-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); example 3B, rituximab-(EG2-MI-(10 kDa PHFBA-(AF-HPA-Ala))); and AF-HPA were evaluated for their antiproliferative properties in tumor cell lines in vitro using Cell Titer-Glo (Promega Corp). OVCAR3 (ovarian adenocarcinoma cell line, unamplified, ATCC, Cat. #HTV-161) was cultured in RPMI medium with 20% FBS. TOV-21G (human ovarian adenocarcinoma cell line, unamplified, ATCC, Cat.# CRL-11730) was cultured in a 1:1 mixture of MCDB 105 medium containing a final concentration of 1.5 g/L sodium bicarbonate and 199 medium containing a final concentration of 2.2 g/l sodium bicarbonate with 15% FBS. IGROV1 (ovarian adenocarcinoma cell line, not amplified) was cultured in RPMI medium with 10% FBS. HCC-4006 (human lung cancer cell line, non-amplified, ATCC, Cat. # ATCC® CRL-2871™) was cultured in RPMI medium with 10% FBS.

For the cytotoxicity test, cells were seeded at a density of 5000 cells per well in 96-well plates and allowed to attach during overnight incubation at 37°C in the presence of 5% CO ₂ . The media were then replaced with new media containing a range of conjugates of Examples 1A, 2C, 3A or AF-HPA (100 nM to 0.1 pM) or Examples 1C, 2C, 3B or AF-HPA (100 nM to 0.1 pM) , and the cells were incubated for 72 hours or 6 days at 37°C in the presence of 5% CO ₂ . Cell survival was measured using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, WI) as described in the kit instructions. Cell viability was normalized to the untreated control group and expressed as a percentage. Values were plotted and IC ₅₀ values were calculated using GraphPad Prism software (San Diego, CA) using a suitable 4-parameter slope-variable dose-response curve algorithm. In table VA and VB give illustrative results for the cytotoxicity of the conjugates and AF-HPA.

Table VA

Tumor cell line Number of NaP12b receptors/cell Example 1A (nM) Example 2C (nM) Example FOR (nM) AF-HPA ¹ (nM) OVCAR-3 -32000 0.002 0.014 0.26 4.18 IGROV1 -35000 -50 10, 62 53.04 28.61 TOV-21G -10000 0.04 2.03 2.76 2.07 NSS-4006 -52000 0.13 2.96 1, 83 0.68

Table VB

Tumor cell line Number of receptors NaP±2b/cell Example 1C (nM) Example 2C (nM) Example PV (nM) AFHPA ¹ (nM) OVCAR-3 -32000 0.01 0.11 1.13 1.34 IGROV1 -35000 0.38 2.73 37.3 25.94 TOV-21G -10000 6, 68 41.54 39, 68 12.70 NSS-4006 -52000 0.35 3.17 5.29 7.83

1=Payload equivalent.

As shown in table. VA and VB, antibody-polymer-drug conjugates XMT-1535

- 63 043790 are more potent than antibody-polymer-drug conjugates (10H1.11.4B) or antibody-polymer-drug conjugates with Rituximab in all cell lines tested.

Example 6 Tumor growth response to NaPi2b-targeting antibody-polymer drug conjugates.

Female CB-17 SCID mice were subcutaneously implanted with fragments of OVCAR-3 (n=10 for each group) or non-small cell lung cancer (n=10 for each group). Test compounds or vehicle were administered IV as a single dose on day 1 or as indicated. Tumor size was measured at the time points indicated in Fig. 1-3 using digital calipers. Tumor volume was calculated and used to determine tumor growth delay. Mice were sacrificed when tumors reached a size of 1000 mm ³ . Tumor volumes are presented as means ±SEM for each group.

In fig. 1 shows the results for the carcinogenic effect in mice that were subcutaneously implanted with OVCAR-3 tumor fragments (n=10 for each group) after i.v. administration of the vehicle; XMT-1535; example 3A, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); example 4A ((10H1.11.4B) - (MC-VC-PABA-MMAE)); example 2C (10H1.11.4B)-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) or example 1B, XMT 1535-(EG2-MI-(10 kDa PHF-BA-( AF-HPA-Ala))), each at 3 mg/kg (equivalent to a dose of 0.21 mg/kg auristatin payload) as a single dose on day 1. Vehicle, XMT-1535; The conjugates of Example 3A and Example 4A all showed an increase in tumor volume, none of the treatments showed at least partial regression. The conjugates of Example 2C and Example 1B each showed a reduction in tumor volume. Example 2C conjugate had a single partial regression response, while Example 1B conjugate had 70% regressions consisting of 4 partial regressions and 3 complete regressions. The median time to outcome (TTE) was 57 days, corresponding to a 118% tumor growth delay (TGD) (30.9 days). On Day 23, tumor growth inhibition (TGI) results were 95%. This was statistically different from control (P < 0.001, Mann-Whitney analysis) and was above 60% of the potential threshold for therapeutic activity.

In fig. 2 shows the results for the carcinogenic effect in mice subcutaneously implanted with OVCAR-3 tumor fragments (n=10 for each group) after i.v. administration of vehicle; Example 3B, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at a dose of 3 mg/kg administered weekly for 3 weeks; example 4B ((10H1.11.4B)-(MC-VC-PABA-MMAE)) at a dose of 3 mg/kg administered weekly for 3 weeks; Example 1C, XMT 1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at a dose of 3 mg/kg as a single dose on day 1 or at a dose of 3 mg/kg administered weekly within 3 weeks; or example 1C, XMT 1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at a dose of 5 mg/kg (equivalent to a dose of 0.36 mg/kg auristatin payload) as a single doses on day 1. The vehicle and conjugates of Example 3B and Example 4B all showed an increase in tumor volume, none of the treatments showed at least partial regression. For the conjugate of Example 4B, tumor growth inhibition (TGI) at day 22 was statistically different from control (P<0.001, Mann-Whitney analysis) and was 63%, slightly above the 60% potential threshold for therapeutic activity. The conjugate of Example 1B was the most active in this study at all treatment doses, producing 80-100% regression. For this conjugate on day 22, the TGI result was statistically different from the control (P < 0.001, Mann-Whitney analysis) and was 96-97%.

In fig. Figure 3 shows the results for the carcinogenic effect in a patient-derived xenograft model (CTG-0860) of the KRAS mutant non-small cell lung cancer (n=10 for each group) after i.v. administration of the vehicle; example 3B, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); example 4B ((10H1.11.4B)-(MC-VC-PABA-MMAE)); or Example 1C, XMT 1535-(EG2-MI-(10 kDa PHFBA-(AF-HPA-Ala))), each at a dose of 3 mg/kg administered weekly for 3 weeks. Carrier; the conjugates of Example 3B and Example 4B each showed an increase in tumor volume. Comparison of tumor volumes on day 17 showed that the tumor volume of Example 1C was significantly lower than that of the vehicle control group (p<0.05). Analysis of data from day 0 to day 17 showed that the Example 1C 3 mg/kg group had a significantly lower tumor volume compared to the vehicle control group (p<0.01). There were 2 PRs in the Example 1C group at 3 mg/kg. There were no deaths in either group during the study.

In fig. Figure 4 shows the results for the carcinogenic effect in a patient-derived xenograft model (CTG-0852) of non-small cell lung cancer (n=8 for each group) after IV administration of the vehicle; example 3B, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); example 4B ((10Н1.11.4В) - (MC-VC-PABA-ММАЭ)); Example 1C, XMT 1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPAAla))), each at a dose of 3 mg/kg administered weekly for 3 weeks, or Example 1C, XMT 1535(EG2- MI-(10 kDa PHF-BA-(AF-HPA-Ala))), at a dose of 5 mg/kg as a single dose on day 1. Analysis of data from day 0 to day 62 showed that all groups had significantly more low tumor volume compared to vehicle controls (p<0.01 or p<0.0001). There were 3 PRs in the Example 4B group at 3 mg/kg. There were 7 PRs in the Example 1C 5 mg/kg group and 5 PRs, 1 CR and 2 TFS in the Example 1C 3 mg/kg group. No PRs, CRs or TFS in Example 3B group at 3 mg/kg. There were no deaths

- 64,043,790 EU in any of the groups during the study.

In fig. Figure 5 shows the results for the carcinogenic effect in a patient-derived xenograft model of non-small cell lung cancer (ST1906, BRAF K601E mutation) (n=6 for each group) after i.v. administration of the vehicle; and Example 1C, XMT 1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPAAla))), at a dose of 3 mg/kg or 6 mg/kg administered weekly for 3 weeks. The vehicle showed an increase in tumor volume, while the conjugate of Example 1C showed strong proto-tumor activity. Six of the six showed TFS at day 60 after treatment with the conjugate of Example 1C.

In fig. Figure 7 shows the results for the carcinogenic effect in a patient-derived xenograft model (CTG-0178) of non-small cell lung cancer (n=8 for each group) after i.v. administration of the vehicle; example 3B, rituximab-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))); example 4B ((1OH1.11.4B)-(MC-VC-PABA-MMAE)); or Example 1C, XMT 1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPAAla))), each at a dose of 3 mg/kg administered weekly for 3 weeks, or Example 1C, XMT 1535(EG2 -MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at a dose of 5 mg/kg as a single dose. Carrier; the conjugates of Example 3B and Example 4B each showed an increase in tumor volume. Example 1C conjugate at 5 mg/kg and 3 mg/kg had significantly lower tumor growth compared to vehicle control (p<0.05). There were no deaths in either group during the study.

In fig. Figure 8 shows the results for the carcinogenic effect in a patient-derived xenograft model of non-small cell lung cancer (ST1437, EGFR wild type, Alk mutation wild type) (n=6 for each group) after i.v. administration of the vehicle; and Example 1C, XMT 1535-(EG2-MI-(10 kDa PHFBA-(AF-HPA-Ala))), at a dose of 3 mg/kg or 6 mg/kg administered weekly for 3 weeks. The vehicle showed an increase in tumor volume, while the conjugate of Example 1C showed antitumor activity. One of the six showed TFS at day 60 after treatment with the conjugate of Example 1C.

In fig. Figure 9 shows the results for the carcinogenic effect in a patient-derived xenograft model of non-small cell lung cancer (ST742, EMLK-Alk translocation) (n=6 for each group) after i.v. administration of the vehicle; and Example 1C, XMT 1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at a dose of 3 mg/kg or 6 mg/kg administered weekly for 3 weeks. The vehicle showed an increase in tumor volume, while the conjugate of Example 1C showed antitumor activity. Five of the six showed TFS at day 60 after treatment with the conjugate of Example 1C.

In fig. 10 shows the results for the carcinogenic effect in a patient-derived xenograft model of non-small cell lung cancer (ST1243, EGFR amplification, KIT amplification, CDKN2A deletion) (n=6 for each group) after i.v. administration of vehicle; and Example 1C, XMT 1535-(EG2MI-(10 kDa PHF-BA-(AF-HPA-Ala))) at a dose of 3 mg/kg or 6 mg/kg administered weekly for 3 weeks. The vehicle showed an increase in tumor volume, while the conjugate of Example 1C showed antitumor activity.

Example 7. Cell binding affinity of XMT-1535 and NaPi2b-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) conjugates using flow cytometry.

Cell surface binding of Example 1C, XMT 1535-(EG2-MI-(10 kDa PHF-BA-(AF-HPA-Ala))) and anti-NaPi2B antibody XMT 1535 to antigen expressing cells was assessed by flow cytometry. TOV-21G or OVCAR3 cells were grown overnight to approximately 90% confluency in the media, then released from the plate surface by treatment with Trypsin-EDTA (GibcoThermo-Fisher-Scientific, USA). Individual cells were washed once with ice-cold media containing 6% goat serum and resuspended in the same media. Aliquots of 50,000 cells were added to the wells of a 96-well plate with a V-shaped bottom and incubated with a range of concentrations of test compounds (0.23-500 nM) in 100 μl of medium (1:1 mixture of MCDB105 (1.5 g/l sodium bicarbonate) and medium 199 (2.2 g/l sodium bicarbonate plus 15% FBS) with 6% goat serum on ice for 3 hours. Cells were then washed once with ice-cold PBS and resuspended in 150 μl medium with 2 % goat serum and 6 μg/ml secondary fluorescently labeled antibody, Alexa Fluor® 647, labeled goat anti-human IgG (Life Technologies, Cat# A-21445) for 1 hour on ice. Cells were washed once with ice-cold PBS and suspended in 150 μl of ice-cold PBS with 1% paraformaldehyde. The amount of fluorescence bound per cell was determined by running 5000 cells for each treatment on a MACSQuant flow cytometer (Miltenyi Biotec, Bergisch Gladbach, Germany). The mean fluorescence value for each treatment was plotted and the binding constant, Kd, was calculated for each test compound by GraphPad Prism software with nonlinear regression using a single site specific binding model.

Table VI

Connection under test K _D (nM) TOV-21G K _D (nM) OVCAR3 XMT 1535 2.46 6, 59 Example 1C 3, 97 3, 84

As shown in table. VI, Naked XMT-1535 antibody and XMT-1535-antibody-polymer-drug conjugate have similar binding affinities for the cell line tested.

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Example 8: Toxicological and pharmacokinetic study in primates.

A single dose toxicity study was performed in cynomolgus monkeys with Example 1C, XMT-1535-(EG2-MI-(1O kDa PHF-BA-(AF-HPA-Ala))). The conjugate was administered to groups of 2 animals/sex at doses of 0 mg/kg (group 1), 1.25 mg/kg (1074 μg/ ^m2 equivalent of auristatin payload; group 2), 2.5 mg/kg (2147 μg/m ^m2 equivalent auristatin payload; Group 3) or 5 mg/kg (or 4294 mcg/ ^m2 equivalent auristatin payload; Group 4) as a single IV infusion. Blood was collected for hematology, coagulation and serum chemistry analysis from all groups on days -7, 3, 8, 15 and 22. Blood samples were also collected pre-dose, 10 min, 1, 6, 24, 48, 72, 96 and 168 hours after the end of the IV infusions, and on days 15 and 22 to determine pharmacokinetics.

The conjugate of Example 1C was well tolerated in cynomolgus monkeys when administered at doses up to 5 mg/kg (ie, 4294 μg/m ² equivalent of auristatin payload), with no observed target-mediated toxicity and limited adverse effects. There was no evidence of bone marrow toxicity. No morbidity was observed in groups 2, 3 and 4. The results of the study are shown in table. VII, below.

Table VII

Final showdown Autopsy after recovery Organ 1.25 mg/kg 2.5 mg/kg 5 mg/kg 1.25 mg/kg 2.5 mg/kg 5 mg/kg Bone brain Absence no Absences cozy Absent Absences cozy Absences cozy Absent Liver* Absence no Absences cozy Minimal apoptosis of hepatocytes (1 female) Absences cozy Absences cozy Absent Seeds Absence no Absences cozy Absent Absences cozy Absences cozy Absent Lung Absence no Absences cozy Minimal mixed infiltration of inflammatory cells (1 male) Absences cozy Absences cozy Minimal mixed infiltration of inflammatory cells (1 male) Uric bubble Absence no Absences cozy Minimal apoptosis in the mucosa; random mitotic figures (1 male) Absent Absences cozy Absent Stomach Minimal infiltration of neutrophils into the mucosa (1 female) Absences cozy Soft focal ulceration (1 male) Absent Absences cozy Absent Cecum Absence no Soft focal ulceration (1 female) Absent Absent Absent Absent

* Minimal Kupffer cell hypertrophy with occasional mitotic figures was seen in all test compound-treated animals at final necropsy and post-mortem examination (no side effects observed).

One animal in all groups suffered adverse changes affecting the gastrointestinal tract, as determined by final necropsy. In recovered animals, test compound-related signatures (one in each tissue) were detected in the liver (groups 2-4), spleen (groups 3 and 4) and lung (group 4), but none were considered adverse. There was a transient increase in neutrophil counts and monocytosis in association with increased globulin concentrations, decreased albumin levels, and a decreased albumin-to-globulin ratio. In addition, increased levels of creatine kinase (CK) and aspartate aminotransferase (AST) were observed. None of the detected alterations persisted during the recovery period. There was no myelosuppression or neutropenia. In addition, there were no significant abnormalities at necropsy and no weight loss. Based on efficacy and this toxicology study in primates, the therapeutic index for the conjugate of Example 1C, XMT-1535-(EG2-MI-(1O kDa PHF-BA-(AF-HPAAla))), was approximately 6.

Blood samples were analyzed by LC-MS/MS to determine total AF-HPA concentration and ELISA to determine total antibody concentration at each time point. Fig. 6A and 6B show plasma pharmacokinetics for total antibody and total drug

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Claims (24)

agents, respectively, in cynomolgus monkeys after administration of a single dose of the conjugate of Example 1C in an amount of 1.25 mg/kg (1074 μg/m ² equivalent of auristatin payload; group 2), 2.5 mg/kg (2147 μg/m ² equivalent auristatin payload; group 3) or 5 mg/kg (or 4294 μg/m ² equivalent auristatin payload; group 4). Table VIII gives a calculated AUC _{of 0-504 h} and half-life for total antibody and total AF-HPA. No free drugs (ie, unconjugated AF and unconjugated AF-HPA) were detected (MS/MS LC method LLOQ was 1 ng/mL). Table VIII Exposure on a glass AUCq-504 h Normalized dose (mcg"h/ml/mg/kg) Plasma half-life T, Average dose range (Days) Total antibody General medicine Total antibody General medicine Drug release 1407+99 157+13 8.8+0.7 5.2+0.2 11.6+0.9 The XMT-1535 polymer-drug conjugate had a half-life of ~9 days with an AUC _{of 0-504 h} ~1.4 mg-h/ml/mg/kg. Total AF-HPA had a half-life of ~5 days with an AUC _{of 0-504 h} ~157 μg-h/ml/mg/kg. The conjugate demonstrated good stability in plasma with minimal detection or absence of free drugs. Other embodiments. Although the invention has been described in conjunction with the detailed description, the foregoing description is intended to illustrate and not to limit the scope of the invention within the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the following claims. CLAIM 1. A conjugate for alleviating a symptom of cancer in a patient, comprising an isolated antibody that specifically binds to the extracellular region of SLC34A2, and one or more polymer backbones associated with the isolated antibody, wherein each one or more polymer backbones independently has the formula (Id) wherein the backbone comprises poly(1-hydroxymethylethylenehydroxymethylformal) (PHF) having a molecular weight ranging from 2 to 40 kDa; m is an integer from 1 to 300; m1 is an integer from 1 to 140; m ₂ is an integer from 1 to 40; m _3a is an integer from 0 to 17; m _3b is an integer from 1 to 8; the sum of m, m1, _m2 , _m3a and _m3b varies from 15 to 300 and the ratio between PHF and isolated antibody is 10 or less; terminal indicates direct attachment of one or more polymer backbones to the isolated antibody; one of Xa and _Xb is H and the other is a water-soluble protecting group for the maleimido group, or Xa and _Xb , together with the carbon atoms to which they are attached, form a carbon-carbon double bond; X is CH ₂ , O or NH and wherein the isolated antibody that specifically binds to the extracellular region of SLC34A2 includes a complementarity determining region heavy chain 1 (CDRH1) variable region comprising the amino acid sequence GYTFTGYNIH (SEQ ID NO: 5), a variable region of the heavy chain complementarity heavy chain region 2 (CDRH2), comprising the amino acid sequence AIYPGNGDTSYKQKFRG (SEQ ID NO: 6), variable region complementarity determining region heavy chain 3 (CDRH3), comprising the amino acid sequence GETARATFAY (SEQ ID NO: 7), variable region complementarity determining region light chain 1 (CDRL1), comprising the amino acid sequence SASQDIGNFLN (SEQ ID NO: 8), light chain complementarity determining region 2 variable region - 67 043790 (CDRL2), comprising the amino acid sequence YTSSLYS (SEQ ID NO: 9), complementarity determining region light chain variable region 3 (CDRL3), comprising the amino acid sequence QQYSKLPLT (SEQ ID NO: 10). 2. The conjugate of claim 1, wherein the isolated antibody that specifically binds to the extracellular region of SLC34A2 comprises a heavy chain variable sequence comprising the amino acid sequence of SEQ ID NO: 3 and a light chain variable sequence comprising the amino acid sequence of SEQ ID NO: 4. 3. The conjugate according to any one of claims 1 and 2, wherein the isolated antibody that specifically binds to the extracellular region of SLC34A2 comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 1 and a light chain comprising the amino acid sequence of SEQ ID NO: 2. 4. The conjugate according to any one of claims 1 to 3, wherein the isolated antibody that specifically binds to the extracellular region of SLC34A2 is a monoclonal antibody. 5. The conjugate according to any one of claims 1 to 4, wherein the isolated antibody that specifically binds to the extracellular region of SLC34A2 is a rabbit, mouse, chimeric, humanized or fully human monoclonal antibody. 6. The conjugate according to any one of claims 1 to 5, wherein the isolated antibody that specifically binds to the extracellular region of SLC34A2 is an IgG isotype. 7. The conjugate according to any one of claims 1 to 6, wherein the isolated antibody that specifically binds to the extracellular region of SLC34A2 is an IgG1 isotype. 8. The conjugate according to any one of claims 1 to 7, in which the sum of m, m _b m ₂ , m _3a and m _3b is from 15 to 150, m ₁ is an integer from 1 to 70, m ₂ is an integer from 1 to 20, m _3a is an integer from 0 to 9, m _3b is an integer from 1 to 8, PHF has a molecular weight ranging from 2 to 20 kDa, and the ratio between PHF and isolated antibody is an integer from 2 to 8 . 9. The conjugate according to any one of claims 1 to 8, in which the sum of m, m1, m ₂ , m _3a and m _3b is from 20 to 110, m1 is an integer from 2 to 50, m ₂ is an integer from 2 to 15, m _3a is an integer from 0 to 7, m _3b is an integer from 1 to 8, PHF has a molecular weight ranging from 3 to 15 kDa, and the ratio between PHF and isolated antibody is an integer from 2 to 6. 10. The conjugate according to any one of claims 1 to 9, in which the sum of m, m1, m ₂ , m ₃ and m ₄ is from 40 to 75, m1 is an integer from 2 to 35, m ₂ is an integer from 2 to 10, m _3a is an integer from 0 to 4, m _3b is an integer from 1 to 5, PHF has a molecular weight ranging from 5 to 10 kDa, and the ratio between PHF and isolated antibody is an integer from 2 to 4. 11. The conjugate according to claim 1, wherein each one or more polymer backbones independently has the formula (If) in which m is an integer from 1 to 300; m1 is an integer from 1 to 140; m ₂ is an integer from 1 to 40; m _3a is an integer from 0 to 17, m _3b is an integer from 1 to 8; the sum of m _3a and m _3b varies from 1 to 18 and the sum of m, m1, m ₂ , m _3a and m _3b varies from 15 to 300; terminal denotes the attachment of one or more polymer backbones to an isolated antibody that specifically binds to SLC34A2, wherein the isolated antibody that specifically binds to SLC34A2 is an isolated antibody comprising a complementarity determining region light chain 1 (CDRL1) variable region comprising the amino acid sequence SASQDIGNFLN (SEQ ID NO: 8); complementarity determining region light chain 2 (CDRL2) variable region comprising the amino acid sequence YTSSLYS (SEQ ID NO: 9); complementarity determining region light chain 3 (CDRL3) variable region comprising the amino acid sequence QQYSKLPLT (SEQ ID NO: 10); a complementarity determining region heavy chain 1 (CDRH1) variable region comprising the amino acid sequence GYTFTGYNIH (SEQ ID NO: 5); a complementary determining region heavy chain 2 (CDRH2) variable region comprising the amino acid sequence AIYPGNGDTSYKQKFRG (SEQ ID NO: 6); and a complementarity determining region heavy chain 3 (CDRH3) variable region comprising the amino acid sequence GETARATFAY (SEQ ID NO: 7); And - 68 043790 the ratio between PHF and antibody is 10 or less. 12. The conjugate according to claim 11, in which the PHF in formula (If) has a molecular weight ranging from 2 to 20 kDa, the sum of m, m1, _m2 , _m3a and _m3b is from 15 to 150, m1 is an integer from 1 to 70, m ₂ is an integer from 1 to 20, m _3a is an integer from 0 to 9, m _3b is an integer from 1 to 8, the sum of m _3a and m _3b is from 1 to 10, and the ratio between PHF and the isolated antibody that specifically binds to SLC34A2 is an integer from 2 to 8. 13. The conjugate according to claim 11 or 12, in which the PHF in formula (If) has a molecular weight ranging from 3 to 15 kDa, the sum of m, m1, _m2 , _m3a and _m3b is from 20 to 110, m1 is an integer from 2 to 50, m ₂ is an integer from 2 to 15, m _3a is an integer from 0 to 7, m _3b is an integer from 1 to 8, the sum of m _3a and m _3b is from 1 to 8, and the ratio between PHF and the isolated antibody that specifically binds to SLC34A2 is an integer from 2 to 8. 14. The conjugate according to any one of claims 11-13, in which the PHF in formula (If) has a molecular weight ranging from 5 to 10 kDa, the sum of m, m1, _m2 , _m3a and _m3b is from 40 to 75, m1 is an integer from 2 to 35, m ₂ is an integer from 2 to 10, m _3a is an integer from 0 to 4, m _3b is an integer from 1 to 5, the sum of m _3a and m _3b is from 1 to 5 , and the ratio between PHF and the isolated anti-antibody that specifically binds to SLC34A2 is an integer from 2 to 8. 15. The conjugate according to any one of claims 11-14, in which the PHF in formula (If) has a molecular weight ranging from 5 to 10 kDa, the sum of m, m1, _m2 , _m3a and _m3b is from 40 to 75, m1 is an integer from 2 to 35, m ₂ is an integer from 2 to 10, m _3a is an integer from 0 to 4, m _3b is an integer from 1 to 5, the sum of m _3a and m _3b is from 1 to 5 , and the ratio between PHF and the isolated antibody specifically binding to SLC34A2 is an integer from 2 to 6. 16. A pharmaceutical composition comprising a conjugate according to any one of claims 1 to 15 and a pharmaceutically acceptable carrier. 17. The method of obtaining a conjugate according to claim 1, including introducing an isolated antibody that specifically binds to SLC34A2, in reaction with a polymer backbone of formula (Id), such that a conjugate is formed in which m is an integer from 1 to 300; m1 is an integer from 1 to 140; m ₂ is an integer from 1 to 40; m _3a is an integer from 0 to 17; m _3b is an integer from 1 to 8 and the sum of m, m1, m ₂ and m _3a and m _3b varies from 15 to 300; terminal indicates direct attachment of one or more polymer backbones to the isolated antibody; one of _Xa and _Xb is H and the other is a water-soluble protecting group for the maleimido group, or Xa and _Xb , together with the carbon atoms to which they are attached, form a carbon-carbon double bond; And X represents CH ₂ , O or NH. 18. A method of relieving a symptom of cancer in a patient, comprising administering to the patient a conjugate according to any one of claims 1 to 15 in an amount sufficient to relieve the symptom of cancer. 19. The method of claim 18, wherein the patient is a human. 20. The method of claim 18 or 19, wherein the cancer is selected from the group consisting of ovarian cancer, thyroid cancer, colorectal cancer, lung cancer, non-small cell lung cancer (NSCLC), breast cancer, kidney cancer and cancer salivary duct. 21. The method according to any one of claims 18 to 20, wherein the cancer is selected from the group consisting of non-small cell lung cancer (NSCLC) and ovarian cancer. 22. The method of claim 21, wherein the non-small cell lung cancer is non-squamous non-small cell lung cancer. 23. The method of claim 21, wherein the ovarian cancer is epithelial ovarian cancer. 24. The method according to any one of claims 18-23, further comprising administering a therapeutic agent to the patient. -