EA045483B1 - COMPOUNDS INTERACTING WITH GLYCANS AND METHODS OF THEIR APPLICATION - Google Patents

Description

Cross reference to related applications

This patent application claims priority to U.S. Provisional Patent Application No. 62/466,766, filed March 3, 2017, entitled GLYCAN-INTERACTING COMPOUNDS AND METHODS OF USE, U.S. Provisional Patent Application No. 62/480,126, filed March 31, 2017. and entitled GLYCAN-INTERACTING COMPOUNDS AND METHODS OF USE, US Provisional Patent Application Number 62/486826, filed April 18, 2017 and entitled GLYCAN-INTERACTING COMPOUNDS AND METHODS OF USE, US Provisional Patent Application Number 62/563718, filed September 27, 2017 entitled GLYCAN-INTERACTING COMPOUNDS AND METHODS OF USE, and US Provisional Patent Application Number 62/577830, filed October 27, 2017, entitled GLYCAN-INTERACTING COMPOUNDS AND METHODS OF USE, the contents of each of which are incorporated herein by reference. in full.

List of sequences

The present invention contains a sequence listing that has been filed electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy in question, created on March 2, 2018, is named 2033_1030PCT_SL.txt and is 24876 bytes in size.

Prerequisites for creating the invention

Aberrant glycosylation accompanies some of the other mutations commonly seen in carcinomas. It is estimated that cells in approximately 80% of all carcinomas express truncated glycans, the Tn antigen, and the sialylated form, sialyl-Tn (STn). With some exceptions, Tn and STn are not expressed in normal, healthy tissues. In addition, an immunogenic sialic acid absent in humans, N-glycolylneuraminic acid (Neu5Gc), appears to be differentially expressed on carcinomas such as breast cancer in the form Neu5Gc-STn (GcSTn).

Multiple aberrant forms of glycosylation have been described in various forms of human cancer, allowing the identification of certain glycans as a class of cell surface molecules suitable for specific tumor targeting of drugs (Cheever, M.A. et al., Clin Cancer Res. 2009 Sep 1; 15(17 ):5323-37). For example, various human cancers (such as bladder, breast, cervical, colon, lung, and ovarian cancers, among others) show high expression of the STn antigen, which is rarely found in normal human tissues (Karlen, P. et al ., Gastroenterology. 1998 Dec; 11 5(6): 1395-404; Ohno, S. et al., Anticancer Res. 2006 NovDec; 26(6A):4047-53). Additionally, the presence of STn on tumor-associated mucins is associated with poor disease prognosis and, as a consequence, it is considered a promising epitope for cancer detection and targeted therapy (Cao, Y. et al., Virchows Arch. 1997 Sep; 431(3): 159-66; Julien, S. et al., Br J Cancer. 2009 Jun 2; 100(11): 1746-54; Itzkowitz, S. H. et al., Cancer. 1990 Nov 1; 66(9): 1960-6 ; Motoo, Y. et al., Oncology. 1991; 48(4):321-6; Kobayashi, H. et al., J Clin Oncol. 1992 Jan; 10(1):95-101). The formation of Tn and STn is associated with somatic mutations in the Cosmc gene, which encodes a molecular chaperone necessary for the formation of active T synthase (Ju, T. et al., Nature. 2005 Oct 27; 437(7063):1252; Ju, T. et al., Cancer Res. 2008 Mar 15;68(6):1636-46). It may also result from increased expression of the sialyl transferase, ST6GalNAc I (Ikehara, Y. et al., Glycobiology. 1999 Nov; 9(11): 1213-24; Brockhausen, I. et al., Biol Chem. 2001 Feb; 382(2):219-32). De novo expression of STn can lead to changes in carcinoma cells, changes in the malignant phenotype and lead to more aggressive cell behavior (Pinho, S. et al., Cancer Lett. 2007 May 8; 249(2): 157-70). Although STn is expressed at high levels in malignant tissues, low levels of expression have also been found in healthy human cells (Jass, J.R. et al., J Pathol. 1995 Jun; 176(2): 143-9; Kirkeby, S. et al. , Arch Oral Biol 2010 Nov;55(11):830-41). STn itself has attracted attention as a target for cancer detection and therapy (Cheever, M.A. et al., Clin Cancer Res. 2009 Sep 1; 15(17):5323-37). STn is also present in mucins associated with cancer stem cells (Engelmann et al., Cancer research, 2008, 68, 2419-2426), and STn is involved in immunosuppression (Carrascal, M.A., et al., Molecular Oncology. 2014. 8( 3): 753-65).

In addition to the presence of STn, other glycosylation changes have been noted in cancer. One of them includes Neu5Gc. N-acetylneuraminic acid (Neu5Ac) and Neu5Gc are the two major sialic acids on mammalian cell surfaces. Neu5Ac and Neu5Gc differ only in that Neu5Gc contains an additional oxygen atom associated with a chemical group attached to carbon 5. Due to the loss of a functional gene, sialic acid in the human body can only be synthesized in the form of Neu5Ac, but not Neu5Gc. However, Neu5Gc can be metabolically absorbed into the human body from animal foods such as red meat (Tangvoranuntakul, P. et al., Proc Natl Acad Sci USA. 2003 Oct 14; 100(21): 12045-50; Nguyen, D.H. et al., J Immunol. 2005 Jul 1; 175(1):228-36; US7682794, US8084219, US2012/0142903, WO2010030666 and WO2010030666). Neu5Gc is present in significant excess in human tumors (Higashi, H. et al., Cancer Res. 1985 Aug; 45(8):3796-802; Miyoshi I. et al., Mol Immunol. 1986. 23: 631-638; Hirabayashi, Y. et al., Jpn J Cancer Res. 1987. 78: 614-620; Kawachi. S, et al., Int Arch Allergy Appl Immunol. 1988. 85: 381-383;

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Devine, P. L. et al., Cancer Res. 1991. 51: 5826-5836; Malykh, Y.N. et al., Biochimie. 2001. 83: 623-634 and Inoue, S. et al., 2010. Glycobiology. 20(6): 752-762) and is found at very low levels in normal human tissue, which has been overlooked for several decades (Diaz, S.L. et al., PloS One. 2009. 4: e4241; Tangvoranuntakul, P. et al., Proc Natl Acad Sci U S A. 2003. 100: 12045-12050; Varki, A. et al., Glycoconj J. 2009. 26: 231-245). The increased metabolic accumulation of dietary Neu5Gc in cancer tissues compared to healthy human tissues can most likely be explained by at least three factors: rapid growth with insufficient production of competing endogenous Neu5Ac, increased macropinocytosis induced by growth factors (Dharmawardhane, S. et al., Mol Biol Cell. 2000 Oct; 11(10):3341-52; Simonsen, A. et al., Curr Opin Cell Biol. 2001 Aug; 13(4):485-92; Johannes, L. et al., Curr Opin Cell Biol. 2001 Aug; 13(4):485-92; Johannes, L. et al. al., Traffic. 2002 Jul; 3(7):443-51; Amyere, M. et al., Int J Med Microbiol. 2002 Feb; 291(6-7):487-94), and stimulation of lysosomal gene expression the sialic acid transporter sialin as a result of hypoxia (Yin, J. et al., Cancer Res. 2006 Mar 15; 66(6):2937-45). In addition, all humans tested to date have a reservoir of polyclonal antibodies against the non-human Neu5Gc, making it the first example of a xeno-autoantigen (PadlerKaravani, V. et al., Glycobiology. 2008 Oct; 18(10):818- 30; Varki, N.M. et al., Annu Rev Pathol. 2011;6:36593). The accumulation of dietary Neu5Gc in malignant tumors, despite the anti-Neu5Gc response, has been shown to promote tumor progression by inducing low-grade chronic inflammation (Hedlund, M. et al., Proc Natl Acad Sci USA. 2008 Dec 2; 105(48): 18936-41). Thus, Neu5Gc containing glycan epitopes on human tumors provides a valuable drug targeting opportunity. A recent study found antibodies against Neu5Gc-containing STn (GcSTn) but not Neu5Ac-STn (AcSTn) in cancer patients and examined their potential as a specific biomarker for cancer detection (Padler-Karavani, V. et al ., Cancer Res. 2011 May 1;71(9):3352-63).

There remains a need in the art for therapeutic antibodies capable of binding glycans, including glycans associated with disease and disease-affected cells and tissues. In addition, there remains a need for more effective methods for producing such antibodies and methods for using such antibodies to target diseased cells and tissues. The present invention satisfies this need by providing suitable compounds and methods.

The essence of the invention

In some embodiments, the present invention provides a method of treating cancer in a subject by administering an antibody that is administered at a dose of from about 1 mg/kg to about 10 mg/kg, wherein the antibody has a terminal half-life of from about 50 hours to about 200 hours, and wherein the antibody binds the sialyl-Tn (STn) antigen.

The antibody may comprise a heavy chain variable domain (VH) with an amino acid sequence selected from the group consisting of SEQ ID NOs: 7 and 9; and a light chain variable domain (VL) with an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 and 10. The antibody may be an antibody-drug conjugate. The antibody may be conjugated to monomethyl auristatin E (MMAE). The antibody can be administered intravenously.

In some embodiments, the present invention provides a method of treating colorectal cancer by administering an anti-STn antibody to a subject with colorectal cancer, wherein the anti-STn antibody comprises a heavy chain variable domain (VH) with an amino acid sequence selected from the group consisting of SEQ ID NO: 7 , 9 and 11; and a light chain variable domain (VL) with an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 10 and 12. Colorectal cancer may be resistant to treatment with at least one of cetuximab, panitumumab, bevacizumab, ramucirumab, trastuzumab, ponatinib, sorafenib, 5-fluorouracil, cisplatin, docetaxel, gemcitabine, irinotecan, paclitaxel and oxaliplatin. The anti-STn antibody may be an antibody-drug conjugate (ADC). The ADC may include at least one conjugate agent, wherein the at least one conjugate agent is selected from one or more of auristatin, maytansine, tubulisin, vinca alkaloid, pyrrolobenzodiazepine dimer, camptothecin, duocarmycin, amanitine, phosphoinositide 3-kinase inhibitor (PI3K ) and mitogen-activated protein kinase (MEK) inhibitor. The ADC may include one or more polymers, wherein the one or more polymers combine the anti-STn antibody and at least one conjugate agent. The one or more polymers may include one or more of poly(ethylene glycol) (PEG), poly(2-hydroxypropyl) methacrylamide (polyHPMA), poly(amino acid), carbohydrate polymer, glycopolysaccharide, glycolipid, glycoconjugate, polyglycerol, polyvinyl alcohol, poly(acrylic acid), polyketal and polyacetal. The one or more polymers may include poly(1-hydroxymethylethylene hydroxymethylformal) (PHF). An anti-STn antibody can kill STn-expressing cells with a half-maximal inhibitory concentration of from about 0.1 nM to about 50 nM.

Administration of the anti-STn antibody may be performed in combination with at least one other treatment. Another treatment method may include standard treatment. Another method of treatment can be selected from administering one or more of cetuximab, panitumumab, bevacizumab, ramucir

- 2 045483 mab, trastuzumab, ponatinib, sorafenib, 5-fluorouracil, cisplatin, docetaxel, gemcitabine, irinotecan, paclitaxel and oxaliplatin. Another method of treatment may also include administration of at least one cell cycle inhibitor. The cell cycle inhibitor may be a cyclin-dependent kinase (CDK) inhibitor. A CDK inhibitor may inhibit CDK4 and/or CDK6. The CDK inhibitor can be selected from palbociclib, ribociclib and abemaciclib. Anti-STn antibody can be administered simultaneously with another treatment. The anti-STn antibody can be administered sequentially with another treatment.

In some embodiments, the present invention provides a method of treating cancer comprising administering an anti-STn antibody to a subject suffering from cancer, wherein the anti-STn antibody comprises a heavy chain variable domain (VH) with an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 9 and 11; and a light chain variable domain (VL) with an amino acid sequence selected from the group consisting of SEQ ID NOs: 8, 10 and 12, wherein administration of the anti-STn antibody is performed in combination with at least one cell cycle inhibitor. The cell cycle inhibitor may be a cyclin-dependent kinase (CDK) inhibitor. A CDK inhibitor may inhibit CDK4 and/or CDK6. The CDK inhibitor can be selected from palbociclib, ribociclib and abemaciclib. Anti-STn antibody can be administered simultaneously with a cell cycle inhibitor. The anti-STn antibody can be administered sequentially with a cell cycle inhibitor.

In some embodiments, the present invention provides an ADC that includes an anti-STn antibody comprising a VH with an amino acid sequence selected from SEQ ID NOs: 7, 9 and 11; VL with an amino acid sequence selected from SEQ ID NO: 8, 10 and 12; and one or more cytotoxic agents. The cytotoxic agent may be selected from at least one of auristatin, maytansine, tubulisin, vinca alkaloid, pyrrolobenzodiazepine dimer, camptothecin, duocarmycin, amanitine, a PI3K inhibitor, and a MEK inhibitor. The anti-STn antibody can be conjugated to one or more cytotoxic agents using a linker. The ADC may include one or more polymers linking an anti-STn antibody and one or more cytotoxic agents. The one or more polymers may include one or more of PEG, polyHPMA, poly(a-amino acid), carbohydrate polymer, glycopolysaccharide, glycolipid, glycoconjugate, polyglycerol, polyvinyl alcohol, poly(acrylic acid), polyketal, and polyacetal. One or more polymers may include PHF. One or more polymers can be attached to the anti-STn antibody using a linker. One or more cytotoxic agents can be attached to one or more polymers using a linker. The linker may be a cleavable linker.

In some embodiments, the present invention provides a method of treating cancer in a subject, wherein the subject has at least one cancer cell expressing STn, comprising administering an anti-STn antibody-drug conjugate described herein. The at least one cancer cell may be an ovarian cancer cell. The at least one cancer cell may be resistant to treatment with at least one chemotherapeutic agent. The chemotherapy agent may be cisplatin. The anti-STn antibody-drug conjugate can be administered at a dose of from about 0.1 mg/kg to about 25 mg/kg. Administration can be done by intravenous injection. The anti-STn antibody-drug conjugate can be administered daily, weekly or monthly.

Some of the methods of the present invention include methods of treating cancer by administering an ADC to a subject, the ADC comprising: VH comprising SEQ ID NO: 7; VL containing SEQ ID NO: 8; at least one human IgG constant region; and a cytotoxic conjugate agent, wherein the cytotoxic conjugate agent is conjugated to at least one human IgG constant region via a linker. The at least one human IgG constant region may be selected from one or more of SEQ ID NOs: 15 and 16. The cytotoxic conjugate agent may include MMAE. The ADC can be administered at a dose of from about 1 mg/kg to about 6 mg/kg. ADCs can be administered by intravenous bolus injection. The ADC can be administered as part of a composition, wherein the composition contains at least one excipient. The composition can be administered in a volume of from about 0.1 ml/kg to about 10 ml/kg. The composition can be administered in a volume of approximately 1.2 ml/kg. The composition may contain an ADC at a concentration of from about 0.5 mg/ml to about 10 mg/ml. An ADC may have an apparent terminal half-life of from about 2 days to about 8 days. ADCs may have an apparent clearance rate of about 10 mL/kg/day to about 20 mL/kg/day. The ADC may have an apparent volume of distribution at steady state from about 50 to about 100 mL/kg. The ADC may have a maximum observed concentration of from about 10 μg/ml to about 200 μg/ml. The ADC may have an area under the concentration-time curve (AUC) from the start of administration to the last observed quantifiable concentration of from about 50 to about 500 days*μg/mL.

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Brief description of drawings

The foregoing and other objects, features and advantages will be apparent from the following description of specific embodiments of the invention, illustrated by the accompanying drawings, in which references refer to like parts in different drawings. Drawings are not necessarily to scale; instead, attention is given to illustrating the principles of various embodiments of the invention.

Fig. 1A is a schematic representation of α2,6-sialylated N-acetylgalactosamine (STn), with the largest ellipse indicating the specific region of STn recognized by group 1 antibodies.

Fig. 1B is a schematic representation of α2,6-sialylated N-acetylgalactosamine (STn), with the largest ellipse indicating the specific region of STn recognized by group 2 antibodies.

Fig. 1C is a schematic representation of α2,6-sialylated N-acetylgalactosamine (STn), with the largest ellipse indicating the specific region of STn recognized by group 3 antibodies.

Fig. 1D is a schematic representation of α2,6-sialylated N-acetylgalactosamine (STn), with the largest ellipse indicating the specific region of STn recognized by group 4 antibodies.

Fig. 2 is a graph showing mean serum antibody concentrations over time following administration of an initial dose of HSIAIOI-MMAE to cynomolgus monkeys.

Fig. 3 is a graph showing mean serum antibody concentrations over time after administration of the second dose (on day 22 of treatment) of hSIA101-MMAE to cynomolgus monkeys.

Fig. 4 is a set of graphs showing patient-derived xenograft tumor volumes during treatment with hSIA101-ADC compared to other treatments. The tumors shown in the top panel were derived from an ovarian tumor from a chemotherapy-naïve patient, and the tumors presented in the bottom panel were derived from an ovarian tumor from a patient who had previously been treated with chemotherapy agents.

Detailed description

Introduction.

The present invention provides antibodies that are specific for or react with epitopes containing carbohydrate groups, referred to herein as glycans. Some of the glycan-interacting antibodies described herein may be used as biotherapeutics. Other embodiments relate to methods for producing such glycan-interacting antibodies.

In nature, STn can be stalated with N-acetylneuraminic acid (Neu5Ac) or Nglycolylneuraminic acid (Neu5Gc). The glycan-interacting antibodies of the present invention can be directed to glycans having any STn (anti-pan-STn antibodies), glycans having an STn that specifically include Neu5Ac (AcSTn), or glycans having an STn that specifically include Neu5Gc (GcSTn) . In some embodiments, the glycan-interacting antibodies of the present invention are directed to cancer-associated glycan antigens, such as α2,6-sialylated N-acetylgalactosamine (STn).

In some embodiments, the present invention relates to methods for producing glycan-reactive antibodies. Such methods may include the use of mice to induce an immune response to one or more antigens, including STn (eg, AcSTn and/or GcSTn). As described herein, a number of methods can be used to target the antibodies produced by immunization of mice. Such methods may include varying the strain and/or sex of the mice immunized, varying the antigen used, varying the type and dose of adjuvant used when administering the antigen, and the dynamics of immunization before performing the fusion to produce hybridomas.

In some embodiments, the present invention provides methods for treating cancer using glycan-interacting antibodies. Such methods may include the use of antibody-drug conjugates (ADCs). ADCs may include cytotoxic conjugate agents attached to glycan-reactive antibodies directly or via a linker. Glycan-interacting antibodies can bind STn. In some embodiments, methods of treating cancer include killing cancer stem cells. In some aspects, glycan-interacting antibodies may be used alone. In other aspects, glycan-interacting antibodies are used in combination with chemotherapeutic agents. Glycan-reactive antibodies can be formulated as compositions containing one or more excipients for administration to subjects. The compositions and routes of administration may provide the glycan-interacting antibodies at concentrations and dosages suitable to achieve the bioavailability, therapeutic window, and/or volume of distribution required for effective treatment.

Optimized, humanized and conjugated forms of the glycan-interacting antibodies disclosed herein are also provided. In addition, kits, assays and reagents are provided, including antibodies and/or methods of the present invention.

Definitions.

Adjacent: As used herein, the term adjacent refers to anything adjacent to, adjacent to, or close to a particular property. In some embodiments, adjacent residues are sugar residues on a glycan chain that are linked to each other. In some embodiments, adjacent glycans are chains of glycans located adjacent to each other, either in direct contact or in close proximity, and without any other glycan in between.

Administered in combination: As used herein, the terms administered in combination or combined administration mean that a subject is simultaneously exposed to two or more agents administered at the same time or within some interval of time such that the subject at some point in time is simultaneously exposed to both agents, and/or such that the patient may experience overlapping effects from each agent. In some embodiments, at least one dose of one or more agents is administered within about 24 hours, 12 hours, 6 hours, 3 hours, 1 hour, 30 minutes, 15 minutes, 10 minutes, 5 minutes, or 1 minute from administration of at least at least one dose of one or more other agents. In some embodiments, administration occurs in an overlapping dose schedule. As used herein, the term dosage regimen refers to multiple doses spaced apart over time. Such doses may be administered at regular intervals, or there may be one or more omissions in administration. In some embodiments, the administration of separate doses of one or more glycan-interacting antibodies described herein occurs at sufficiently short time intervals such that a combinatorial (eg, synergistic) effect is achieved.

Amino acid: As used herein, the terms amino acid and amino acids refer to all naturally occurring L-alpha amino acids as well as non-naturally occurring amino acids. Amino acids are designated by either a one-letter or three-letter code, as follows: aspartic acid (Asp:D), isoleucine (Ile:I), threonine (Thr:T), leucine (Leu:L), series (Ser:S), tyrosine ( Tyr:Y), glutamic acid (Glu:E), phenylalanine (Phe:F), proline (Pro:P), histidine (His:H), glycine (Gly:G), lysine (Lys:K), alanine ( Ala:A), arginine (Arg:R), cysteine (Cys:C), tryptophan (Trp:W), valine (Val:V), glutamine (Gln:Q), methionine (Met:M), asparagine (Asn :N), where the amino acid is given first, then the three-letter and one-letter code are given in brackets, respectively.

Animal: As used herein, the term animal refers to any member of the animal kingdom. In some embodiments, animal means humans at any stage of development. In some embodiments, animal means non-human animals at any stage of development. In specific embodiments, the non-human animal is a mammal (eg, rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cattle, primate, or pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, and worms. In some embodiments, the animal is a transgenic animal, a genetically modified animal, or a clone.

Antibody: As used herein, the term antibody is used in the broadest sense and specifically covers various embodiments, including, but not limited to, monoclonal antibodies, polyclonal antibodies, polyspecific antibodies (e.g., bispecific antibodies formed from at least two intact antibodies), and also antibody fragments such as diabodies, provided they have the desired biological activity. Antibodies are primarily amino acid-based molecules, but may also have one or more modifications, such as containing sugar moieties.

Antibody fragment: As used herein, the term antibody fragment means a portion of an intact antibody, preferably containing its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules and polyspecific antibodies formed from antibody fragments. Papain cleavage of antibodies results in two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site. A residual Fc fragment is also formed, the name of which reflects its ability to readily crystallize. Treatment with pepsin results in the production of an F(ab')2 fragment, which has two antigen-binding sites and is still capable of binding antigen. Glycan-interacting antibodies may include one or more of these moieties. For purposes of the present invention, an antibody may contain a heavy and light chain variable domain, as well as an Fc region.

Antigen-binding region: As used herein, the term antigen-binding region means a portion of an antibody, antibody fragment, or related molecule that directly interacts with a target molecule or epitope. Antigen binding regions typically contain a pair of variable domains, as in the Fab region of an antibody or as regions linked together in a scFv.

Approximately: As used herein, the term approximately, or about, when applied to one or more values of interest, means a value that is similar to a specified reference value. In specific embodiments, the term about, or about, means a range of values that are in the range of 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1% or less, in either direction (more or less) of the stated reference value, unless otherwise indicated or the context otherwise requires (except for a number that would exceed 100% of the possible value) .

Related: As used herein, the terms associated, conjugated, associated, attached, and tethered, when applied to two or more moieties, mean that the moieties are physically associated or connected to each other, either directly or through one or more additional moieties that serve as linking moieties. , forming a structure that is sufficiently stable that the fragments remain physically associated under the conditions under which the structure is used, such as physiological conditions. Bonding does not have to occur strictly through direct covalent chemical bonding. It may also involve the formation of ionic or hydrogen bonds, or hybridization-based bonding that is sufficiently stable such that the linked moieties remain physically associated.

Bifunctional: As used herein, the term bifunctional refers to any substance, molecule or moiety that is capable of performing, or maintaining, at least two functions. Functions can lead to the same result, or to different results. The structures that provide the functions may be the same or different.

Biomolecule: As used herein, the term biomolecule means any naturally occurring molecule based on amino acids, based on nucleic acids, based on carbohydrates or based on lipids, and the like.

Bispecific Antibody: As used herein, the term bispecific antibody means an antibody capable of binding two different antigens. Such antibodies typically contain regions of at least two different antibodies. Bispecific antibodies may include any of the antibodies described in Riethmuller, G. 2012. Cancer Immunity. 12:12-18, Marvin, J.S. et al., 2005. Acta Pharmacologica Sinica. 26(6):649-58 and Schaefer, W. et al., 2011. PNAS. 108(27): 11187-92, each of which is incorporated herein by reference in its entirety.

Branch: As used herein, the term branch means a molecule, fragment, or appendage that is related to, or extends from, a parent molecule or source. In some embodiments, the branch chain or branch chain comprises one or more residues (including, but not limited to, sugar residues) that extend from the parent chain. As used herein, the term parent chain generally means a chain of residues (including, but not limited to, sugar residues) to which a branch chain is associated. In the case of a glycan with multiple chains, the parent chain may also be called the parent chain to which all such branches are directly or indirectly linked. In the case of a polysaccharide having a chain of hexose residues, the bonds in the parent chain, as a rule, take place between carbon atoms 1 and 4 of neighboring residues, while the branch chains are linked to the parent chain by a bond between carbon atom 1 of the branching residue and carbon atom 3 of the parent residue the chain from which the branch extends. As used herein, the term branching residue refers to a residue attached to the parent chain in a branched chain.

Cancer Stem Cells: As used herein, the term cancer stem cells (CSC) refers to a subset of tumor cells that have the ability to self-renew. CSCs may be able to regenerate different types of cells. In some cases, these cells are difficult or impossible to remove by surgically or chemically targeting the tumor.

Compound: As used herein, the term compound means a single chemical molecule. In some embodiments, a particular compound may exist in one or more isomeric or isotopic forms (including, but not limited to, stereoisomers, geometric isomers, and isotopes). In some embodiments, the compound is provided or used in only one such form. In some embodiments, the compound is provided or used as a mixture of two or more such forms (including, but not limited to, a racemic mixture of stereoisomers). Those skilled in the art will appreciate that certain compounds exist in different such forms and exhibit different properties and/or activities (including, but not limited to, biological activities). In such cases, one skilled in the art may select, or avoid, particular forms of the compound for use in the present invention. For example, compounds containing asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods for preparing optically active forms from optically active starting materials are known in the art, for example by resolution of racemic mixtures or by stereoselective synthesis.

Cyclic or Looped: As used herein, the term cyclic means the presence of a closed loop. Cyclic molecules do not necessarily have to be circular, only connected, forming an unbroken chain of subunits.

Cytidine monophosphate-M-acetylneuraminic acid hydroxylase.

As used herein, the term cytidine monophosphate-N-acetylneuraminic acid hydroxylase, or CMAN, refers to an enzyme not found in humans but present in most other mammals (including, but not limited to, mice, pigs, and chimpanzees) that catalyzes the formation of N-glycolylneuraminic acid from N -acetylneuraminic acid. The absence of this enzyme in humans is a consequence of a frameshift mutation, leading to premature termination of the CMAN transcript and the production of a nonfunctional protein.

Cytotoxic: As used herein, the term cytotoxic refers to an agent that destroys or has a damaging, toxic or detrimental effect on a cell (e.g., mammalian cell (e.g., human cell)), bacterium, virus, fungus, protozoan, parasite, prion, or their combination.

Delivery: As used herein, the term delivery means the act or method of transporting a compound, substance, molecule, fragment, cargo or payload to the intended site.

Delivery Vehicle: As used herein, the term delivery vehicle means any substance that facilitates, at least partially, in vivo delivery of a compound, substance, molecule, fragment, cargo or payload.

Detectable Label: As used herein, the term detectable label means one or more markers, signals, or moieties that are attached, incorporated, or linked to another molecule, which markers, signals, or moieties can be readily detected by methods known in the art, including fluoroscopy, fluorescence, chemiluminescence, enzymatic activity, absorption and the like. Detectable labels include radioactive isotopes, fluorophores, chromophores, enzymes, dyes, metal ions, ligands such as biotin, avidin, streptavidin and haptens, quantum dots and the like. Detectable labels can be located in any position in the molecule with which they are connected, embedded or bound. For example, when attached, incorporated, or bound to a peptide or protein, it may be contained within amino acids, peptides, or proteins, or located at the N- or C-terminus.

Display Library: As used herein, the term display library refers to a tool used in scientific research to identify biomolecular interactions. There are various variations of display libraries that include the use of bacteriophages, yeast and ribosomes. In each case, the proteins in a particular library (also referred to herein as library members) are linked (physically or through association with a host) to a nucleic acid encoding the protein. When a target molecule is incubated with display library members, any library members that bind to the target can be isolated and the bound protein coding sequences can be determined by analyzing the bound nucleic acid. In some embodiments, the display libraries are phage display libraries, in which case the display library is derived from bacteriophage viral particles (also referred to herein as phage particles), wherein the nucleic acids have been incorporated into the phage genome, resulting in the production of viral envelope proteins, fused to proteins encoded by the introduced nucleic acids. Such fusion proteins are displayed on the outer surface of the assembled phage particles, where they can interact with a specific target.

Distal: As used herein, the term distal means located away from the center, or away from the point or area of interest.

Recombinant: As used herein, embodiments of the invention are recombinant if they are designed to have a quality or property, structural or chemical, different from that of the parent, wild type, or naturally occurring molecule. Thus, recombinant agents or molecules are those whose design and/or production involves manipulation by the human hand.

Epitope: As used herein, the term epitope means a surface or region on a molecule capable of interacting with components of the immune system, including, but not limited to, antibodies. In some embodiments, the epitope may include a target site. Epitopes may include a region on an antigen, or between two or more antigens, that is specifically recognized and bound by the corresponding antibody. Some epitopes may include one or more sugar residues along the entire length of one or more glycans. Such epitopes may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 sugar residues. Epitopes may also include one or more interaction regions between molecules. In some embodiments, epitopes may

- 7 045483 include a junction region between two sugar residues, between a branch chain and a parent chain, or between a glycan and a protein.

Ester Bond: As used herein, the term ester bond means a chemical bond that involves an oxygen atom bonded between two carbon atoms. In some embodiments, ester linkages link sugar moieties to other moieties, including, but not limited to, other sugar moieties, to form a glycan chain. Such bonds are also called glycosidic bonds or glycosidic bridges. In the context of at least one sugar moiety, the terms linkage and/or bridge are also used herein to refer to a glycosidic linkage. In some embodiments, bridges can link glycans to other molecules, including, but not limited to, proteins, lipids, phospholipids, and sphingolipids. In some embodiments, sugar residues may be associated with a protein, typically forming a bond between the sugar residue and an amino acid residue. Such amino acid residues include serine and threonine. In some embodiments, ester linkages link the glycans to the glycan matrix via a carbohydrate linker that participates in the formation of the linkage. Glycosidic bonds may differ in their stereochemical properties. In some embodiments, alpha-oriented glycosidic bonds (also referred to herein as alpha bonds) result in an axial orientation between the bound oxygen atom of the ether linkage and the cyclohexane ring of the sugar moiety. In some embodiments, beta-oriented glycosidic bonds (also referred to herein as beta bonds) result in an equatorial orientation between the bound oxygen atom of the ether linkage and the cyclohexane ring of the sugar moiety.

Expression: As used herein, the term expression of a nucleotide sequence means one or more of the following events: (1) production of an RNA template from a DNA sequence (eg, by transcription); (2) processing of the RNA transcript (eg, by splicing, editing, 5' cap formation, and/or 3' end processing); (3) translation of RNA into polypeptide or protein; (4) polypeptide or protein folding; and (5) post-translational modification of the polypeptide or protein.

Feature: As used herein, the term feature means a characteristic, property, or characteristic element.

Drug: As used herein, the term drug means a material, or mixture, prepared in accordance with the formula, which may include at least one antibody, compound, substance, molecule, fragment, cargo or payload and a delivery vehicle, carrier or excipient.

Functional: As used herein, the term functional biological molecule means a biological molecule having the structure and form in which it exhibits the property and/or activity by which it is characterized. As used herein, the term functional group, or chemical group, means a characteristic group of atoms or chemical bonds that are part of a larger molecule. In some embodiments, the functional groups may be associated with different molecules, but may participate in similar chemical reactions regardless of the molecule of which they are part. Common functional groups include, but are not limited to, carboxyl groups (-COOH), acetyl groups (-COH), amino groups ( _-NH2 ), methyl groups ( _-CH3 ), sulfate groups (-SO3H) and acyl groups. In some embodiments, the addition of one or more functional groups to a molecule can be described using terms that modify the name of the functional group by ending -ylated, such as acetylated, methylated, and sulfated.

Glycan: As used herein, the terms glycan, oligosaccharide and polysaccharide are used interchangeably and refer to polymers composed of sugar monomers typically linked by glycosidic bonds, also referred to herein as linkages. In some embodiments, the terms glycan, oligosaccharide, and polysaccharide may be used to refer to the carbohydrate moiety of a glycoconjugate (eg, glycoprotein, glycolipid, or proteoglycan).

Glycan chain: As used herein, the term glycan chain means a sugar polymer containing two or more sugars. In some embodiments, the glycan chains are covalently linked to proteins through serine or threonine residues of the protein.

Glycan-rich composition: As used herein, the term glycan-rich composition means a mixture containing a high percentage of glycans. In some embodiments, the glycans in the glycan-rich composition may comprise from about 1% to about 10%, from about 5% to about 15%, from about 20% to about 40%, from about 30% to about 50%, from about 60% to about 80%, from about 70% to about 90%, or at least 100% based on the total weight of the composition.

Glycosidic Bond: As used herein, the term glycosidic bond refers to a covalent bond formed between a carbohydrate and another chemical group. In some embodiments, glycosidic bonds are formed between the reducing end of one sugar molecule and the non-reducing end of a second sugar molecule or polysaccharide chain. Such glycosidic bonds are also known as O-glycosidic bonds due to the presence of an oxygen atom (or ester bond) between the linked sugars. In some embodiments, a glycosidic bond between two sugars or between a sugar and a linker may also be referred to as a bridge.

In vitro: As used herein, the term in vitro refers to events that occur in an artificial environment, such as a test tube or reaction vessel, a cell culture, a Petri dish, etc., rather than in an organism (such as an animal, plant, or microorganism).

In vivo: As used herein, the term in vivo refers to events that occur in an organism (eg, an animal, plant or microorganism, or in its cells or tissues).

Separated: As used herein, the term segregated is synonymous with separated, however it is implied that the separation was effected by human hand. In one embodiment, the isolated substance or molecule is a substance or molecule that has been separated from at least some of the components with which it was previously associated (either in nature or under experimental conditions). The isolated substances may have different degrees of purity relative to the compounds with which they were previously associated. The isolated substances and/or molecules may be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or more than other components with which they were originally associated. In some embodiments, the isolated substances have a purity greater than about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more than about 99%. As used herein, a substance is pure if it is substantially free of other components.

Kit: As used herein, the term kit means a kit containing one or more components intended to be used together and instructions for their use.

Knockout. As used herein, the term knockout is used to refer to an organism in which an existing gene has been inactivated through human manipulation. In a knockout organism, a gene that has been inactivated is said to have been knocked out. In some embodiments, a knockout gene may be inactivated by insertion of a nucleotide sequence into the gene or by complete replacement of the gene.

Linker. As used herein, the term linker means a fragment that connects two or more domains, fragments or molecules. In one embodiment, the linker may contain 10, 11, 12, 13, 14, 15 or more atoms. In a further embodiment, the linker may contain a group of atoms, for example 10-1000 atoms. Such atoms or groups thereof may include, but are not limited to, carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl, carbonyl and imine. In some embodiments, the linker may include an amino acid, peptide, polypeptide, or protein. In some embodiments, the moiety linked by the linker may include, but is not limited to, an atom, a chemical group, a nucleoside, a nucleotide, a nucleic base, a sugar, a nucleic acid, an amino acid, a peptide, a polypeptide, a protein, a protein complex, a payload (e.g. drug) or marker (including, but not limited to, a chemical, fluorescent, radioactive or bioluminescent marker). The linker can be used for any useful purpose, for example, to produce multimers or conjugates, as well as to introduce a payload, as described herein. Examples of chemical groups that may be included in the linker include, but are not limited to, alkyl, alkenyl, alkynyl, amido, amino, ether, thioester, ester, alkylene, heteroalkylene, aryl or heterocyclyl, each of which may optionally be substituted as described herein. Examples of linkers include, but are not limited to, unsaturated alkanes, polyethylene glycols (eg, ethylene or propylene glycol monomer units, eg, diethylene glycol, dipropylene glycol, triethylene glycol, tripropylene glycol, tetraethylene glycol or tetraethylene glycol) and dextran polymers. Other examples include, but are not limited to, cleavable moieties in the linker, such as, for example, a disulfide bond (-SS-) or a nitrogen bond (-N=N-), which can be cleaved using a reducing reagent or photolysis. Non-limiting examples of selectively cleavable bonds include an amide bond, which can be cleaved, for example, through the use of tris(2carboxyethyl)phosphine (TCEP) or other reducing reagents, and/or photolysis, and an ester bond, which can be cleaved, for example, by using acid or alkaline hydrolysis. In some embodiments, the linker is a carbohydrate moiety used to link glycans to a substrate, such as a glycan matrix. Such carbohydrate linkers include, but are not limited to, -O(CH ₂ ) ₂ CH ₂ HN ₂ and -O(CH ₂ ) ₃ NHCOCH ₂ (OCH ₂ CH ₂ ) ₆ NH ₂ .

mRNA: As used herein, the term mRNA refers to the messenger RNA produced by transcription of a gene and processing of the resulting transcript. In some variants

- 9 045483 implementation of mRNA that has left the cell nucleus can be extracted from a cell or group of cells and analyzed to determine which genes were transcribed at a particular time or under a particular set of conditions.

Mucin: As used herein, the term mucin refers to a family of proteins that are highly glycosylated. In some embodiments, mucins are produced by the submandibular glands and are present in saliva and mucous secretions.

Negative selection: As used herein, the term negative selection means the selection of library members from a display library based on their ability to bind molecules and/or composition components that do not include the target antigen. In some embodiments, negative selection is used before positive selection to remove elements that may bind nonspecifically to the target.

Off-target: As used herein, the term off-target refers to any unintended effect on one or more targets, genes or cellular transcripts.

Patient: As used herein, the term patient means a subject who desires, or needs, treatment, requires treatment, is receiving treatment, will receive treatment, or is under the care of a qualified (e.g., licensed) professional for a specific disease or condition. .

Peptide: As used herein, the term peptide means a protein or polypeptide that is less than or exactly 50 amino acids in length, such as about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

Pharmaceutically Acceptable: As used herein, the expression pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms that, in the judgment of sound medical judgment, are suitable for use in contact with tissues of humans and animals without causing excessive toxicity, irritation, or allergic reaction. , or other problems and complications, in accordance with a reasonable benefit/risk ratio.

Pharmaceutically Acceptable Excipients: As used herein, the expression pharmaceutically acceptable excipient means any ingredient other than active agents (eg, those described herein) present in a pharmaceutical composition that is substantially non-toxic and does not cause inflammation in a patient. In some embodiments, the pharmaceutically acceptable excipient is a medium in which the active agent can be suspended or dissolved. Excipients may include, for example: release agents, antioxidants, binders, coatings, compression aids, disintegrants, colorants, emollients, emulsifiers, fillers (diluents), film or coating agents, flavoring agents, flavoring agents , glidants (substances that prevent caking and caking), lubricants, preservatives, printing inks, sorbents, suspending or dispersing agents, sweeteners and water for hydration. Exemplary excipients include, but are not limited to: butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate , maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn ), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C and xylitol.

Pharmaceutically Acceptable Salts: Pharmaceutically acceptable salts of the compounds described herein are forms of the disclosed compounds in which the acidic or basic moiety is in its salt form (eg, prepared by reacting the free base moiety with a suitable organic acid). Examples of pharmaceutically acceptable salts include, but are not limited to, salts of mineral or organic acids with basic compounds such as amines; salts of alkalis or organic bases with acidic compounds such as carboxylic acids; etc. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentane propionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate , hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxyethane sulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalene sulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3- phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate, and the like. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium and the like salts, as well as non-toxic ammonium, quaternary ammonium and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, tri- 10 045483 methylamine, triethylamine, ethylamine, and the like. Pharmaceutically acceptable salts include conventional non-toxic salts, for example, from non-toxic inorganic or organic acids. In some embodiments, a pharmaceutically acceptable salt is prepared from a parent compound containing a basic or acidic moiety by conventional chemical methods. Typically, such salts can be prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the corresponding base or acid in water or an organic solvent, or a mixture of these; generally, a non-aqueous medium such as ether, ethyl acetate, ethanol, isopropanol or acetonitrile is preferred. A list of appropriate salts can be found in Remington's Pharmaceutical Sciences, ^17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, Pharmaceutical Salts: Properties, Selection, and Use, P. H. Stahl and C. G. Wermuth (eds.), Wiley-VCH, 2008, and Berge et al., Journal of Pharmaceutical Science, 66, 1-19 (1977), contents of each of which are incorporated herein by reference in their entirety.

Pharmaceutically Acceptable Solvate: As used herein, the term pharmaceutically acceptable solvate means a crystalline form of a compound in which molecules of the appropriate solvent are embedded in the crystal lattice. For example, solvates can be prepared by crystallization, recrystallization, or precipitation from a solution containing organic solvents, water, or a mixture thereof. Examples of suitable solvents are ethanol, water (eg mono-, di- and trihydrates), N-methylpyrrolidinone (NMP), dimethyl sulfoxide (DMSO), NN'-dimethylformamide (DMF), NN'-dimethylacetamide (DMAC), 1,3 -dimethyl-2-imidazolidinone (DMEU), 1,3-dimethyl-3,4,5,6-tetrahydro-2-(1H)-pyrimidinone (DMPU), acetonitrile (ACN), propylene glycol, ethyl acetate, benzyl alcohol, 2 -pyrrolidone, benzyl benzoate, and the like. If the solvent is water, the solvate is called a hydrate. In some embodiments, the solvent included in the solvate is, or is at a level that is physiologically tolerated by the organism to which the solvate is administered (eg, in a unit dosage form of a pharmaceutical composition).

Pharmacokinetic: As used herein, the term pharmacokinetic refers to any one or more properties of a molecule or compound that are associated with determining the fate of substances introduced into a living organism. Pharmacokinetic properties are divided into several areas, including the extent and rate of absorption, distribution, metabolism and excretion. The abbreviation ADME is commonly used, where (A) absorption is the process of a substance entering the bloodstream; (D) distribution is the dispersion or dispersion of substances throughout body fluids and tissues; (M) metabolism (or biotransformation) is the irreversible transformation of the parent compound into daughter metabolites; and (E) excretion (or elimination) is the elimination of substances from the body. In rare cases, some drugs accumulate irreversibly in body tissues.

Physicochemical: As used herein, the term physicochemical means physical and/or chemical properties.

Positive selection: As used herein, the term positive selection means the selection of a particular element from a group of individual elements. Such elements and groups thereof may be, for example, antibodies. In some cases, they may be antibody fragments or antibody fragments expressed in association with an agent capable of expressing such fragments (eg, members of a display library). Selection may be based on the ability of the selected elements to bind to a desired target or epitope. In some embodiments, positive selection can be used with a display library to identify phage particles expressing scFv that bind to the desired target. In other embodiments, positive selection may be selection of candidate antibodies from a pool of antibodies. In other cases, the elements may be cells, cell lines, or clones, such as when selecting clones for hybridoma selection. In such cases, positive selection may be clonal selection based on one or more antibody traits (eg, specificity for one or more desired epitopes) exhibited by such clones. In some cases, desired epitopes in positive selection methods may include STn (eg, AcSTn and/or GcSTn).

In contrast, as used herein, the term negative selection includes the same principles and examples described for positive selection, but the defining characteristic is used to remove undesirable elements from a group of individual elements.

Prevention: As used herein, the term prevention means partially or completely delaying the onset of an infection, disease, disorder and/or condition; partial or complete delay in the onset of one or more symptoms, signs or clinical manifestations of a particular infection, disease, disorder and/or condition; partially or completely delaying the onset of one or more symptoms, signs or manifestations of a particular infection, disease, disorder and/or condition; partial or complete delay in the progression of an infection, specific disease, disorder and/or condition; and/or reduction in rice

- 11 045483 development of pathology associated with infection, disease, disorder and/or condition.

Prodrug: The present invention also includes prodrugs of the compounds described herein. As used herein, the term prodrug means any substance, molecule or element that is in the form of a precursor to that substance, molecule or element, the therapeutic effect of which will occur after a chemical or physical change. Prodrugs may be covalently linked or sequestered in a specific manner and released or converted to active drug before, during, or after administration to a mammalian subject. Prodrugs can be prepared by modifying functional groups present in compounds such that the modified groups are cleaved, either by conventional manipulation or in vivo, to form the parent compounds. Prodrugs include compounds in which hydroxyl, amino, sulfhydryl or carboxyl groups are linked to any group that, when administered to a mammalian subject, is cleaved to form a hydroxyl, amino, sulfhydryl or carboxyl group, respectively. The preparation and use of prodrugs are described in T. Higuchi and V. Stella, Pro-drugs as Novel Delivery Systems, Vol. 14 of the A.C.S. Symposium Series, and Bioreversible Carriers in Drug Design, ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987, both of which are incorporated herein by reference in their entirety.

Proximal: As used herein, the term proximal means located near the center of, or from, a point or area of interest.

Interaction Area: As used herein, the term interaction area means the area in which two or more elements interact or overlap. In some embodiments, the interaction region may include one or more sugar residues along the glycan chain that contact the second glycan chain. In some embodiments, the glycan chains are chains that branch from the same parent chain. In some embodiments, the interaction region may be between two glycan chains, with one chain being a branch chain and the second chain being a parent chain. For glycan chains, the interaction regions may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 sugar residues. In some embodiments, regions of interaction may also be between glycans and proteins or between glycans and lipids.

Residue: As used herein, the term residue means a monomer associated with, or capable of being associated with, a polymer. In some embodiments, the moieties include sugar molecules, including, but not limited to, glucose, galactose, N-acetylglucosamine, N-acetylgalactosamine, sialic acids. In some embodiments, the residues include amino acids.

Sample: As used herein, the term sample means an aliquot or portion taken from a source and/or made available for analysis or processing. In some embodiments, the sample is obtained from a biological source (also referred to herein as a biological sample), such as a tissue, cell, or component (e.g., body fluid, including, but not limited to, blood, plasma, serum, mucous secretions, lymph fluid, synovial fluid, cerebrospinal fluid, saliva, amniotic fluid, amniotic cord blood, urine, vaginal fluid and seminal fluid). In some embodiments, the sample may be, or include, a homogenate, lysate, or extract obtained from a whole organism or a subset of tissues, cells, or components thereof, or a fraction or portion thereof, including, but not limited to, for example, plasma, serum, spinal cord fluid, lymphatic fluid, external secretions of the skin, fluid from the respiratory tract, intestines and genitourinary tract, tears, saliva, milk, blood cells, tumors, organs. In some embodiments, the sample includes a medium, such as a nutrient broth or gel, which may contain cellular components, such as proteins or nucleic acid molecules. In some embodiments, the primary sample is an aliquot from a source. In some embodiments, the primary sample is subjected to one or more processing steps (eg, separation, purification, etc.) to obtain a sample for analysis or other use.

Sialyl: The prefix sialyl as used herein, as well as the term stalylated, refer to compounds containing sialic acid.

Single Unit Dose: As used herein, the term unit unit dose means a dose of any therapeutic agent administered in one dose/at one point in time/by one route of administration/at one point of contact, that is, during one administration event. In some embodiments, the unit dose is provided in a discrete dosage form (eg, tablet, capsule, patch, prefilled syringe, ampoule, etc.).

Split Dose: As used herein, the term split dose means dividing a single unit dose or total daily dose into two or more doses.

Stable: As used herein, the term stable refers to the connection

- 12 045483 or an element that has sufficient strength to be retained when isolated to a sufficient purity for use from the reaction mixture, and preferably capable of being formulated into an effective drug.

Stabilized: As used herein, the terms stabilize, stabilized, and stabilized domain refer to imparting, or acquiring, stability. In some embodiments, stability is measured relative to an absolute value. In some embodiments, stability is measured relative to a reference compound or element.

Standard Treatment: As used herein, standard treatment refers to methods of therapeutic treatment consistent with those practiced by most practitioners in providing such treatment.

Subject: As used herein, the term subject, or patient, refers to any organism to which a composition of the invention may be administered, for example, for experimental, diagnostic, prophylactic and/or therapeutic purposes. Typical subjects include animals (eg, mammals such as mice, rats, rabbits, primates and humans) and/or plants.

Submandibular Glands: As used herein, the term submandibular glands, or submandibular glands, refers to the mucus-producing glands located under the floor of the mouth. These glands are capable of producing mucins and, in some embodiments, can be extracted from mammals as a source of mucin.

Suffering from: an individual suffering from a disease, disorder and/or condition, has been diagnosed with, or has one or more symptoms of the disease, disorder and/or condition.

Susceptible: An individual who is susceptible to a disease, disorder and/or condition has not been diagnosed and/or may not have symptoms of the disease, disorder and/or condition, but is predisposed to developing the disease or its symptoms. In some embodiments, an individual susceptible to a disease, disorder and/or condition (eg, cancer) may be characterized by having one or more of the following: (1) a genetic mutation associated with the development of the disease, disorder and/or condition; (2) genetic polymorphisms associated with the development of a disease, disorder and/or condition; (3) increased and/or decreased expression and/or activity of a protein and/or nucleic acid associated with a disease, disorder and/or condition; (4) habits and/or lifestyle associated with the development of the disease, disorder and/or condition; (5) family history of disease, disorder, and/or condition; and (6) exposure to and/or infection with a microorganism associated with the development of the disease, disorder and/or condition. In some embodiments, an individual susceptible to the disease, disorder and/or condition will develop the disease, disorder and/or condition. In some embodiments, an individual susceptible to the disease, disorder and/or condition will not develop the disease, disorder and/or condition.

Synthetic: The term synthetic means manufactured, derived and/or manufactured by man. Synthesis of the polynucleotides or polypeptides or other molecules of the present invention may be chemical or enzymatic.

Target: As used herein, the term target refers to the object or element upon which an action will be taken. In some embodiments, targets refer to antigens that will be used to produce antibodies that specifically bind the antigens.

Screening by target. As used herein, the term target screening means the use of a target substance to identify binding partners for that substance.

Target Site: As used herein, the term target site means a region on, or in, one or more glycans, glycoproteins, biomolecules and/or biostructures on, or in, a cell, extracellular space, tissue, organ and/or organism that recognizes a binder or effector molecule (such as an antibody). In some embodiments, glycan target sites may be located exclusively on a single sugar residue, may be formed by two or more residues, or may include both glycan and non-glycan components. In some embodiments, the target sites are formed between two or more glycans or glycoproteins. In some embodiments, the target sites are formed between branch chains of the same glycan, or between one or more branch chains and the parent chain.

Target cells: As used herein, the term target cell means one or more cells of interest. Cells can be in vitro, in vivo, in situ, or in a tissue or organ of an organism. The organism may be an animal, mammal, or human (eg, a human patient).

Terminal: As used herein, the term terminal refers to the last residue in a polymer chain. In some embodiments, the terminal residues are sugar residues located at the non-reducing end of the polysaccharide chain.

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Therapeutic: As used herein, the term therapeutic refers to any substance or procedure that is used to treat a disease, disorder or condition. For example, therapeutic treatment means any treatment used to treat a disease, disorder or condition.

Therapeutic agent: The term therapeutic agent means any substance that, when administered to a subject, produces a therapeutic, diagnostic and/or prophylactic effect, and/or produces a desired biological and/or pharmacological effect.

Therapeutically effective amount: As used herein, the term therapeutically effective amount means an amount of a delivered agent (e.g., nucleic acid, drug, therapeutic agent, diagnostic agent, prophylactic agent, etc.) that is sufficient when administered to a subject suffering from, or susceptible to , infections, diseases, disorders and/or conditions, to treat, relieve symptoms, diagnose, prevent and/or delay the onset of an infection, disease, disorder and/or condition. In some embodiments, a therapeutically effective amount is provided in a single dose. In some embodiments, a therapeutically effective amount is administered in a dosage regimen that includes multiple doses. Those skilled in the art will understand that, in some embodiments, a unit dosage form may be considered to contain a therapeutically effective amount of a particular agent or element if it contains an amount that is effective when administered as part of such a dosage regimen.

Therapeutically effective result: As used herein, the term therapeutically effective result means a result that is sufficient in the case of a subject suffering from, or susceptible to, an infection, disease, disorder and/or condition to treat, alleviate symptoms, diagnose, prevent and/or delaying the onset of an infection, disease, disorder and/or condition.

Total Daily Dose: As used herein, the term total daily dose means the amount administered or prescribed to be administered over a 24-hour period of time. It may be administered as a single unit dose.

Transgenic: As used herein, the term transgenic refers to an organism that contains one or more genes introduced into the organism's genome that are not naturally present in that organism.

Treatment: As used herein, the term treatment means the partial or complete alleviation, cure, amelioration, improvement, alleviation, delay of onset, inhibition of progression, reduction in severity, and/or reduction in the incidence of one or more symptoms or signs of a particular infection, disease, disorder and/or condition. For example, treating cancer may mean inhibiting the survival, growth and/or spread of a tumor. The treatment may be administered to a subject who has no signs of the disease, disorder and/or condition, and/or to a subject who has only early signs of the disease, disorder and/or condition, to reduce the risk of developing pathology associated with the disease, disorder and/or condition.

Variable region. As used herein, the term variable region, or variable domain, refers to specific domains of an antibody, highly variable in sequence among different antibodies, that are involved in binding and determining the specificity of each particular antibody for its particular antigen.

Whole IgG: As used herein, the term whole IgG means whole IgG molecules. In some embodiments, entire IgG molecules comprise regions naturally found in two or more other organisms.

Wild type: As used herein, the term wild type refers to an organism that has a naturally occurring genome (does not have genes from other organisms).

I. Compounds and compositions.

In some embodiments, the present invention provides compounds, as well as compositions, comprising at least one glycan-interacting antibody. In a glycan, all monosaccharide monomers may be the same or may be different. Common monomers include, but are not limited to, trioses, tetroses, pentoses, glucose, fructose, galactose, xylose, arabinose, lyxose, allose, altrose, mannose, gulose, iodose, ribose, mannoheptulose, sedoheptulose, and talose. Amino sugars can also be monomers in a glycan. Glycans containing such sugars are referred to herein as aminoglycans. As used herein, amino sugars refer to sugar molecules that contain an amino group instead of a hydroxyl group or, in some embodiments, a sugar derived from such a sugar. Examples of amino sugars include, but are not limited to, glucosamine, galactosamine, N-acetylglucosamine, N-acetylgalactosamine, sialic acids (including, but not limited to, N-acetylneuraminic acid and N-glycolylneuraminic acid) and Ldownosamine.

- 14 045483

As used herein, the term glycan-interacting antibody means an antibody that can interact with a glycan moiety. Such antibodies may bind to a single glycan moiety, to multiple glycan moieties, or to epitopes that include both glycan and non-glycan components. Non-glycan components may include, but are not limited to, proteins, protein-associated moieties (eg, post-translational modifications), cells, and cell-associated molecules/structures. Glycan-interacting antibodies can bind, modify, activate, inhibit, stabilize, degrade, and/or modulate a glycan or glycan-linked molecule or element. Due to this, antibodies interacting with glycans can act as a therapeutic, or palliative, prophylactic or therapeutic composition. In some embodiments, glycan-interacting antibodies may include conjugates or combinations with other molecules. In some embodiments, glycan-interacting antibodies are directed against glycans having one or more amino sugars. In a further embodiment, one or more amino sugars are sialic acid. In a further embodiment, one or more sialic acids are N-acetylneuraminic acid and/or N-glycolylneuraminic acid.

Antibodies.

Glycan-interacting antibodies may include whole antibodies or fragments thereof. As used herein, the term antibody is used in the broadest sense and specifically covers a variety of formats, including, but not limited to, monoclonal antibodies, polyclonal antibodies, polyspecific antibodies (e.g., bispecific antibodies formed from at least two intact antibodies), antibody conjugates (including but not limited to, antibody-drug conjugates), antibody variants [including, but not limited to, antibody mimetics, chimeric antibodies (e.g., antibodies with amino acid sequences from more than one species), and synthetic variants], and antibody fragments, provided that they have the desired biological activity (eg, the activity of binding, activating, inhibiting, stabilizing, disrupting and/or modulating one or more targets). Antibodies are primarily amino acid-based molecules, but may also have one or more post-translational or synthetic modifications. Post-translational modifications may include glycosylation.

As used herein, the term antibody fragment means a portion of an intact antibody or fusion protein thereof, in some cases including at least one antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv, single chain variable fragments (scFv); diabodies; triatela; linear antibodies; single-chain antibody molecules and polyspecific antibodies formed from antibody fragments. Papain cleavage of antibodies results in two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site. A residual Fc fragment is also formed, the name of which reflects its ability to readily crystallize. Treatment with pepsin results in the production of an F(ab') ₂ fragment that has two antigen-binding sites and is still capable of binding antigen. Glycan-interacting antibodies may include one or more of these moieties and may, for example, be produced by enzymatic digestion of the whole antibody or by recombinant expression.

Natural antibodies are typically heterotetrameric glycoproteins of approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. The genes encoding the heavy and light chains of antibodies are known, and the segments composing each are well characterized and described (Matsuda, F. et al., 1998. The Journal of Experimental Medicine. 188(11); 2151-62 and Li , A. et al., 2004. Blood. 103 (12: 4602-9, the contents of each publication are incorporated herein by reference in their entirety.) Each light chain is linked to the heavy chain by one covalent disulfide bond, the number of disulfide bonds varies among immunoglobulin heavy chains of different isotypes.Each heavy and light chain also has regularly spaced intrachain disulfide bridges.Each heavy chain has a variable domain (VH) at one end, followed by a number of constant domains.Each light chain has a variable domain at one end (VL) and a constant domain at the other end thereof; the light chain constant domain is aligned with the first heavy chain constant domain, and the light chain variable domain is aligned with the heavy chain variable domain.

As used herein, the term variable domain refers to specific domains of an antibody, found on both the heavy and light chains of an antibody, which vary widely in sequence between antibodies, and which are involved in binding and determining the specificity of each particular antibody for its particular antigen. . Variable domains include hypervariable regions. As used herein, the term hypervariable region means a region in the variable domain that contains amino acid residues responsible for antigen binding. The amino acids present in the hypervariable regions determine the structure of the complementarity determining regions (CDRs), which are part of

- 15 045483 antigen-binding site of the antibody. As used herein, the term CDR means a region of an antibody that contains a structure complementary to its target antigen, or epitope. Other parts of the variable domain that do not interact with the antigen are called framework (FW) regions. The antigen-binding site (also known as the antigen receptor or paratope) contains the amino acid residues necessary for interaction with a specific antigen. The exact residues that form the antigen-binding site are typically determined by crystallography by co-crystallizing with the bound antigen, but computer calculations based on comparisons with other antibodies can also be used (Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch 3, p47-54, the contents of the publication are incorporated herein by reference in their entirety). Identification of the residues constituting a CDR may involve the use of numbering systems, including, but not limited to, those described by Rabat [Wu, T.T. et al., 1970, JEM, 132(2):211-50 and Johnson, G. et al., 2000, Nucleic Acids Res. 28(1): 214-8, the contents of each publication are incorporated herein by reference in their entirety], Chothia [Chothia and Lesk, J. Mol. Biol. 196, 901 (1987), Chothia et al., Nature 342, 877 (1989) and Al-Lazikani, B. et al., 1997, J. Mol. Biol. 273(4):927-48, the contents of each publication are incorporated herein by reference in their entirety], Lefranc (Lefranc, M.P. et al., 2005, Immunome Res. 1:3) and Honegger (Honegger, A. and Pluckthun , A. 2001. J. Mol. Biol. 309(3):657-70, contents of publications are incorporated herein by reference in their entirety).

The VH and VL domains each have three CDR regions. The VL CDRs are herein referred to as CDR-L1, CDR-L2, and CDR-L3, in order of their N- to C-terminal order along the variable domain polypeptide. The VH CDRs are herein referred to as CDR-H1, CDR-H2, and CDRH3, in order of their N- to C-terminal order along the variable domain polypeptide. Each of the CDRs has a favorable canonical structure, with the exception of CDR-H3, which contains amino acid sequences that can be highly variable in sequence and length among different antibodies, resulting in different three-dimensional structures in the antigen binding domains (Nikoloudis, D. et al., 2014 Peer J 2:e456). In some cases, CDR-H3 regions can be analyzed among a panel of related antibodies to assess antibody diversity. Various methods for determining CDR sequences are known in the art and can be applied to known antibody sequences ((Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, pp. 47-54, contents of the publication are incorporated herein by reference in full).

As used herein, the term Fv means an antibody fragment that contains the minimum fragment on the antibody necessary to form a complete antigen-binding site. These regions consist of a dimer of one heavy chain variable domain and one light chain variable domain in a tight, non-covalent association. Fv fragments can be obtained by proteolytic cleavage, but are very unstable. Recombinant methods for producing stable Fv fragments are known in the art, typically by inserting a flexible linker between a light chain variable domain and a heavy chain variable domain [to form a single chain Fv (scFv)], or by introducing a disulfide bridge between the heavy and heavy chain variable domains. light chains (Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 3, p46-47, the contents of the publication are incorporated herein by reference in their entirety).

Antibody light chains from any vertebrate species can be classified into one of two distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, antibodies can be classified into different classes. There are five main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, and some of them can also be divided into subclasses (isotypes), such as IgG1, IgG2a, IgG2b, IgG2c, IgG3, IgG4, IgA and IgA2.

As used herein, the term single chain Fv, or scFv, means a fusion protein of the VH and VL domains of an antibody, the domains being linked together into a single polypeptide chain by a flexible peptide linker. In some embodiments, the Fv polypeptide linker allows the scFv to form the structure necessary for antigen binding. In some embodiments, scFvs are used in combination with phage display, yeast display, or other display methods, where they can be expressed in association with a surface component (eg, a phage coat protein) and used to identify high-affinity peptides for a particular antigen.

The term diabodies refers to small antibody fragments with two antigen-binding sites, the fragments containing a heavy chain variable domain VH linked to a light chain variable domain VL in a single polypeptide chain. By using a linker that is too short to allow pairing between two domains on one chain, the domains are forced to pair with complementary domains on the other chain to form two antigen-binding sites. Diabodies are described in more detail, for example, in EP 404097; WO 93/11161 and Hollinger et al.

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Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993), the contents of each of which are incorporated herein by reference in their entirety.

The term intrabody refers to a form of antibody that is not secreted from the cell in which it is produced, but instead targets one or more intracellular proteins. Intrabodies can be used to influence many cellular processes, including, but not limited to, intracellular directed migration, transcription, translation, metabolic processes, proliferative signaling and cell division. In some embodiments, the methods of the present invention may include intrabody based therapy. In some such embodiments, the variable domain sequences and/or CDR sequences disclosed herein may be included in one or more intrabody therapy constructs. In some cases, the intrabodies of the invention may target one or more glycated intracellular proteins or may modulate the interaction between one or more glycated intracellular proteins and an alternative protein.

As used herein, the term chimeric antigen receptor, or CAR, refers to artificial receptors that are engineered to be expressed on the surface of immune effector cells, resulting in the specific targeting of such immune effector cells to cells expressing elements that bind with high affinity to the artificial receptors. CARs can be designed to include one or more segments of an antibody, an antibody variable domain, and/or an antibody CDR such that when such CARs are expressed on immune effector cells, the immune effector cells bind to and kill any cells that recognize the antibody portions of the CAR. In some cases, CARs are engineered to specifically bind to cancer cells, resulting in immune-regulated killing of the cancer cells.

As used herein, the term monoclonal antibody means an antibody derived from a population of substantially homogeneous cells (or clones), that is, the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variations that may occur in During the production of a monoclonal antibody, such variants are usually present in small quantities. Unlike polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), all monoclonal antibodies are directed against a single determinant on an antigen.

The designation monoclonal indicates the nature of the antibody as being derived from a substantially homogeneous population of antibodies and does not imply that the antibody must be produced by any particular method. As used herein, monoclonal antibodies include chimeric antibodies (immunoglobulins) in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibodies, while the remainder chain(s) identical or homologous to corresponding sequences in antibodies derived from another biological species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies.

Humanized forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequences of non-human immunoglobulins. Generally, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region of the recipient antibody are replaced by residues from the hypervariable region of an antibody from a non-human species (donor antibody), such as mouse, rat, rabbit, or primacy, having the desired specificity, affinity and efficiency.

Humanized antibodies may have one or more reverse mutations that result in one or more amino acids being replaced back to those of the donor antibody. Conversely, residues from a donor antibody included in humanized antibodies may be mutated to match residues present in human recipient antibodies.

In some embodiments, the glycan-interacting antibodies of the present invention may be antibody mimetics. The term antibody mimetic means any molecule that mimics the function or effect of an antibody and that binds specifically and with high affinity to its molecular targets. In some embodiments, antibody mimetics may be monobodies engineered to include a fibronectin type III domain (Fn3) as a protein scaffold (US 6,673,901; US 6,348,584). In some embodiments, antibody mimetics may be mimetics known in the art, including, but not limited to, affibody molecules, affilins, affitins, anticalins, avimers, darpins, finomers, as well as Kunitz domain peptides. In other embodiments, antibody mimetics may include one or more non-peptide regions.

As used herein, the term antibody variant means a biomolecule that is similar to an antibody in structure, sequence and/or function, but has some differences in amino acid sequence, composition or structure compared to another antibody or a naturally occurring antibody.

Obtaining antibodies.

The glycan-interacting antibodies of the present invention are designed to bind the antigens described herein. As used herein, the term antigen means an element that induces or causes an immune response in the body. The immune response is characterized by the reaction of cells, tissues and/or organs of the body to the presence of a foreign element. Such an immune response typically results in the body producing one or more antibodies against a foreign element, such as an antigen or part of an antigen. In some cases, immunization methods may be modified depending on one or more desired immunization outcomes. As used herein, the term immunization result means one or more desired effects of immunization. Examples include high titers of the antibody and/or increased specificity of the antibody for the target of interest.

Antigens of the invention may include glycans, glycoconjugates (including, but not limited to, glycoproteins and glycolipids), peptides, polypeptides, fusion proteins, or any of the foregoing, and may be conjugated or complexed with one or more separate adjuvants or heterologous proteins. In some embodiments, antigens used in the methods of the present invention may include stacked glycans, such as STn. Antigens having STn may include mucins. Mucins are a family of proteins that are highly glycosylated. They are a component of many tumors arising from epithelial cells (Ishida, A. et al., 2008. Proteomics. 8: 3342-9, incorporated herein by reference in its entirety). They are expressed at high levels by the submandibular glands and can be found in large quantities in saliva and mucous secretions. Animal-derived submandibular gland mucins can be used as antigens to generate anti-STn antibodies in immunogenic hosts. Mucins of the submandibular glands from different biological species differ in the STn they contain in terms of the forms AcSTn and GcSTn. Porcine submandibular mucin (PSM) has a particularly high GcSTn content, which accounts for approximately 90% of total STn. STn from bovine submandibular mucin (BSM) contains approximately equal percentages of GcSTn and AcSTn. Ovine submandibular mucin (OSM) has a particularly high AcSTn content, which accounts for approximately 90% of total STn. In some cases, solutions prepared for immunization may be modified to contain one or more of PSM, BSM and OSM depending on the desired target of the antibodies resulting from immunization. PSM can be used in immunizations to generate antibodies in immunogenic hosts that are more likely to be specific for GcSTn. PSM is rich in Neu5Gc-containing mucin-type glycoproteins on which GcSTn is located. Sources currently known to be high in Neu5Gc include red meat; in particular, the submandibular glands have been previously described as an abundant source of Neu5Gc due to the high expression of the enzyme CMAN, which catalyzes the reaction to form the Neu5Gc precursor, CMPNeu5Ac. In some cases, PSM can be used to prevent a pan-anti-Neu5Gc response and induce a more specific immune response against GcSTn. OSM can be used in immunizations to produce antibodies in immunogenic hosts that are more likely to be specific for AcSTn.

In one embodiment, the present invention provides a glycan-interacting antibody that is GcSTn-specific. The antibody has little cross-reactivity with Neu5Ac-STn or Tn. The antibody can bind GcSTn, but has reduced affinity for AcSTn.

In some embodiments, antigens may be enzymatically digested prior to immunization to modulate the resulting immune response in immunogenic hosts. In one example, submandibular gland mucins can be treated with trypsin or protease K enzymes prior to immunization. The activity of such enzymes may assist in the cleavage and thereby reduce the percentage and variability of non-STn epitopes. Glycan moieties can shield the regions of the peptide to which they are attached from enzymatic proteolysis, which thereby remain intact.

Antibody titers resulting from immunizations may have different levels depending on the type and amount of antigen used in such immunizations. In some cases, certain antigens may be selected for use in immunizations based on the expected titer.

As used herein, the term adjuvant means a pharmacological or immunological agent that modifies the effect of other agents. Adjuvants of the present invention include, but are not limited to, chemical compositions, biomolecules, therapeutic agents and/or therapeutic regimens. Adjuvants may include Freund's adjuvant (complete and/or incomplete), immunostimulatory oligonucleotides [e.g., CpG oligodeoxynucleotides (ODN), mineral-containing compositions, bacterial ADP-ribosylating toxins, bioadhesives, mucoadhesives, microparticles, lipids, liposomes, muramyl peptides, N-oxidized polyethylene piperazine derivatives, saponins and/or immunostimulating complexes (ISCO). In some embodiments, adjuvants may include oil-in-water emulsions (eg, submicron oil-in-water emulsions). The adjuvants of the present invention may also include any of the adjuvants disclosed in US Patent Publication No. US20120027813 and/or US Patent Publication No. US8506966, the contents of each publication being incorporated herein by reference in their entirety.

The antibodies of the present invention may be polyclonal or monoclonal, or recombinant, produced by methods known in the art or described herein. In some embodiments, the antibodies of the present invention may be labeled for detection purposes with a detectable label known to those skilled in the art. The label may be a radioactive isotope, a fluorescent compound, a chemiluminescent compound, an enzyme or an enzyme cofactor, or any other labels known in the art. In some aspects, the antibody that binds to the antigen of interest is not labeled, but can be detected by binding of a labeled secondary antibody that specifically binds to the primary antibody.

Antibodies of the present invention (e.g., glycan-interacting antibodies) include, but are not limited to, polyclonal, monoclonal, polyspecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab' fragments), fragments generated by expression Fab libraries, anti-idiotypic (anti-id) antibodies (including, for example, anti-id antibodies to antibodies of the invention), intracellularly derived antibodies (ie intrabodies), and epitope-binding fragments of any of the above. The antibodies of the present invention (eg, glycan-interacting antibodies) can be from any animal, including birds and mammals. Preferably, such antibodies are human, mouse (eg mouse and rat), donkey, sheep, rabbit, goat, guinea pig, camel, horse or chicken. Antibodies of the present invention may be monospecific or polyspecific (eg, bispecific, trispecific, or greater specificity). Polyspecific antibodies may be specific for different epitopes of a target antigen of the present invention, or may be specific for both a target antigen of the present invention and a heterologous epitope, such as a heterologous glycan, peptide, or solid support material. (See, for example, WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tub, A. et al., Trispecific F(ab)3 derivatives that use cooperative signaling via the TCR/CD3 complex and CD2 to activate and redirect resting cytotoxic T cells. J Immunol. 1991 Jul 1; 147(1):60-9; US Patent Nos. 4474893; 4714681; 4925648; 5573920; 5601819; and Kostelny, S.A. et al., Formation of a bispecific antibody by the use of leucine zippers. J. Immunol. 1992 Mar 1; 148(5): 1547-53).

The glycan-reactive antibodies of the present invention can be produced using commonly used methods known in the art for producing monoclonal antibodies. In one embodiment, monoclonal antibodies are produced using hybridoma technology (Kohler, G. et al., Continuous cultures of fused cells secreting antibody of predefined specificity. Nature. 1975 Aug 7; 256(5517):495-7). To produce hybridomas, a mouse, hamster, or other suitable host animal is typically immunized with an immunizing agent (eg, a target antigen of the invention) to stimulate lymphocytes that produce, or are capable of producing, antibodies that will specifically bind the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused to immortalized cell lines using an appropriate fusion agent such as polyethylene glycol to produce hybridoma cells (Goding, J.W., Monoclonal Antibodies: Principles and Practice. Academic Press. 1986; 59-1031). Immortalized cell lines are typically transformed mammalian cells, particularly rodent, rabbit, bovine and human myeloma cells. Typically, rat or mouse myeloma cell lines are used. Hybridoma cells can be cultured in an appropriate culture medium, which preferably contains one or more substances that inhibit the growth or survival of non-fused immortalized cells. For example, if the parent cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), hybridoma culture medium will typically contain hypoxanthine, aminopterin, and thymidine (HAT medium), which prevent the growth of HGPRT-deficient cells.

Preferred immortalized cell lines are those that undergo fusion efficiently, maintain high levels of antibody expression by the selected antibody-producing cells, and are sensitive to a medium, such as a HAT medium. 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 from the American Type Culture Collection, Manasas, Virginia. Cell lines of human myeloma and mouse-human heteromyeloma used for the production of human monoclonal antibodies have also been described (Kozbor, D. et al., A human hybrid myeloma for production of human monoclonal antibodies. J Immunol. 1984 Dec; 133( 6):3001-5; Brodeur, B. et al., Monoclonal Antibody

Production Techniques and Applications. Marcel Dekker, Inc., New York. 1987; 33:51-63).

In some embodiments, the myeloma cells may be genetically manipulated. Such manipulation can be accomplished using zinc finger nuclease (ZFN) mutagenesis as described herein. Alternatively, transfection methods known in the art can be used. NS0 myeloma cells or other murine myeloma cell lines can be used. For example, Sp2/0-Agl4 may be an alternative cell line for hybridoma production.

Transcription activator-like effector nucleases (TALEN)-induced genome editing is an alternative method for gene knockout. TALENs are artificial restriction enzymes produced by fusing the DNA binding domain of the TAL effector to a DNA cleavage domain. Like ZFN, TALEN induces double-strand breaks at desired loci, which can be repaired using reduced fidelity NHEJ, producing insertions/deletions at the break sites (Wood, A.J. et al., Targeted genome editing across species using ZFNs and TALENs. Science. 2011 Jul 15;333(6040):307). Cellectis Bioresearch (Cambridge, MA) provides TALEN design and plasmid construction services. The culture medium in which the hybridoma cells are cultured can then be analyzed for the presence of monoclonal antibodies. Preferably, the binding specificity (ie, specific immunoreactivity) of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or an in vitro binding assay such as a radioimmunoassay (RIA) or an enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known to those skilled in the art. The binding specificity of a monoclonal antibody can, for example, be determined using the Scatchard assay (Munson, P. J. et al., Ligand: a versatile computerized approach for characterization of ligand binding systems. Anal Biochem. 1980 Sep 1; 107(1):220-39). In some cases, the specificity of an antibody for regions of a particular antigen can be characterized by chemically modifying the antigens before performing an antibody binding assay. In one example, periodate treatment can be used to disrupt the C6 side chain of sialic acids. Assays can be performed with or without periodate treatment to determine whether binding in untreated samples is specific to sialic acid. In some cases, antigens having 9-O-acetylated sialic acid can be treated with a weak base (eg, 0.1 M NaOH) to destroy the 9-O-acetyl groups. Assays can be performed with or without weak base treatment to determine whether binding in untreated samples is dependent on sialic acid 9-O-acetylation.

Once the desired hybridoma cells have been identified, clones can be subcloned by serial dilution and grown using standard methods. Suitable culture media for this purpose include, for example, Dulbecco's modified Eagle's medium or RPMI-1640 medium. Alternatively, hybridoma cells can be grown in vivo as ascites in a mammal.

Alternative methods for cloning hybridomas may include methods for which kits are available from STEMCELL Technologies (Vancouver, BC, Canada), such as the ClonaCell™-HY kit, which includes a semi-solid methylcellulose-based medium and other media and reagents to support the selection and growth of hybridoma clones . However, the media in this kit contain ETS, which is an exogenous source for Neu5Gc incorporation. Although the machinery for endogenous Neu5Gc synthesis is disrupted in the Cmah'/' hybridoma, Neu5Gc incorporated from the culture medium may also pose a problem in some cases (Bardor, M. et al., Mechanism of uptake and incorporation of the non-human sialic acid N -glycolylneuraminic acid into human cells. J Biol Chem. 2005. 280: 4228-4237). In such cases, Neu5Ac can be added to the culture medium to eliminate the incorporation of Neu5Gc due to metabolic competition (Ghaderi, D. et al., Implications of the presence of N-glycolylneuraminic acid in recombinant therapeutic glycoproteins. Nat Biotechnol. 2010. 28: 863- 867).

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

In another embodiment, the monoclonal antibodies of the present invention can also be produced by recombinant DNA methods, such as those described in US Pat. No. 4,816,567, the contents of which are incorporated herein by reference in their entirety. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced by conventional methods (eg, using oligonucleotide probes that are capable of specifically binding to the genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of DNA. Once isolated, the DNA can be placed into expression vectors, which are then transfected into host cells. Host cells may include, but are not limited to, HEK293 cells, HEK293T cells,

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Monkey COS, Chinese hamster ovary (CHO) cells, and myeloma cells that do not otherwise produce immunoglobulin protein are used to synthesize monoclonal antibodies in recombinant host cells. The DNA can also be modified, for example, by replacing the coding sequences for the human antibody heavy and light chain constant domains with homologous murine sequences (U.S. Pat. No. 4,816,567) or by covalently linking all or part of the coding sequence of a non-immunoglobulin polypeptide to an immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can replace the constant domains of an antibody of the invention, or the variable domains of one antigen binding site of an antibody of the invention can be replaced to create a chimeric divalent antibody.

In some embodiments, the antibodies of the present invention (eg, glycan-interacting antibodies) can be produced by various methods known to those skilled in the art. To obtain polyclonal antibodies in vivo, animal hosts such as rabbits, rats, mice, cows, horses, donkeys, chickens, monkeys, sheep or goats are immunized with either free or carrier-bound antigens, for example, by intraperitoneal and/or intradermal injections. In some embodiments, the injection material may be an emulsion containing about 100 μg of antigen or carrier protein. In some embodiments, the injectable materials may include a glycan-rich composition, such as non-human mammalian submandibular gland mucin, in solution. Various adjuvants can also be used to enhance the immune response, depending on the host species. Adjuvants include, but are not limited to, Freund's adjuvant (complete and incomplete), mineral gels such as aluminum hydroxide, surfactants such as lysolecithin, Pluronic polyols, polyanions, peptides, oil emulsions, TITERMAX® (CytRx Corp, Los Angeles , CA), snail hemocyanin, dinitrophenol, and potentially beneficial human adjuvants such as BCG (Bacillus Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art. Several booster injections may be required, for example at intervals of approximately two weeks, to achieve the desired antibody titer, which can be detected, for example, in an ELISA assay using glycans and/or free peptide adsorbed on a solid surface. The antibody titer in serum from an immunized animal can be increased by antibody selection, for example, by adsorption of antigens to a solid support and elution of the selected antibodies by methods well known in the art.

Glycan-interacting antibodies, variants and fragments thereof can be selected and produced using high-throughput research methods. In one embodiment, glycan-interacting antibodies, which include synthetic antibodies, variants and fragments thereof, are produced using display libraries. As used herein, the term display means the expression or display of proteins or peptides on the surface of a particular host. As used herein, the term library means a collection of unique cDNA sequences and/or the proteins encoded by them. A library can contain from as little as two unique cDNAs to hundreds of billions of unique cDNAs. In some embodiments, glycan-reactive antibodies, which are synthetic antibodies, are produced using antibody display libraries or antibody fragment display libraries. As used herein, the term antibody fragment display library means a display library in which each member encodes an antibody fragment containing at least one antibody variable region. Such antibody fragments are preferably Fab fragments, but other antibody fragments such as single chain variable fragments (scFv) are also contemplated. In an antibody Fab fragment library, all encoded Fabs may be identical except for the amino acid sequence contained in the variable loops of the complementarity determining regions (CDRs) of the Fab fragment. In an alternative or additional embodiment, the amino acid sequences in the individual VH and/or VL regions may also differ.

Display libraries can be expressed in a number of possible hosts, including, but not limited to, yeast, bacteriophages, bacteria and retroviruses. Additional display technologies that may be used include ribosomal display, microbead display, and protein-DNA coupling methods. In a preferred embodiment, Fab display libraries are expressed in yeast or bacteriophages (also referred to herein as phages or phage particles). When expressed, Fabs are located on the surface of the phage or yeast, where they can interact with a specific antigen. An antigen that includes a glycan or other antigen from a desired target can be used to select phage particles or yeast cells that express antibody fragments with the highest affinity for that antigen. The DNA sequence encoding the CDR of the bound antibody fragment can then be determined by sequencing using the bound particle or cell. In one embodiment, positive selection is used in the production of antibodies. In some embodiments, negative selection is used to produce antibodies. In some embodiments, both positive and negative selection techniques are used during multiple rounds of selection to produce antibodies using display libraries.

In yeast display, cDNAs encoding different antibody fragments are introduced into yeast cells where they are expressed, and the antibody fragments are displayed on the cell surface, as described by Chao et al. (Chao, G. et al., Isolating and engineering human antibodies using yeast surface display. NatProtoc. 2006; 1(2):755-68). When displayed on the surface of yeast, the expressed antibody fragments may contain an additional domain that includes the yeast agglutinin protein, Aga2p. This domain allows the antibody fragment fusion protein to attach to the outer surface of the yeast cell by forming disulfide bridges with surface-expressed Aga1p. The result is a yeast cell coated with a specific antibody fragment. Initially, cDNA display libraries encoding these antibody fragments are used, in which each antibody fragment has a unique sequence. Such fusion proteins are expressed on the cell surface of millions of yeast cells, where they can interact with the desired target antigen incubated with the cells. Target antigens may be covalently or otherwise modified with a chemical or magnetic moiety to allow efficient cell sorting upon successful binding to the appropriate antibody moiety. Retrieval can be accomplished using magnetically activated cell sorting (MACS), fluorescence-activated cell sorting (FACS), or other cell sorting methods known in the art. Once a subpopulation of yeast cells has been selected, the corresponding plasmids can be analyzed to determine the CDR sequence.

Bacteriophage display technology typically uses filamentous phage, including, but not limited to, fd, F1, and M13 virions. Such strains are non-lytic, which allows the host to continuously multiply and increase viral titers. Examples of phage display methods that can be used to produce antibodies of the present invention include those described in Miersch et al. (Miersch, S. et al., Synthetic antibodies: Concepts, potential and practical considerations. Methods. 2012 Aug; 57(4):486-98), Bradbury et al. (Bradbury, A.R. et al., Beyond natural antibodies: the power of in vitro display technologies. Nat Biotechnol. 2011 Mar; 29(3):245-54), Brinkman et al. (Brinkmann, U. et al., Phage display of disulfide-stabilized Fv fragments. J Immunol Methods. 1995 May 11; 182(1):41-50); Ames et al. (Ames, R.S. et al., Conversion of murine fobs isolated from a combinatorial phage display library to full length immunoglobulins. J Immunol Methods. 1995 Aug 18; 184(2):177-86); Kettleborough et al. (Kettleborough, C.A. et al., Isolation of tumor cell-specific single-chain Fv from immunized mice using phage-antibody libraries and the re-construction of whole antibodies from these antibody fragments. Eur J Immunol. 1994 Apr; 24(4) : 952-8); Persic et al. (Persic, L. et al. An integrated vector system for the eukaryotic expression of antibodies or their fragments after selection from phage display libraries. Gene. 1997 Mar 10; 187(1):9-18); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401 and US patents No. 5698426; 5223409; 5403484; 5580717; 5427908; 5750753; 5821047; 5571698; 5427908; 5516637; 5780225; 5658727; 5733743 and 5969108, all of which are incorporated herein by reference in their entirety. Expression of an antibody fragment on bacteriophages can be achieved by inserting a cDNA encoding the fragment into the gene expressing the viral envelope protein. The viral envelope of filamentous bacteriophages consists of five envelope proteins encoded in a single-stranded genome. The pill envelope protein is the protein of choice for antibody fragment expression, typically at the N-terminus. If expression of an antibody fragment disrupts pill protein function, viral function can be restored by coexpression of wild-type pill protein, although such expression will reduce the number of antibody fragments expressed on the viral envelope but may increase the availability of the antibody fragment to the target antigen. Alternatively, expression of viral proteins, as well as antibody fragments, can be encoded on multiple plasmids. This method can be used to reduce the overall size of the infectious plasmid and increase the efficiency of transformation.

As described above, once a host expressing a high-affinity antibody or antibody fragment (e.g., a glycan-interacting antibody) has been selected, coding regions from the antibody or antibody fragment can be isolated and used to produce whole antibodies, including human antibodies, or any other antigen-binding fragment desired. , and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast and bacteria, for example, as described in detail below.

The DNA sequence encoding a high-affinity antibody can be mutated in additional rounds of selection, in a process known as affinity maturation. As used herein, the term affinity maturation refers to a method by which antibodies with increased affinity for a particular antigen are produced through successful rounds of mutation and selection of the cDNA sequence encoding the antibody or antibody fragment. In some cases, this process is carried out in vitro. To accomplish this, the CDR coding sequence can be amplified using reduced precision PCR, yielding millions

- 22,045,483 copies containing mutations, including, but not limited to, point mutations, region mutations, insertion mutations, and deletion mutations. As used herein, the term point mutation means a mutation of a nucleic acid in which one nucleotide in a nucleotide sequence is replaced by another nucleotide. As used herein, the term region mutation means a mutation of a nucleic acid in which two or more consecutive nucleotides are replaced by different nucleotides. As used herein, the term insertion mutation means a mutation of a nucleic acid in which one or more nucleotides are inserted into a nucleotide sequence. As used herein, the term deletion mutation means a nucleic acid mutation in which one or more nucleotides are deleted from a nucleotide sequence. Insertion or deletion mutations can involve a complete replacement of an entire codon or the replacement of one codon with another by changing one or two nucleotides of the original codon.

It is possible to mutagenesis the CDR-encoding cDNA sequence, producing millions of mutants with single mutations in the heavy and light chain CDR regions. In another approach, random mutations are introduced only at CDR residues that are more likely to result in increased affinity. These newly generated mutant libraries can be used to repeat the process to screen for clones that encode antibody fragments with even higher affinity for the target antigen. Continuous rounds of mutation and selection promote the synthesis of clones with higher and higher affinities (Chao, G. et al., Isolating and engineering human antibodies using yeast surface display. Nat Protoc. 2006; 1(2):755-68).

Examples of methods that can be used to produce antibodies and antibody fragments, such as Fab and scFv, include methods described in US patent No. 4946778 and 5258498; Miersch et al. (Miersch, S. et al., Synthetic antibodies: Concepts, potential and practical considerations. Methods. 2012 Aug; 57(4):486-98), Chao et al. (Chao, G. et al., Isolating and engineering human antibodies using yeast surface display. Nat Protoc. 2006; 1(2):755-68), Huston et al. (Huston, J.S. et al., Protein engineering of single-chain Fv analogs and fusion proteins. Methods Enzymol. 1991; 203:46-88); Shu et al. (Shu, L. et al., Secretion of a singlegene-encoded immunoglobulin from myeloma cells. Proc Natl Acad Sci USA. 1993 Sep 1; 90(17):7995-9); and Skerra et al. (Skerra, A. et al., Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science. 1988 May 20; 240(4855): 1038-41), the contents of all of which are incorporated herein by reference in their entirety.

For certain applications, including in vivo use of antibodies (eg, glycan-interacting antibodies) in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different parts of the antibody are derived from different animal species, for example, antibodies having a variable region from a murine monoclonal immunoglobulin and a constant region from a human immunoglobulin. Methods for producing chimeric antibodies are known in the art. (Morrison, S.L., Transfectomas provide novel chimeric antibodies. Science. 1985 Sep 20; 229(4719):1202-7; Gillies, S.D. et al., High-level expression of chimeric antibodies using adapted cDNA variable region cassettes. J Immunol Methods 1989 Dec 20; 125(l-2):191-202.; and US Patent Nos. 5,807,715, 4,816,567 and 4,816,397, the contents of which are incorporated herein by reference in their entirety).

Humanized antibodies are antibody molecules of a non-human species that bind an antigen of interest and have one or more complementarity determining regions (CDRs) from non-human species antibodies and framework regions from a human immunoglobulin molecule. Often, framework residues in the framework regions of a human antibody are replaced with corresponding residues from the CDRs and framework regions of the donor antibody to alter, preferably improve, antigen binding. These framework residue substitutions are determined by methods well known in the art, for example, by modeling the interactions of CDRs and framework residues to identify framework residues important for antigen binding, and by comparing sequences to identify unusual framework residues at specific positions. (U.S. Patent Nos. 5,693,762 and 5,585,089; Riechmann, L. et al., Reshaping human antibodies for therapy. Nature. 1988 Mar 24; 332(6162):323-7, the contents of which are incorporated herein by reference in their entirety). Antibodies can be humanized using various methods known in the art, including, for example, CDR grafting (EP 239400; PCT publication WO 91/09967; US patent No. 5225539; 5530101 and 5585089); masking of surface residues or modification of the surface (EP 592106; EP 519596; Padlan, E.A., A possible procedure for reducing the immunogenicity of antibody variable domains while preserving their ligand-binding properties. Mol Immunol. 1991 Apr-May; 28(4-5) :489-98; Studnicka, G.M. et al., Human-engineered monoclonal antibodies retain full specific binding activity by preserving non-CDR complementarity-modulating residues. Protein Eng. 1994 Jun; 7(6): 805-14; Roguska, M.A. et al., Humanization of murine monoclonal antibodies through variable domain resurfacing. Proc Natl Acad Sci USA. 1994 Feb 1; 91(3):969-73); and chain shuffling (US Patent No. 5565332); the contents of all of which are incorporated herein by reference in their entirety. The humanized antibodies of the present invention can be developed to achieve the desired

- 23 045483 binding specificity, complement-dependent cytotoxicity and antibody-dependent cell-mediated cytotoxicity, and so on.

In some cases, human antibody framework sequences are selected by aligning donor antibody sequences with human antibody framework sequences to identify candidate human antibody frameworks with the highest level of homology. In some cases, framework regions may be selected from more than one candidate human antibody framework (eg, framework regions 1-3 may be selected from one candidate and framework region 4 may be selected from an alternative candidate). In some cases, framework regions can be selected from human consensus sequences to avoid the risk of including immunogenic epitopes resulting from somatic mutations. Consensus sequences are sequences obtained by comparing many sequences and selecting the most frequently occurring residues at each position. In some cases, human antibody framework sequences may be selected from human antibody germline sequences. They can be obtained by database searching (eg, using the NCBI Protein Database or other databases).

The framework sequences of the light and heavy chains of a human antibody can be selected from the same or from different clones. Light and heavy chains derived from the same clone are more likely to bind, producing binding sites that are functional; however, the conserved nature of the interface between the heavy and light chains generally allows light and heavy chains from different lineages to bind and be functional. Information on the frequency of pairing between framework sequences of the light and heavy chains of human antibodies can be obtained, for example, from Tiller et al., 2013. MAbs. 5(3): 445-70, the contents of the publication are incorporated herein by reference in their entirety.

The possibility of backmutating residues in the humanized antibody sequences may be considered to increase or restore antibody affinity lost during the humanization process. Back mutation involves changing residues modified during the humanization process back to residues present in the original non-human antibody sequence. Residues that are candidates for back mutation can be identified, for example, by comparison with standard conformations found in canonical antibody structures (see Al-Lazikani, et al., 1997. J. Mol. Biol. 273: 927- 48, the contents of the publication are incorporated herein by reference in their entirety). Unusual canonical residues can be identified and targeted for reverse mutation. In some cases, residues that are candidates for reverse mutation may be vernier residues, a term generally referring to residues in contact with a CDR. These residues are more likely to influence the location and conformation of the CDR, and therefore the affinity and/or specificity of the antibody (Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 6, p117). In some cases, the framework regions of the human antibody are not altered, but reverse-mutated to the CDRs from the donor antibody, matching the human CDR regions while maintaining binding by empirical means.

Fully human antibodies (eg, glycan-interacting antibodies) are particularly preferred for the therapeutic treatment of human patients to avoid, or reduce, an immune response to a foreign protein. Human antibodies can be produced by a variety of methods known in the art, including the antibody display methods described above using antibody libraries derived from human immunoglobulin sequences. See also US Patent Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741; the contents of all of which are incorporated herein by reference in their entirety.

Human antibodies (eg, glycan-interacting antibodies) can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins, but which can express human immunoglobulin polynucleotides. For example, polynucleotide complexes of human immunoglobulin heavy and light chains can be introduced randomly, or by homologous recombination, into mouse embryonic stem cells. Alternatively, the variable region, constant region and variability region of a human antibody can be introduced into murine embryonic stem cells, in addition to the heavy and light chain polynucleotides of the human antibody. Mouse immunoglobulin heavy and light chain polynucleotides can be rendered nonfunctional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. Modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then crossed to produce homozygous offspring that express human antibodies. Transgenic mice are immunized in the usual manner with a selected antigen, for example all or part of a glycan molecule, glycoconjugate and/or polypeptide of the invention.

Thus, useful human IgG, IgA, IgM, IgD and IgE antibodies can be obtained using this method. For a review of technologies for producing human antibodies, see Lonberg and Huszar (Lonberg, N. et al., Human antibodies from transgenic mice. Int Rev Immunol. 1995; 13(1):65-93). For a detailed description of the technology for producing human antibodies and human monoclonal antibodies, as well as protocols for producing such antibodies, see, for example, PCT publication WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; US Patents No. 5,413,923; 5625126; 5633425; 5569825; 5661016; 5545806; 5814318; 5885793; 5916771; 5939598; 6075181 and 6114598, all of which are incorporated herein by reference in their entirety. In addition, companies such as Abgenix, Inc. can be involved. (Fremont, Calif.), Protein Design Labs, Inc. (Mountain View, Calif.) and Genpharm (San Jose, Calif.) to produce human antibodies directed against a selected antigen using technology similar to the technologies described above.

Once the antibody molecule of the present invention has been produced using an animal, cell line, chemically synthesized, or recombinantly expressed, it can be purified (i.e., isolated) by any method known in the art for purifying an immunoglobulin molecule or polypeptide, such as chromatography. (eg, ion exchange, affinity, particularly by affinity for a specific antigen, protein A and size exclusion chromatography column), centrifugation, differential solubility, or any other standard protein purification method. In addition, antibodies of the present invention, or fragments thereof, can be fused to heterologous polypeptide sequences described herein or known in the art to facilitate purification.

The affinity between an antibody and a target or ligand (such as the antigen used to make a particular antibody) can be measured as K _D using one or more of the binding assays described herein. Depending on the desired use of a particular antibody, different KD values may be desirable. High affinity antibodies typically form bonds with ligands with a KD value of about 10 ^-5 M or less, for example, about 10 ^-6 M or less, about 10 ^-7 M or less, about 10 ^-8 M or less, about 10 ^{- 9} M or less, about 10 ^-10 M or less, about 10 ^-11 M or less, or about 10 ^-12 M or less.

In some embodiments, antibodies of the invention can be characterized by their half-maximal effective or inhibitory concentration (EC ₅₀ or IC ₅₀ , respectively). In some cases, this value may represent the concentration of antibody required to inhibit cells expressing STn (e.g., kill, reduce proliferation rate, and/or eliminate one or more cellular functions) at a level equal to half the maximum inhibition observed at the highest concentrations antibodies. Such IC ₅₀ values may range from about 0.001 nM to about 0.01 nM, from about 0.005 nM to about 0.05 nM, from about 0.01 nM to about 1 nM, from about 0.05 nM to about 5 nM, from about 0.1 nM to about 10 nM, from about 0.5 nM to about 25 nM, from about 1 nM to about 50 nM, from about 5 nM to about 75 nM, from about 10 nM to about 100 nM, from about 25 nM to about 250 nM, about 200 nM to about 1000 nM, or greater than 1000 nM.

In some embodiments, the antibodies described herein can be tested for their ability to target patient-derived cancer cells and/or cancer stem cells (CSCs). In such embodiments, patient-derived cancer cells can be cultured in vitro, and the antibodies of the present invention can be used to target such cells.

In other embodiments, patient-derived tumor cells or tumor fragments can be used to produce patient-derived tumor xenografts (PDX). In some cases, fragments of primary or metastatic solid tumors that preserve tissue structure can be obtained through a surgical procedure or biopsy. In some cases, fluid collected from malignant ascites or pleural effusions may be used. Tumors can be implanted as fragments or single-cell suspensions, either alone or, in some studies, coated with Matrigel® (Coming Life Sciences, Coming, NY) or mixed with human fibroblasts or mesenchymal stem cells. Implantation sites may include the dorsal region of the mouse (subcutaneous implantation), although one option may be implantation in the same organ as the original tumor (orthotopic implantation, i.e. pancreas, oral cavity, ovary, mammary fat pad, brain and so on). Additionally, regardless of tumor origin, some approaches may involve implanting primary tumors into the renal capsule in an attempt to increase the implantation success rate. Such studies can use different strains of mice that have varying degrees of immunosuppression. For hormone-dependent tumors, some studies may use the addition of hormones with the intention of increasing graft engraftment rates. In some embodiments, PDX tumors can be generated in non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. Antibodies can be administered to mice bearing PDX tumors and the effect on tumor volume analyzed. In some cases, PDX tumors can be excised, dissociated into cells, and the resulting cells grown in culture. The ability of the antibodies of the present invention to target these cells can be assessed in vitro.

The production of antibodies, monoclonal or polyclonal, is known in the art. Techniques for producing antibodies are well known in the art and are described, for example, in Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988 and Harlow and Lane Using Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory Press, 1999.

Targets.

The glycan-interacting antibodies of the present invention may exert their effect by binding (reversibly or irreversibly) to one or more glycans, or glycan-associated or glycan-linked targets. In some embodiments, glycan-interacting antibodies can be generated against any target region described herein. In some embodiments, the targets of the present invention include glycans. Glycans used to produce antibodies may include a sugar chain having at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least at least 19 or at least 20 residues. Some glycans used to make antibodies can range from about 2 residues to about 5 residues.

In some embodiments, the target antigens of the glycan-interacting antibodies include sialic acids. N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) are the main sialic acids on mammalian cell surfaces. Of these, Neu5Ac is naturally produced in humans. Neu5Gc is naturally produced in most mammals, with the exception of humans due to a mutation in the cytidine monophosphate (CMP)-N-acetylneuraminic acid (CMAN) hydroxylase gene responsible for the production of CMP-Neu5Gc from CMP-Neu5Ac. Neu5Gc in humans is, in fact, immunogenic, and almost all humans express anti-Neu5Gc antibodies. Although not produced, most systems in the human body contain some amount of Neu5Gc due to dietary absorption. Such foreign products are subsequently incorporated into human glycoproteins. Such glycoproteins are provided as targets according to the invention.

Glycan target antigens of the present invention may include, but are not limited to, those listed in Table 1. 1. Abbreviations used include: Glc - glucose, Gal - galactose, GlcNAc - N-acetylglucosamine, GalNAc - N-acetylgalactosamine, GlcNAc6S - 6-sulfo-Nacetylglucosamine, KDN - 2-keto-3-deoxy-O-glycero-O- galactonononic acid, Neu5,9Ac2 - Nacetyl-9-O-acetylneuraminic acid, Fuc - fucose and Neu5GcOMe - 2-O-methyl-Nglycolylneuraminic acid. O-glycosidic bonds are present between all residues in the listed glycans, with α and β indicating the relative stoichiometry between the two residues linked by the bond, with α indicating axial orientation and β indicating equatorial orientation. The numbers following α and/or β, in the format x, x, indicate the carbon number of each of the carbon atoms from each of the adjacent residues involved in the formation of the bond. Although the listed glycans represent the individual glycan target antigens provided, the present invention also includes embodiments in which the above glycans contain different combinations of α and β-oriented O-glycosidic linkages than those shown. R represents a moiety to which a glycan can be linked. In some embodiments, R is a protein, wherein the glycan is typically linked to a serine or threonine residue. In some embodiments, R is a linker molecule used to connect the glycan to a substrate, for example, in a glycan matrix. In some embodiments, R may be a linker with the formula -(CH ₂ ) ₂ CH ₂ NH ₂ or -(CH ₂ ) ₃ NHCOCH ₂ (OCH ₂ CH ₂ ) ₆ NH ₂ . In some embodiments, R may be biotin, albumin, ProNH ₂ , -CH-, -OH, -OCH ₃ , -OCH2CH3, -H, hydrido, hydroxy, alkoxy, oxygen, carbon, sulfur, nitrogen, polyacrylamide, phosphorus, NH2, ProNH2=O(CH2)2CH2NH2, (OCH2CH2)6NH2, O(CH2)3NHCOCH2(OCH2CH2)6NH2, fluorescent labels 2-aminobenzamide (AB) and/or 2-aminobenzoic acid (AA), an analogue of 2-aminobenzamide containing alkylamine (AEAB), aminooxy groups, methylaminooxy groups, hydrazide groups, aminolipid 1,2-dihexadecyl-sn-glycero-3-phosphoethanolamine (DHPE), aminooxy (AO) functionalized DHPE and glycosylphosphatidylinositol (GPI). Without intending to limit the source or nature of R, this may include structures that affect the physical location of the glycan residue. In some embodiments, the R group may include a combination of the R groups provided herein, for example, biotinylated polyacrylamide. In some embodiments, the R group in combination with underlying substrates may influence the location of the glycan residue.

- 26 045483

Table 1

Glycan target antigens ________________Glycan___________

GalNAca-R

Galal,3Galfl,4GlcNAc3-R

Gal31,3GalNAc3-R

Galfl,3GlcNAca-R

Gaipi,3GlcNAc31,3Gaipi,4Glc3-R

Galfl,3GlcNAc3-R

Galfl,4GlcNAc6S3-R

Galfl,4GlcNAc3-R

Gaφl,4Glcβ-R

KDNa2,8Neu5Aca2,3Galfl,4Glc3-R

KDNa2,8Neu5Gca2,3Galfl,4Glc3-R

Neu5,9Ac2a2,3Gal31,3GalNAca-R

Neu5,9Ac2a2,3Galfl,3GalNAc3-R

Neu5,9Ac2a2,3Galfl,3GlcNAc3-R

Neu5.9Ac2a2.3Gal31.4GlcNAc3-R

Neu5,9Ac2a2,3Galfl,4Glc3-R

Neu5.9Ac2α2,ЗGaφ-R

Neu5.9Ac2a2.6GalNAca-R

Neu5,9Ac2a2,6Gaipi,4GlcNAc3-R

Neu5,9Ac2a2,6Galfl,4Glc3-R

Neu5.9Ac2a2.6Gaφ-R

Neu5 Aca2.3 Saf 1.3GalNAca-R

Neu5 Aca2,3 Caf1,3GalN Αοβ-R

Neu5 Aca2,3 Saf1,3GlcN Αοβ 1,3 Saf1,4Gktf-R

Neu5 Aca2.3 Ga^ 1.3GlcN Αοβ-R

Neu5Acα2,ЗGalβl,4(Fucαl,3)GlcNAc6Sβ-R

Νευ5Αοα2,3θ31β1,4(Ρυοα1,3)01οΝΑοβ-Κ

Neu5 Aca2.3 Ga^ 1.4GlcNAc6Sβ-R

Neu5 Asa2,3 Saf 1,4GlcN Αοβ-R

Neu5 Aca2,3 Saf 1,4Glcβ-R

Neu5Aca2,3 Saf-R

Νευ5Αοα2,6(ΚΟΝα2,3)Οαφ1,401οβ^

Neu5Acα2,6(Neu5Acα2,3)Gaφl,4Glcβ-R

Neu5Aca2,6(Neu5Gca2,3)Gaφl,4Glcβ-R

Neu5Aca2,6GalNAca-R

Neu5Aco^6Gai|L4GlcNAc^R

Hei5Asa2.6C!af1.4Okf^

Neu5Acα2,6Gaφ-R

Hei5Asa2.8KOHA2.6S!af1.4Okf^

Neu5Aca2^8Neu5Aca2^

- 27 045483

Neu5Aca2,8Neu5Aca2,3Gal31,4Glc3-R

Neu5Aca2,8Neu5Aca2,6Gal31,4Glc3-R

Neu5Aca2,8Neu5Aca2,8Neu5Aca2,3Gal31,4Glc3-R

Neu5Aca2,8Neu5Aca2,8Neu5Aca2,3Gal31,4Glc3-R

Neu5Aca2,8Neu5Gca2,3Gal31,4Glc3-R

Neu5Aca2.8Neu5Gca2.6Gal31.4Glc3-R

Neu5Gc9Aca2,3Gal31,4Glc3-R

Neu5Gc9Aca2.6Gal31.4Glc3-R

INeu5 Gc9 Aca2.3 Gal β 1,3 GalNAc a-R

Neu5 Gc9 Aca2.3 Gal β 1,3 GalN Ac β-R

Neu5Gc9Aca2,3Gal31,3GlcNAc3-R

Neu5Gc9Aca2,3Gal31,4GlcNAc3-R

Neu5Gc9Aca2,3Gal3-R

Neu5Gc9Aca2,6GalNAca-R

Neu5Gc9Aca2.6Gal31.4GlcNAc3-R

Neu5Gc9Aca2,6Gal3-R

Neu5GcOMea2,8Neu5Aca2,3Gal31,4Glc3-R

Neu5Gca2,3Gal31,3GalNAca-R

Neu5Gca2,3Gal31,3GalN Αοβ-R

Neu5Gca2,3 Gal β1,3GlcN Αοβ 1,3 Saf 1,4Glc3-R

Neu5Gca2,3Gal31,3GlcNAc3-R

Neu5Gca2,3Gaipi,4(Fucal,3)GlcNAc6SP-R

Neu5Gca2,3Gal31,4(Fucal,3)GlcNAcP-R

Neu5Gca2,3Gaipi,4GlcNAc6SP-R

Neu5Gca2,3Gal31,4GlcN Αοβ-R

Neu5Gca2,3Gal31,4Glc3-R

Neu5Gca2,3Gal3-R

Neu5Gca2,6GalNAca-R

Neu5Gca2,6Gal31,4GlcNAc3-R

Neu5Gca2,6Gal31,4Glc3-R

Neu5Gca2,6Gal3-R

Neu5Gca2,8Neu5Aca2,3Gal31,4Glc3-R

Neu5Gca2,8Neu5Gca2,3Gal31,4Glc3-R

Glycan targets of the present invention may include one or more antibody recognition regions. As used herein, the term antibody recognition region means a segment located on any portion of a molecule, an attached moiety, or located on an interaction region between a glycan and another molecule, including, but not limited to, another glycan, protein, membrane, cell surface structure, or extracellular component. matrix. In some embodiments, antibody recognition regions are located at intrachain target sites, wherein the term intrachain means located within the actual polymer chain. Intrastrand target sites may include antibody recognition regions having 1, 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 residues, bonds between residues, or combinations of residues and bonds. In some embodiments, antibody recognition regions are located in regions of interaction between one or more glycan chains. Such regions may be between 2, 3, 4 or at least 5 glycan chains.

In some embodiments, antibody recognition regions are located in regions of interaction between branching glycan chains associated with a common parent chain. In some embodiments, antibody recognition regions are located in regions of interaction between the branch glycan chain and the parent chain. In some embodiments, antibody recognition regions are located in regions of interaction between glycans and proteins. Such interaction areas may include chemical bonds between the glycan and the protein, including, but not limited to, covalent bonds, ionic bonds, hydrostatic bonds, hydrophobic bonds, and hydrogen bonds. In some embodiments, antibody recognition regions are located in regions of interaction between glycans and other biomolecules, including, but not limited to, lipids and nucleic acids. Such interaction areas may include chemical bonds between the glycan and the biomolecule, including, but not limited to, covalent bonds, ionic bonds, hydrostatic bonds, hydrophobic bonds, and hydrogen bonds.

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In some embodiments, the glycan targets of the present invention are components of glycoconjugates. As used herein, the term glycoconjugate means an element associated with a glycan moiety. In some embodiments, the glycoconjugates are glycolipids. As used herein, the term glycolipid refers to a class of lipids having a covalently attached carbohydrate moiety. In some embodiments, the carbohydrate moieties found on the glycolipids may be glycans. In some embodiments, the lipid components of the glycolipids include ceramide moieties. Examples of glycolipids contemplated as targets of the present invention include, but are not limited to, glyceroglycolipids (including, but not limited to, galactolipids and sulfolipids), glycosphingolipids (including, but not limited to, cerebrosides (e.g., galactocerebrosides, glucocerebrosides, and sulfatides), gangliosides , globosides and glycophosphophosphingolipids) and glycosylphosphatidylinositols. When located in cell membranes, the glycan moieties of glycolipids are located on the extracellular side of the membrane, where they can interact with other cells as well as cell signaling ligands (Maccioni, H.J. et al., Organization of the synthesis of glycolipid oligosaccharides in the Golgi complex. FEBS Lett. 2011 Jun 6;585(11):1691-8).

In some embodiments, the glycoconjugate targets of the present invention are glycoproteins and/or proteoglycans. The term glycoproteins refers to any proteins that are covalently linked to glycans. Proteoglycans are a class of proteins that are highly glycosylated with glycans that often carry a negative charge. This property makes them very hydrophilic and important components of connective tissue.

Cancer-related targets.

In some embodiments, the targets of the present invention are cancer-related antigens or epitopes. As used herein, the term cancer-related is used to describe elements that may be associated in any way with cancer, cancer cells and/or cancer tissues. Many cancer-associated antigens or epitopes containing glycans whose expression is correlated with tumor cells have been identified (Heimburg-Molinaro, J. et al., Cancer vaccines and carbohydrate epitopes. Vaccine. 2011 Nov 8; 29(48):880226). These are referred to herein as tumor-associated carbohydrate antigens or TACAs. TACAs include, but are not limited to, mucin-associated antigens [including, but not limited to, Tn, sialyl-Tn (STn), and Thomson-Friedenreich antigen], Lewis blood group-related antigens [including, but not limited to, ^LewisY ), Lewisx (Lex), sialyl-Lewisx (SLex) and sialyl-Lewis ^A (SLeA)], glycosphingolipid-associated antigens [including, but not limited to, Globo H, stage-specific embryonic antigen 3 (SSEA-3) and glycosphingolipids, which include sialic acid], ganglioside-associated antigens [including, but not limited to, gangliosides GD2, GD3, GM2, fucosyl GM1 and Neu5GcGM3] and polysialic acid-associated antigens. Many of these antigens are described in International Publication No. WO 2015054600, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the TACA targets of the present invention include Lewis blood group-related antigens. Lewis blood group-related antigens contain a fucose residue linked to a GlcNAc α1-3 linkage or an α1-4 linkage. They can be found on both glycolipids and glycoproteins. Lewis blood group-related antigens can be found in the body fluids of individuals who secrete these antigens. Their presence on red blood cells is a consequence of the absorption of Lewis antigens from serum by red blood cells.

In some embodiments, the TACA targets of the present invention include Le ^Y . Le ^Y (also known as CD174) consists of Gale1,4GlcNAC having α1,2- as well as α1,3-linked fucose residues, producing the epitope Fuca(1,2)Gale(1,4)Fuca(1,3 )GlcNAc. It is synthesized from Nantigen by α1,3-fucosyltransferases, which add α1,3-fucose to the GlcNAc residue of the parent chain. Le ^Y can be expressed in a variety of cancers, including, but not limited to, ovarian, breast, prostate, colon, lung, and epithelial cancers. Due to its low expression level in normal tissues and increased expression levels in many cancers, Le ^Y antigen is a promising target for therapeutic antibodies.

In some embodiments, the TACA targets of the present invention include Lex. Lex includes the Gale1-4(Fuca1-3)GlcNAce-R epitope. It is also known as CD15 and stage-specific embryonal antigen 1 (SSEA-1). This antigen was first identified as immunoreactive with serum obtained from mice immunized with F9 teratocarcinoma cells. A correlation of Lex with embryonic development at certain stages has also been established. It is also expressed in various tissues, both in the presence and absence of cancer, but can also be found in breast and ovarian cancer cells, where it is expressed only by malignant cells.

In some embodiments, the TACA targets of the present invention include SLe ^A

- 29 045483 and/or SLex. SLe ^A and SLex contain the structures Neu5Aca2-3Gale1-3(Fuca1-4)GlcNAce-R and Neu5Aca23Gaiei-4(Fuca1-3)GlcNAce-R, respectively. Their expression is increased in cancer cells. The presence of these antigens in serum correlates with malignancy and poor prognosis. SLex is primarily found as a terminal epitope of mucin. It is expressed in a number of different cancers, including breast, ovarian, melanoma, colon, liver, lung and prostate cancers. In some embodiments of the present invention, the targets SLe ^A and SLex contain Neu5Gc (herein referred to as GcSLe and GcSLex, respectively).

In some cases, cancer-related targets of the invention may include mucins. Ishida et al. demonstrated that MUC2 interaction with dendritic cells (with antitumor activity) leads to dendritic cell apoptosis (Ishida, A. et al., 2008. Proteomics. 8: 3342-9, incorporated herein by reference in its entirety volume). In some aspects, the present invention relates to anti-mucin antibodies used to prevent dendritic cell apoptosis and maintain antitumor activity.

In some embodiments, the TACA targets of the present invention include glycolipids and/or epitopes found on glycolipids, including, but not limited to, glycosphingolipids. Glycosphingolipids contain the lipid ceramide linked to a glycan by the hydroxyl group of the ceramide. On the cell membrane, glycosphingolipids form clusters called lipid rafts.

In some embodiments, the TACA targets of the present invention include Globo H. Globo H is a cancer-associated glycosphingolipid first identified in breast cancer cells. The glycan moiety of Globo H includes Fuca(1-2)Gale(1-3)GalNAce(13)Gala(1-4)Gale(1-4)Glce(1). Although it can be found in a variety of normal epithelial tissues, Globo H has been found to be associated with many tumor tissues, including, but not limited to, small cell lung cancer, breast, prostate, lung, pancreatic, gastric, ovarian, and endometrial tumors.

In some embodiments, cancer-related glycosphingolipid targets of the present invention include gangliosides. Gangliosides are glycosphingolipids containing one or more sialic acids. According to ganglioside nomenclature, G is used as an abbreviation for ganglioside. This abbreviation is followed by the letters M, D, or T, indicating the number of sialic acid residues attached (1, 2, or 3, respectively). Finally, the numbers 1, 2 or 3 are used to indicate the order of the distance each travels when analyzed by thin layer chromatography (with 3 traveling the longest distance, followed by 2, and then 1). Gangliosides are known to be involved in cancer growth and metastasis and can be expressed on the cell surface of tumor cells. Gangliosides expressed on tumor cells may include, but are not limited to, GD2, GD3, GM2, and Fucosyl-GMI (also referred to herein as Fuc-GM1). In some embodiments of the present invention, the glycan-interacting antibodies are directed against GD3. GD3 is a cell growth regulator. In some embodiments, GD3-directed antibodies are used to modulate cell growth and/or angiogenesis. In some embodiments, GD3-directed antibodies are used to modulate cell attachment. GD3 associated with some tumor cells may contain 9-O-acetylated sialic acid residues (Mukheijee, K. et al., 2008. J Cell Biochem. 105: 724-34 and Mukherjee, K. et al., 2009. Biol. Chem. 390: 325-35, the contents of each publication are incorporated herein by reference in their entirety). In some cases, the antibodies of the invention are selective for 9-O-acetylated sialic acid residues. Some antibodies may be specific for 9-O-acetylated GD3. Such antibodies can be used to target tumor cells expressing 9-O-acetylated GD3. In some embodiments of the present invention, the glycan-interacting antibodies are directed against GM2. In some embodiments, GM2-directed antibodies are used to modulate cell-cell contacts. In some embodiments, the ganglioside targets of the present invention contain Neu5Gc. In some embodiments, such targets may include a GM3 variant containing Neu5Gc (herein referred to as GcGM3). The glycan component of GcGM3 is Neu5Gca2-3Gale1-4Glc. GcGM3 is a known component of tumor cells (Casadesus, A.V. et al., 2013. Glycoconj J. 30(7):687-99, incorporated herein by reference in its entirety).

In some embodiments, the TACAs of the present invention contain at least one Neu5Gc residue.

Recombinant antibodies.

Recombinant antibodies (eg, glycan-interacting antibodies) of the invention can be produced by standard methods known in the art. In some embodiments, the recombinant antibodies may be anti-glycan antibodies. In addition, the antibodies may be anti-STn antibodies (for example, anti-GcSTn or anti-AcSTn an

- 30 045483 title). Recombinant antibodies of the invention can be produced using variable domains from antibodies obtained from hybridoma cells by the methods described herein. The cDNA sequences of the variable regions of the heavy and light chains of antibodies can be determined using standard biochemical methods. Total RNA can be extracted from antibody-producing hybridoma cells and converted to cDNA by reverse transcriptase (RT) polymerase chain reaction (PCR). PCR amplification of the resulting cDNA can be performed to amplify variable region genes. Such amplification may involve the use of primers specific for amplification of heavy and light chain sequences. In other embodiments, recombinant antibodies can be produced using variable domains obtained from other sources. This includes the use of variable domains selected from one or more antibody fragment libraries, for example, a scFv library used for antigen panning. The resulting PCR products can then be subcloned into plasmids for sequence analysis. Once sequenced, the antibody coding sequences can be placed into expression vectors. For the purpose of humanization, the coding sequences of the constant domains of the heavy and light chains of a human antibody can be used to replace homologous murine sequences. The resulting constructs can then be transfected into mammalian cells for large-scale translation.

Anti-Tn antibodies.

In some embodiments, the recombinant antibodies of the invention (eg, glycan-interacting antibodies) may be anti-Tn antibodies. Such antibodies can bind targets containing Tn. Anti-Tn antibodies may be specific for Tn or may bind other modified forms of Tn, for example, Tn bound to other moieties, including, but not limited to, additional carbohydrate residues. In some cases, anti-Tn antibodies may be anti-sialyl-Tn antibodies. Such antibodies can bind Stalated Tn containing Neu5Ac and/or Stalated Tn containing Neu5Gc. Some anti-Tn antibodies can specifically bind Tn antigen clusters.

Anti-STn antibodies.

In some embodiments, antibodies of the invention (eg, glycan-interacting antibodies) may specifically bind STn. Anti-STn antibodies of the invention may be categorized based on their binding to certain fragments of STn antigens and/or based on their specificity for AcSTn or GcSTn. In some cases, the anti-STn antibodies of the invention are Group 1 antibodies. The Group 1 antibodies of the invention are antibodies capable of binding AcSTn and GcSTn. Such antibodies may also be referred to herein as anti-pan-STn antibodies due to their ability to bind a wider range of STn structures. In some embodiments, Group 1 antibodies may bind the STn fragment indicated by the largest ellipse in FIG. 1A. In some cases, the anti-STn antibodies of the invention are Group 2 antibodies. The Group 2 antibodies of the invention are antibodies capable of binding STn as well as certain related structures containing O-bonds to serine or threonine residues. In some embodiments, group 2 antibodies can bind glycans containing a sialylated galactose moiety. In some cases, group 2 antibodies may bind the STn fragment indicated by the largest ellipse in FIG. 1B. Some group 2 antibodies preferentially bind AcSTn structures over GcSTn structures. The following anti-STn antibodies may be group 3 antibodies. As used herein, group 3 antibodies are antibodies capable of binding STn, but also capable of binding a broader group of related structures. Unlike group 2 antibodies, group 3 antibodies do not require such structures to be O-bonded to serine or threonine residues. In some embodiments, Group 3 antibodies may bind the STn fragment indicated by the largest ellipse in FIG. 1C. Finally, some anti-STn antibodies of the invention may be group 4 antibodies. As used herein, group 4 antibodies are antibodies capable of binding both AcSTn and GcSTn, as well as non-stalated Tn antigen, and thus have more broad specificity. In some embodiments, group 4 antibodies may bind the STn fragment indicated by the largest ellipse in FIG. 1D.

In some cases, anti-STn antibodies of the invention may specifically bind STn clusters on a particular antigen or cell surface. Some such antibodies can recognize epitopes generated by STn cluster formation, including epitopes involving contact regions between adjacent STn structures. Such epitopes can be formed by clustering 2, 3, 4, 5, 6, 7, 8, 9, 10 or more STn structures.

In some embodiments, the anti-STn antibodies of the present invention can be used to bind cellular proteins bearing STn. Such antibodies may be useful for targeting cellular proteins associated with cancer cells, which can be distinguished from similar proteins in non-cancerous cells by STn expression. In some cases, such proteins may include cell surface proteins. Proteins on the surface of cancer cells that carry STn can

- 31 045483 be sent anti-STn antibodies during the treatment and/or diagnosis of cancer. Cell surface proteins bearing STn can be identified by mass spectrometry and/or immunological methods (eg, using FACS analysis, immunoprecipitation, immunoblotting, ELISA, etc.). In some cases, the cellular proteins carrying STn may include cancer cell markers, cancer stem cell markers, and/or cancer stem cell signaling proteins. In some embodiments, STn-bearing cellular proteins may include, but are not limited to, CD44, CD133, CD117, integrins, Notch, and Hedgehog.

Antibody components.

In some cases, the antibodies, or antigen-binding fragments thereof, of the invention may comprise the variable domain and/or CDR amino acid sequences provided herein. In some cases, antibodies may contain any of the antibody or antibody fragment sequences set forth in International Patent Publication Number WO2017083582 (the entire contents of which are incorporated herein by reference), including: any of the variable domain sequences shown in Table. 2 specified documents; any of the CDR sequences given in table. 3 of the specified document; any of the groups of VH CDR sequences given in table. 4 of the specified document; any of the groups of VL CDR sequences given in table. 5 of the specified document; any of the nucleotide sequences of the variable domains given in table. 6 of the specified document; or any of the sequences of humanized variable domains shown in table. 11 of the specified document. Some antibodies, or antigen binding fragments, may include various combinations of such sequences or variants having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98 %, at least 99%, or at least 99.5% sequence identity. In some cases, the antibodies, or antigen binding fragments, of the invention may contain one or more of the variable domain sequences set forth in Table 1 below. 2. The following light chain variable domains can be expressed with or without the C-terminal arginine residue. This residue typically links light chain variable domains to light chain constant domains and may be expressed as part of a light chain constant domain instead of a light chain variable domain. In some cases, antibodies, or antigen binding fragments thereof, may comprise an amino acid sequence having from about 50% to about 99.9% sequence identity (e.g., from about 50% to about 60%, from about 55% to about 65%, from about 60% to about 70%, about 65% to about 75%, about 70% to about 80%, about 75% to about 85%, about 80% to about 90%, about 85% to about 95%, about 90% to about 99.9%, about 95% to about 99.9%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%, about 99.6%, about 99.7%, or about 99.8%) with one or more variable domain sequences shown in table. 2. In some cases, antibodies, or antigen-binding fragments thereof, of the invention may contain an amino acid sequence containing one or more fragments of any of the above sequences.

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table 2

Variable domain sequences

Domain Subsequence SEQ ID NO mSIAlOl, VH domain QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPE QGLEWIGYFSPGNDDIKYNEKFRGKATLTADKSSSTAYMQLN SLSSDDSAVYFCKRSLSTPYWGQGTLVTVSA 1 mSIAlOl, VL domain DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNRGNHKNYLTWY RQKPGLPPKLLIYWASTRESGVPDRFTGSGSGTDFALTISSVQA EDLAVYYCQNDYTYPYTFGGGTKLEIKR 2 mSIA102, VH domain QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPE QGLEWIGYFSPGNDDIKYNEKFKVKATLTADKSSSTAYMQLT SLTSEDSAVYFCKRSYYGDwgqgttltvss 3 mSIA102, VL domain DIQMTQSPASLSVSVGETVTITCRASENIYSHLAWYQQKQGKS PQLLVYGATNLADGVPSRFSGSGSGTQFSLKIHSLQSEDFGSY YCQHFWGAPFTFGSGTKLEIK 4 mSIA103, VH domain QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQKPE QGLDWIGYISPGNGDIKYNEKFKDKVTLTADKSSSTACMHLN SLTSEDSAVYFCKRSLLALDywgogttltvss 5 mSIA103, VL domain DIVMTQSHKFMSTSVGDRVSITCKASQDVGTNIAWYQQKPGR SPKVLIYSASTRHTGVPDRFTGSGSGTDFTLTISNVQSEDLTDY FCQQYSSFPLTFGVGTKLELK 6 hSIAlOl, VH domain EVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAP GQGLEWMGYFSPGNDDIKYNEKFRGRVTMTADKSSSTAYME LRSLRSDDTAVYFCKRSLSTPYWGOGTLVTVSS 7 hSIAlOl, VL domain DIVMTQSPDSLAVSLGERATINCKSSQSLLNRGNHKNYLTWY QQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQA EDVAVYYCQNDYTYPYTFGOGTKVEIK 8 hSIA102, VH domain QVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVRQAPG QGLEWIGYFSPGNDDIKYNEKFKVRATLTADKSSSTAYMELRS 9 LRSDDTAVYFCKRSYYGDWGQGTLVTVSS hSIA102, VL domain DIQMTQSPSSLSASVGDRVTITCRASENIYSHLAWYQQKPGKA PKLLVYGATNLASGVPSRFSGSGSGTQFTLTISSLQPEDFATYY CQHFWGAPFTFGQGTKVEIK 10 hSIA103, VH domain QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVRQAP GQGLEWMGYISPGNGDIKYNEKFKDRVTMTADKSSSTAYMQ LRSLRSDDTAVYFCKRSLLAldywgogtlvtvss eleven hSIA103, VL domain DIQMTQSPSSLSSASVGDRVTITCKASQDVGTNIAWYQQKPGK APKVLIYSASTRHTGVPSRFSGSGSGTDFTLTISSLOPEDFATYF COOYSSFPLTFGOGTKVEIK 12

In some cases, the antibodies, or antigen binding fragments, of the invention may contain any of the IgG sequences listed in Table 1. 3. In some cases, antibodies, or fragments thereof, may contain an amino acid sequence having from about 50% to about 99.9% sequence identity (e.g., from about 50% to about 60%, from about 55% to about 65%, from about 60% to about 70%, from about 65% to about 75%, from about 70% to about 80%, from about 75% to about 85%, from about 80% to about 90%, from about 85% to about 95%, about 90% to about 99.9%, about 95% to about 99.9%, about 97%, about 97.5%, about 98%, about 98.5%, about 99%, about 99.5%, about 99.6%, about 99.7%, or about 99.8%) with one or more of the following constant domain sequences. In some cases, antibodies, or fragments thereof, of the invention may contain an amino acid sequence containing one or more fragments of any of the above sequences.

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Table 3

IgG constant domain sequences

Domain Subsequence SEQ ID NO Mouse IgG2a, regions constant heavy chain domain AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTWN SGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSrrCNVA HPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSWIFPPKIK DVLMISLSPIVTCVVVDVSEDDPDVQISWFVNNVEVHTAQTQ THREDYNSTLRVVSALPIQHQ DWMSGKEFKCKVNNKDLPAPI ERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTCMVTDFMP EDIYVEWTNNGKTELNYKNTEPVLDSDGSYFMYSKLRVEKK NWVERNSYSCSWHEGLHNHHTTKSFSRTPGK 13 Mouse RADAAPTVSIFPPSSEQLTSGGASWCFLNNFYPKDINVKWKID 14 IgG2a, kappa light chain constant region GSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSY TCEATHKTSSTSPIVKSFNRNEC Human IgGl, heavy chain constant regions ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNS GALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKP KDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKT KPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 15 Human IgGl, light chain constant regions RTVAAPSWIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKV DNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVY ACEVTHQGLSSPVTKSFNRGEC 16

In some embodiments, the invention provides antibody fragments made using one or more of the antibody sequences or related variants set forth above. Such antibody fragments may include scFv fragments, Fab fragments, or any other antibody fragments, including any of those described herein.

Humanized antibodies.

Humanized forms of non-human (eg, murine) antibodies are chimeric antibodies that contain minimal sequences from non-human immunoglobulin. Generally, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from the hypervariable region of the recipient antibody are replaced by residues from the hypervariable region of an antibody from a non-human species (donor antibody), such as mouse, rat, rabbit, or primacy, having the desired specificity, affinity and efficiency.

To construct expression plasmids encoding fully humanized antibodies with human constant regions, DNA sequences encoding the antibody variable region can be inserted into expression vectors (e.g., mammalian cell expression vectors) between an upstream promoter/enhancer, e.g., the cytomegalovirus immediate early promoter/enhancer (CMV IE), plus an immunoglobulin signal sequence, and a downstream immunoglobulin constant region gene. DNA samples can then be prepared for transfection into mammalian cells.

To generate cell lines and select fully humanized antibodies, pairs of heavy and light chain plasmid DNA can be transfected into cells for expression. In some embodiments, mammalian NS0 cells may be used. Cell lines producing humanized antibodies can be expanded to express antibodies, which can be collected and purified from the cell culture medium.

In some embodiments, humanized antibodies may be cross-reactive with antibodies from non-human species. Species cross-reactivity may allow antibodies to be used in other animals for different purposes. For example, cross-reacting antibodies can be used in preclinical animal studies to obtain information about the effectiveness and/or toxicity of the antibody. Non-human species may include mouse, rat, rabbit, dog, pig, goat, sheep, or a primate such as a cynomolgus macaque.

IgG synthesis.

IgG antibodies (e.g., IgG1, IgG2, IgG3, or IgG4) containing one or more of the amino acid sequences of the variable domains and/or CDRs set forth herein (or fragments or variants thereof) can be synthesized for further testing and/or drug development . Such antibodies can be produced by inserting one or more cDNA segments encoding the desired amino acid sequences into expression vectors suitable for the production of IgG. Expression vectors may include mammalian expression vectors suitable for expression of IgG in mammalian cells. IgG can be expressed in mammalian cells to ensure that the resulting antibodies have modifications (e.g., glycosylation) characteristic of mammalian proteins and/or to ensure that antibody preparations do not contain endotoxin and/or other impurities that may be present in protein proteins. preparations from bacterial expression systems.

Immunogenic hosts.

In some embodiments, the glycan-reactive antibodies of the present invention can be produced using non-human animals, referred to herein as immunogenic hosts, as immunization hosts. In some embodiments, the immunogenic hosts are mammals. In some embodiments, the immunogenic hosts are transgenic knockout mice. Antigens having target sites and/or target epitopes for glycan-interacting antibodies can be used in contact with immunogenic hosts to stimulate an immune response and produce antibodies in the immunogenic host that specifically bind the target sites and/or target epitopes, present on the administered antigens.

Antibodies produced by immunization can be isolated from the serum of immunogenic hosts. Antibody-producing cells from immunogenic hosts can also be used to generate cell lines that produce the desired antibody. In some embodiments, antibodies and/or antibody-producing cells from an immunogenic host can be screened using enzyme-linked immunosorbent assays (ELISAs) and/or glycan arrays.

Antibody sequence, structural analysis and optimization.

In some embodiments, antibodies of the present invention may be subject to sequence analysis and/or structural analysis in which they are analyzed for characteristics that may influence chemical properties, affinity, specificity, protein folding, stability, manufacturing characteristics, expression and/or immunogenicity (i.e., induction of immune responses in subjects treated with such antibodies) of the antibody. Such analysis may include comparisons between antibodies that bind the same or similar epitopes.

Sequences of antibodies that bind the same epitope can be analyzed for variations in light and/or heavy chain sequences. Such analysis may include analysis of germline sequences and/or CDR sequences. The information obtained from such an analysis can be used to identify (and optionally modify, remove, replace or restore) conserved amino acid residues; conserved amino acid segments; amino acid positions with conserved side chain characteristics; conservative length of CDR regions; and other features that are conserved among antibodies that bind the same epitope. This information can be used to design variants or to develop methods for optimizing antibodies to improve the affinity, specificity, protein folding, stability, manufacturing characteristics, expression and/or immunogenicity of the antibody.

Sequence analysis may involve aligning the sequences of two or more antibodies that bind the same or similar epitopes to identify similarities. In such an analysis, the sequence and/or length of antibody regions (eg, CDRs, variable domains, germline segments) can be compared. Amino acid insertions, amino acid deletions and substitutions can be detected and assessed. Sequence differences can be compared in terms of antibody affinity and/or specificity.

In some cases, sequence analysis is performed to identify (and optionally modify, remove, replace or restore) one or more unpaired cysteine residues or unusual disulfide bonds; glycosylation sites (eg, N-linked NXS/T sites); acid cleavage sites, amino acid oxidation sites, similarity to mouse germline sequences; asparagine deamidation sites; aspartate isomerization sites; sites for the formation of N-terminal pyroglutamate and fragments prone to aggregation in the CDR.

In some cases, the present invention relates to sequence-based variants of the antibodies provided herein. As used herein, the term sequence-biased variant means an antibody variant that

- 35 045483 was modified based on one or more findings from analysis of the antibody sequence. In some cases, antibodies of the invention may be modified to produce antibody variants that have modifications in one or more of: affinity, specificity, protein folding, stability, manufacturing characteristics, expression, and/or immunogenicity of the antibody.

Some sequence analysis-derived variants have a modification in the length of one or more CDR regions. Antibodies with a modified CDR length may have one or more amino acids added or deleted in one or more CDR regions relative to the original antibody sequence. In some cases, sequence-based variants may have one or more CDR regions replaced by one or more CDR regions from another antibody (eg, an antibody that binds the same or a similar epitope). In some cases, sequence-based variants may have a heavy or light chain variable domain substitution from another antibody (eg, an antibody that binds the same or a similar epitope). Sequence-based variants may have modifications in one or more germline genes from which the antibody is expressed. Such modifications may include point mutations, region mutations, insertion mutations, or deletion mutations. In some cases, germline gene modifications are performed to move a CDR from one known germline gene to another. Sequence-based variants may include other variants described herein, including, but not limited to, scFv, monobodies, diabodies, intrabodies, CARs, antibody mimetics, and so on.

In some embodiments, sequence and/or structure analysis can be used to provide information for the construction of antibody fragment display libraries (including, but not limited to, scFv libraries, phage display libraries, and yeast display libraries). In one example, sequence alignment can be performed to align the sequences of two or more antibodies having a common antigen or epitope, and amino acid residues that are conserved in the aligned antibodies or variable in the aligned antibodies can be identified. In such cases, it is possible to construct antibody fragment display libraries such that the variability between library members is largely limited to the variable amino acids identified in the sequence analysis. In some cases, such libraries can be used to identify variants with altered affinity and/or specificity for a target antigen (e.g., STn) or a specific epitope of the target antigen (e.g., epitopes recognized by group 1, 2, 3, and 4 antibodies, such as described in example 1, later in this document).

In some embodiments, the antibodies of the invention may be modified to remove, replace, or otherwise eliminate one or more unpaired cysteine residues. In some cases, unpaired cysteine residues may be reactive, and in some cases may affect the affinity and/or specificity of the antibody. Accordingly, some antibodies of the invention have been modified to eliminate unpaired cysteine residues. In some cases, such variants may have modified specificity and/or affinity for the epitope. In some cases, modification of unpaired cysteine residues can lead to modification of antibody folding. In some cases, these variants have a substitution or deletion of one or more cysteine residues. In some cases, these variants have one or more additional amino acid residues (including, but not limited to, the addition of one or more cysteine residues) to prevent or reduce the undesirable effects of unpaired cysteine residues. In some cases, the cysteine residues are replaced with an amino acid having a hydrophobic side chain (eg, such as tyrosine, alanine, valine, isoleucine, leucine, methionine, phenylalanine or tryptophan).

Antibody testing and characterization.

The antibodies described herein can be tested and/or characterized using a variety of methods. Such methods can be used to determine various characteristics, which may include, but are not limited to, the affinity, specificity and activity (eg, activation or inhibition of cellular signaling pathways or other cellular or biological activities) of the antibody. Antibody testing may also include in vivo testing (eg, in animal and/or human studies) of one or more of: toxicity, therapeutic effect, pharmacodynamics, pharmacokinetics, absorption, deposition, metabolism, and excretion. Animal testing may include, but is not limited to, testing on mice, rats, rabbits, guinea pigs, pigs, primates (such as cynomolgus monkeys), sheep, goats, horses and cattle.

Cellular assays.

In some embodiments, the antibodies of the present invention can be tested or characterized using one or more cell-based assays. Such cell-based assays can be performed in vitro with cells in culture. In some cases, cell-based assays can be performed in vivo. Examples of in vivo cell-based assays include tumor models in which tumor cells are injected or otherwise administered to a host.

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In some cases, cells used in cell-based assays may express one or more target glycans recognized by one or more antibodies of the invention. Such glycans may be naturally expressed by cells or, alternatively, cells may be induced to express one or more glycans required for the purposes of a particular assay. Expression can be induced by one or more treatments that result in increased expression of glycosylated proteins or glycosylation-regulating enzymes. In other cases, induced expression may involve transfection, transduction, or other form of introduction of one or more genes or transcripts for endogenous expression of one or more glycosylated proteins or enzymes involved in the regulation of glycosylation.

In some cases, the cellular assays described herein may involve the use of cancer cells. Many cancer cell lines are available for experiments testing the antibodies of the invention. Such cells may express the target glycan or may be induced to express the target glycan. In addition, cancer cell lines that are representative of cancer stem cells can be used to test the antibodies of the invention. Cancer stem cell (CSC) cell lines can be isolated or differentiated from cancer cells growing in culture (eg, by sorting based on cancer stem cell-specific markers). Cell lines used in cell-based assays may include, but are not limited to, breast, colon, ovarian, lymphocyte, bone marrow, and skin cancer cell lines. Specific cell lines may include, but are not limited to, SNU-16 cells, LS-174T cells, MC38 cells, TOV-112D cells, TOV-21G cells, Jurkat E6.1 cells, K-562 cells, B16-F0 cells, B16-F10 cells, LS180 cells, COLO205 cells, TB4 cells, HT29 cells, Pancl cells, HPAC cells, HPAFII cells, RKO cells, SW480 cells and SNU-C2A cells.

In some embodiments, ovarian cancer cell lines may be used. Such cell lines may include, but are not limited to, the SKOV3, OVCAR3, OV90 and A2870 cell lines. In some cases, CSC cells can be isolated from these cell lines by isolating cells expressing the cell markers CD44 and/or CD 133.

OVCAR3 cells were first obtained using malignant ascites obtained from a patient suffering from advanced ovarian adenocarcinoma (Hamilton, T.C. et al., 1983. Cancer Res. 43: 5379-89). Cancer stem cell populations can be isolated from OVCAR3 cell cultures by selection based on specific cell surface markers such as CD44 (involved in cell adhesion and migration), CD133 and CD117 (Liang, D. et al., 2012. BMC Cancer. 12: 201, the contents of the publication are incorporated herein by reference in their entirety). OV90 cells are epithelial ovarian cancer cells that were similarly derived from human ascites (see US Pat. No. 5,710,038). OV-90 cells can also express CD44 when activated (Meunier, L. et al., 2010. Transl Oncol. 3(4): 230-8).

Glycan matrices.

In some embodiments, the glycan-reactive antibodies of the present invention can be produced using glycan matrices. As used herein, the term glycan array refers to a tool used to identify substances that interact with any of a number of different glycans associated with the matrix substrate. In some embodiments, the glycan arrays include a series of chemically synthesized glycans, herein referred to as glycan probes. In some embodiments, the glycan matrices include at least 2, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 350, at least 1000 or at least 1500 glycan probes. In some embodiments, glycan arrays can be custom-made to represent the desired set of glycan probes. In some embodiments, the glycan probes may be attached to the matrix substrate using a linker molecule. Such linkers may include molecules such as, but not limited to, -O(CH ₂ ) ₂ CH ₂ )NH ₂ and O(CH ₂ ) ₃ NHCOCH ₂ (OCH ₂ CH ₂ ) ₆ NH ₂ .

In some embodiments, the glycan matrix contains more than 70 chemically synthesized glycans, most of which are Neu5Ac and Neu5Gc-containing glycan pairs. Some examples of glycan probes may include: Neu5Ac-a-2-6-GalNAc (AcSTn); Neu5Gc-a-2-6-GalNAc (GcSTn); Neu5,9Ac2-a-2,6-GalNAc; Neu9Ac5Gc-a-2,6-GalNAc and GalNAc (Tn). The binding specificity of an antibody to AcSTn versus GcSTn can be determined using a matrix or other specificity determination methods known in the art. In addition, the binding profile of antibodies to O-acetylated STn can be determined. Loss of O-acetylation on STn is relevant to cancer because cancer-associated expression correlates with increased recognition of STn by antibodies (Ogata, S. et al., Tumor-associated sialylated antigens are constitutively expressed in normal human colonic mucosa. Cancer Res. 1995 May 1;55(9):1869-74). In some cases glycan

- 37 045483 matrices can be used to determine recognition of STn versus Tn.

Methods for screening antibody fragment display libraries.

In some embodiments, the antibodies of the present invention can be produced and/or optimized using high-throughput assay methods. Such methods may include any of the display methods (eg, display library screening methods) disclosed in International Patent Application No. WO2014074532, the contents of which are incorporated herein by reference in their entirety. In some embodiments, synthetic antibodies can be designed, selected, or optimized by screening against target antigens using display technologies (eg, phage display technologies). Phage display libraries can contain millions or billions of phage particles, all of which express unique antibody fragments on their viral envelopes. Such libraries can be rich sources that can be used to screen for potentially hundreds of antibody fragments with varying levels of affinity for one or more antigens of interest (McCafferty, et al., 1990. Nature. 348:552-4; Edwards, B.M. et al. ., 2003. JMB. 334: 10318; Schofield, D. et al., 2007. Genome Biol. 8, R254 and Pershad, K. et al., 2010. Protein Engineering Design and Selection. 23: 279-88; contents each publication is incorporated herein by reference in its entirety). Often the antibody fragments present in such libraries include scFv antibody fragments, which are a fusion protein of the VH and VL domains of an antibody connected by a flexible linker. In some cases, scFvs may contain identical sequences except for unique sequences encoding complementarity determining region (CDR) variable loops. In some cases, scFvs are expressed as fusion proteins linked to viral envelope proteins (eg, the N-terminus of the viral envelope pill protein). The VL chains can be expressed separately to assemble with the VH chains in the periplasm before the complex is incorporated into the viral envelope. The precipitated library members can be sequenced from the bound phage to obtain cDNA encoding the desired scFv. Such sequences can be directly incorporated into antibody sequences for recombinant antibody production, or mutated and used for further optimization through in vitro affinity maturation.

Preparation of cytotoxic antibodies.

In some embodiments, the antibodies of the present invention may be capable of inducing antibody-dependent cell-mediated cytotoxicity (ADCC) and/or antibody-dependent cellular phagocytosis (ADCP). ADCC is an immune mechanism that causes cell lysis due to attack by immune cells. Such immune cells may include CD56+ cells, CD3- natural killer (NK) cells, monocytes and neutrophils ((Strohl, W.R. Therapeutic Antibody Engineering. Woodhead Publishing, Philadelphia PA. 2012. Ch. 8, p186, contents of the publication are incorporated herein by reference in full).

In some cases, the antibodies of the present invention can be designed to include a specific isotype depending on whether there is a need for ADCC or ADCP upon binding of the antibody. Such antibodies, for example, can be constructed by any of the methods described in Alderson, K.L. et al., J Biomed Biotechnol. 2011. 2011:379123). In the case of murine antibodies, antibodies of different isotypes are more effective in stimulating ADCC. IgG2a, for example, is more effective at inducing ADCC than IgG2b. Certain antibodies of the present invention, including murine IgG2b antibodies, can be reverse engineered to create IgG2a antibodies. Such redesigned antibodies may be more effective in inducing ADCC by binding to cell-associated antigens. In some embodiments, the antibodies are redesigned by modifying or introducing one or more post-translational modifications to enhance biological activity in the form of ADCC and/or complement-dependent cytotoxicity (CDC).

In some embodiments, the genes encoding the variable regions of antibodies produced by the methods of the present invention can be cloned into mammalian expression vectors encoding the Fc regions of a human antibody. Such Fc regions may be Fc regions from human IgG1K. The Fc regions of IgG1K may have amino acid mutations that are known to cause increased Fc receptor binding and ADCC.

Antibody-drug conjugates.

In some embodiments, antibodies of the invention may be developed for antibody-drug conjugate (ADC) therapeutic applications. ADCs are antibodies to which one or more payloads (eg, drugs) are attached [eg, directly or via a linker (eg, cleavable linker or non-cleavable linker)]. ADCs are useful for delivering drugs (eg, therapeutics or cytotoxic agents) to one or more target cells or target tissues (Panowski, S. et al., 2014. mAbs 6:1, 34-45). In some cases, ADCs may be designed to bind to a surface antigen on a target cell. Once bound, the entire antibody-antigen complex can be internalized and targeted to the cell's lysosome. The ADC can then be destroyed, releasing the associated load. If the load is a cytotoxic agent, the cell

- 38 045483 the target will be destroyed or otherwise inactivated. Cytotoxic agents may include, but are not limited to, cytoskeletal inhibitors [e.g., tubulin polymerization inhibitors and spindle-essential kinesin protein (KSP) inhibitors], DNA damaging agents (e.g., calicheamicins, duocarmycins, and pyrrolobenzodiazepine dimers such as thalirine and tesirine) , topoisomerase inhibitors [e.g., camptothecin compounds or derivatives such as 7-ethyl-10-hydroxycamptothecin (SN-38), and the exatecan derivative DXd], transcription inhibitors (e.g., RNA polymerase inhibitors such as amanitin), and kinase inhibitors [ for example, phosphoinositide 3-kinase (PI3K) inhibitors or mitogen-activated protein kinase ((MEK)] inhibitors.

Tubulin polymerization inhibitors may include, but are not limited to, maytansines (eg, emtansine [DM1] and ravtansine [DM4]), auristatins, tubulisins, and vinca alkaloids, or derivatives thereof. Exemplary auristatins include auristatin E (also known as dolastatin-10 derivative), auristatin EB (AEB), auristatin EFP (AEFP), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), auristatin F and dolastatin. Exemplary tubulisin compounds include natural tubulisins A, B, C, D, E, F, G, H, I, U and V, and tubulisin analogs such as pretubulysin D (PTb-D43) and N ¹⁴ -deacetoxytubulysin H (Tb1) . Exemplary vinca alkaloids include vincristine, vinblastine, vindesine, and navelbine (vinorelbine). In some embodiments, cytotoxic agents may include auristatin derivatives [eg, 1-aminopropan-2yl auristatin F, auristatin F-hydroxypropylamide, auristatin F-propylamide, auristatin F-phenylenediamine (AFP)]; tubulisin derivatives; vinca alkaloid derivatives [eg, N-(3hydroxypropyl)vindesine (HPV)], as well as any of those described in US Pat. No. 8,524,214; 8685383; 8808679 and 9254339; publications of US patent applications US20150314008A1, US20160220696A1 and US20160022829A1; the contents of each publication are incorporated herein by reference in their entirety.

In some embodiments, the antibody-drug conjugates (ADCs) of the invention may further include one or more polymeric carriers connecting the antibody and the drugs (eg, antibody-polymer-drug conjugates). As used herein, the term polymeric carrier means a polymer or modified polymer that can be covalently attached to one or more drugs and/or antibodies. Polymeric carriers can provide additional conjugation sites for drugs, increasing the drug-to-antibody ratio and enhancing the therapeutic effects of ADCs. In some embodiments, the polymeric carriers used in the present invention may be water-soluble and/or biodegradable. Such polymeric carriers may include, but are not limited to, poly(ethylene glycol) (PEG), poly(N-(2hydroxypropyl)methacrylamide) (polyHPMA), poly(a-amino acids) [e.g., poly(L-lysine), poly(L-glutamine acid) and poly((N-hydroxyalkyl)glutamine)], carbohydrate polymers [e.g. dextrins, hydroxyethyl starch (HES) and polysialic acid], glycopolysaccharides (e.g. homopolysaccharide such as cellulose, amylose, dextran, levan, fucoidan, carrageenan, inulin, pectin, amylopectin, glycogen and lyxenan; or homopolysaccharide such as agarose, giluronan, chondroitin sulfate, dermatan sulfate, keratan sulfate, alginic acid and heparin), glycolipids, glycoconjugates, polyglycerols, polyvinyl alcohols, poly(acrylic acid), polyketal and polyacetal [ for example, poly(1-hydroxymethylethylene hydroxymethylformal), also known as PHF or FLEXIMER®, described in US patent No. 5811510; 5863990 and 5958398; the contents of each publication are incorporated herein by reference in their entirety], as well as their derivatives, dendrimers, copolymers and mixtures. For example, the polymeric carrier may include a copolymer of a polyacetal/polyketal (eg, PHF) and a hydrophilic polymer, such as polyacrylates, polyvinyl polymers, polyesters, polyorthoesters, polyamides, polypeptides, and derivatives thereof.

In some embodiments, drugs are attached (eg, covalently linked) to antibodies of the invention directly or through linkers. In some embodiments, drugs are attached to polymeric carriers directly or through linkers, and polymeric carriers are attached to antibodies directly or through linkers. In some embodiments, the linkers may include a moiety of oxalic, malonic, succinic, glutaric, adipic, pimelic, suberic, azelaic, sebacic, phthalic, isophthalic, terephthalic, diglycolic, tartaric, glutamic, fumaric, or aspartic acid, including amide, imide, or cyclic acids. imide derivatives of each of them, and each of them is optionally substituted. Exemplary linkers may include any of the linkers described in US Pat. No. 8,524,214; 8685383; 8808679; 9254339 and/or 9555112, the contents of each of which are incorporated herein by reference in their entirety.

In some embodiments, the linkers may be cleavable linkers. Cleavable linkers can be degraded under certain conditions (such as changes in pH, temperature, or reduction) or cleaved by enzymes (such as proteases and glucuronidases), releasing drugs from the ADC. Such linkers may contain a labile bond, such

- 39 045483 forged as an ester bond, amide bond or disulfide bond. Non-limiting examples of cleavable linkers may include pH-sensitive linkers (eg, hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, thioester, orthoester, acetal, or ketal); reduction-sensitive linkers [e.g. N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), N-succinimidyl-4-(2-pyridyldithio)butanoate (SPDB), N-succinimidyl-4-(2-pyridyldithio)pentanoate ( SPP), N-succinimidyl-S-acetylthioacetate (SATA) and N-succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)toluene or 2,5-dioxopyrrolidin-1-yl-4-(1-(pyridin-2- yldisulfanyl)ethyl)benzoate (SMPT)]; photosensitive linkers and enzymatically cleavable linkers [e.g., peptide linkers such as valine-citrulline, valine-citrulline-p-aminobenzoyloxycarbonyl (vc-PAB), maleimidocaproyl-valine-citrulline-p-aminobenzoyloxycarbonyl (MC-vc-PAB), linkers, glucuronidases, such as glucuronide-MABC, or esterase-cleavable linkers].

In other embodiments, the linkers may be non-cleavable linkers. Non-cleavable linkers may increase the plasma stability of ADCs compared to cleavable linkers. Illustrative examples of non-cleavable linkers include maleinimidalkane and maleimidocyclohexane (MCC).

The antibody-drug conjugates (ADCs) of the invention can be prepared using any method known in the art. For example, drugs can be modified to contain a functional group that can react with a functional group on the antibody. Antibody-drug conjugates (ADCs) can be prepared by reacting two functional groups to form a conjugate. In some cases, polymeric carriers can be modified to contain functional groups that can react with the functional group on the drugs and the functional group on the antibody under different chemical conditions. Antibodies, polymer carriers and drugs can be linked to form antibody-polymer-drug conjugates through a chain of sequential chemical reactions. For conjugation with antibodies, a lysine or cysteine residue can be used as the conjugation site. In some embodiments, antibodies can be engineered to contain additional lysine or cysteine residues. Such approaches may help avoid disruption of antibody structure (eg, interchain disulfide bonds) and maintain stability and/or activity of the antibody.

As described herein, the drug-to-antibody ratio (DAR) is the average number of drugs (eg, therapeutics or cytotoxic agents) conjugated to antibodies. In some embodiments, the drug to antibody ratio of the ADC of the invention is at least 1:1, at least 2:1, at least 4:1, at least 6:1, at least 8:1, at least 10:1, at least 12:1, at least 15:1, at least 20:1 or at least 25:1.

In some embodiments, antibodies of the invention may be tested for their ability to promote cell death when in ADC form. Cell viability assays can be performed in the presence and absence of secondary antibody-drug conjugates. Antibodies that effectively inhibit cell growth can then be used to design direct antibody-drug conjugates (ADCs). The use of such secondary antibody-drug conjugates in cellular cytotoxicity assays may allow rapid prescreening of many ADC candidates. In such assays, an unconjugated candidate antibody is directly added to cells in the presence of a secondary antibody conjugated to one or more cytotoxic agents (referred to herein as 2°ADC). Internalization of the antibody/2°AOC complex into cells that express the target antigen at high levels can lead to dose-dependent drug release into the cells, causing a cytotoxic effect of killing cells (for example, tumor cells), while cells expressing low level of the target antigen are not affected (for example, normal cells).

The ADCs of the invention can be designed to target cancer cells. Such ADCs may include antibodies directed against one or more tumor-associated carbohydrate antigens (TACA). In some cases, the ADCs of the invention are anti-STn antibodies. In some embodiments, the ADCs comprise one or more of the variable domains listed in Table 1. 2. Such ADCs may also contain at least one human IgG constant region, including, but not limited to, any of those listed in Table. 3.

Preparation of chimeric antigen receptors.

In some embodiments, antibody sequences of the invention can be used to create a chimeric antigen receptor (CAR). CARs are transmembrane receptors expressed on immune cells that promote recognition and destruction of target cells (eg, tumor cells). CARs typically have three main parts. They are an ectodomain (also known as a recognition domain), a transmembrane domain, and an intracellular (signaling) domain.

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Ectodomains promote binding to cellular antigens on target cells, while intracellular domains typically serve cell signaling functions to promote killing of bound target cells. In addition, they may have an extracellular domain with one or more antibody variable domains described herein, or fragments thereof. The CARs of the invention also include a transmembrane domain and a cytoplasmic fragment. CARs can be engineered to contain one or more antibody segments, an antibody variable domain, and/or an antibody CDR such that when such CARs are expressed on immune effector cells, the immune effector cells bind and kill any cells that recognize the CAR antibody fragments.

Characteristics of CARs include their ability to redirect T cell specificity and reactivity to a chosen target in an MHC-independent manner, exploiting the antigen-binding properties of monoclonal antibodies. Non-MHC-restricted antigen recognition confers CAR-expressing T cells the ability to recognize antigen independent of antigen processing, thereby bypassing the primary defense mechanism of tumors. In addition, when expressed on T cells, CAR does not preferably dimerize with the alpha and beta chains of the endogenous T cell receptor (TCR).

CARs designed to target tumors may have specificity for one or more tumor-associated carbohydrate antigens (TACAs). In some embodiments, the ectodomains of such CARs may comprise one or more antibody variable domains or fragments thereof. In some embodiments, the CARs are expressed on T cells, which may be referred to as CAR-modified T cells or CAR-T. CAR-Ts can be designed with CAR ectodomains having one or more antibody variable domains.

Structural features of chimeric antigen receptors.

Using gene transfer technology, T cells can be modified to stably express antibodies on their surface that impart the desired antigen specificity. Chimeric antigen receptors (CARs) combine the antigen recognition domain of a particular antibody with the intracellular domain of the CD3 zeta chain or FcyRI protein, which has T-cell activating properties, into a single chimeric fusion protein. CAR technology enables non-MHC-restricted recognition of target cells by T cells. Removing MHC restrictions on T cells facilitates the use of these molecules in any patient and, in addition, in both CD8+ and CD4+ T cells, which are typically restricted to MHC class I or II epitopes, respectively. The use of Ab binding regions allows T cells to respond to epitopes formed not only by protein, but also by carbohydrate and lipid. This chimeric receptor approach is particularly suitable for cancer immunotherapy because it bypasses many of the mechanisms by which tumors evade immune recognition, such as MHC down-regulation, lack of co-stimulatory molecule expression, CTL resistance and induction of T-cell suppression, and In this case, the use of both CD8 ⁺ CTL and CD4 + T cells allows combining effects for optimal antitumor efficacy. This approach has been shown to be applicable to a wide range of tumor antigens, in addition to viruses such as HIV (Finney, et al., J. Immunology, 2004, 172:104-113).

Although chimeric antigen receptors can initiate T cell activation in a manner similar to endogenous T cell receptors, in practice, clinical application of CAR technology has been hampered by insufficient in vivo expansion of chimeric antigen receptor T cells. For example, first-generation CARs included the cytoplasmic region of CD3ξ or the Fc receptor γ chain as their signaling domain. These first-generation CARs were tested in phase I clinical trials in patients with ovarian cancer, kidney cancer, lymphoma and neuroblastoma and were found to produce modest responses, effectively redirecting T cell cytotoxicity but without allowing T cell proliferation and survival with repeated exposure to the antigen. Second generation CAR prototypes included both CD28 and CD3ξ receptors, and second generation CARs have been tested in the treatment of B cell malignancies and other cancers (Sadelain, et al., (2009) Current Opinion in Immunology, 21(2) ):215-223). Thus, CARs have rapidly proliferated into a diverse palette of receptors with different functional properties.

Recently, it was discovered that CAR-mediated T cell responses can be enhanced by the addition of a costimulatory domain. In preclinical studies in models, inclusion of the CD137 (4-1BB) signaling domain was found to significantly increase the antitumor activity and in vivo persistence of chimeric antigen receptors compared with inclusion of the CD3-zeta chain alone (Porter, et al., N. Engl. J Med 2011, 365:725-733).

Thus, in some embodiments of the present invention, antibody sequences of the invention may be used to create a chimeric antigen receptor (CAR). In some embodiments, CARs are transmembrane receptors expressed on immune cells that facilitate recognition and killing of target cells (eg, tumor cells).

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In many cancers, tumor-specific antigens to target have not been identified, but in B-cell malignancies, CD19 is a promising target. CD19 expression is restricted to normal and malignant B cells and B cell precursors. A pilot clinical trial of treatment with autologous anti-CD19 chimeric antigen receptor-expressing T cells (CART 19) was conducted in patients with advanced, p53-deficient chronic lymphoid leukemia (CLL). The development of a CD19-specific immune response in the bone marrow was demonstrated by the transient release of cytokines and ablation of leukemia cells that coincided with the peak of chimeric antigen receptor T cell infiltration. (Porter, et al., N. Engl. J. Med. 2011, 365:725-733).

Additional structural features of the CAR may include any of those described in several PCT publications assigned to City of Hope that share a common inventor, Michael Jensen. For example, PCT publication WO 00/23573 describes genetically modified CD20-specific redirected T cells expressing a cell surface protein having an extracellular domain that contains a CD20-specific receptor, an intracellular signaling domain, and a transmembrane domain. The use of such cells for cellular immunotherapy of CD20+ malignancies and to eliminate any adverse B-cell function. In one embodiment, the cell surface protein is a single chain receptor FvFc:ξ, where Fv represents the VH and VL chains of a single chain anti-CD20 monoclonal antibody linked by peptide, Fc represents the hinge-CH2CH3 region of human IgG1, and ξ represents the intracellular signaling domain zeta- human CD3 chain. A method for producing a redirected T cell expressing a chimeric T cell receptor by electroporation using naked DNA encoding the receptor. Similarly, PCT publication WO 02/077029 describes genetically modified, CD19-specific redirected immune cells expressing a cell surface protein having an extracellular domain containing a CD19-specific receptor, an intracellular signaling domain and a transmembrane domain. The use of such cells for cellular immunotherapy of CD19+ malignancies and to eliminate any adverse B-cell function. In one embodiment, the immune cell is a T cell and the cell surface protein is a scFvFc:ξ receptor, where Fv represents the VH and VL chains of a single chain anti-CD19 monoclonal antibody, Fc represents at least a portion of an IgG1 constant region, and ξ, is the intracellular signaling domain of the T cell antigen receptor complex zeta chain (human CD3 zeta chain). The extracellular scFvFc domain and the intracellular zeta domain are linked by a transmembrane domain, such as the CD4 transmembrane domain. A method for producing a redirected T cell expressing a chimeric T cell receptor by electroporation using naked DNA encoding the receptor. These chimeric antigen receptors, when expressed on T cells, have the ability to redirect antigen recognition depending on the specificity of the monoclonal antibody. Design of scFvFc: receptors with target specificity for epitopes on the surface of tumor cells represents a conceptually promising strategy for generating antitumor immune effector cells for adoptive therapy without relying on pre-existing antitumor immunity. Such receptors are versatile in that they bind antigen in an MHC-independent manner, therefore, a single receptor construct can be used to treat a population of patients with antigen-positive tumors. City of Hope PCT Publications WO 02/088334, WO 2007/059298 and WO 2010/065818 describe zetakins generated from an extracellular domain comprising a soluble receptor ligand bound to a scaffold region capable of tethering the extracellular domain to the cell surface, a transmembrane region and intracellular signaling domain. When expressed on the surface of T lymphocytes, zetakins direct T cell activity to specific cells expressing a receptor for which the soluble receptor ligand is specific.

Additional CAR features may include any of those disclosed in the two PCT publications assigned to the University of Texas and which have a common inventor, Lawrence Cooper. PCT Publication No. WO 2009/091826 describes compositions containing a human CD19-specific chimeric T-cell receptor (or chimeric antigen receptor, CAR) polypeptide (designated hCD19CAR), which contains an intracellular signaling domain, a transmembrane domain and an extracellular domain, wherein the extracellular domain contains the human CD19 binding region. In another aspect, the CD19 binding region is F(ab') ₂ , Fab', Fab, Fv or scFv. The intracellular domain may include the intracellular signaling domain of human CD3ξ and may further include the intracellular segment of human CD28. In some aspects, the transmembrane domain is a CD28 transmembrane domain. PCT Publication No. WO 2013/074916 describes methods and compositions for immunotherapy using CAR+ T cells genetically modified to eliminate T expression

- 42 045483 cellular receptor and/or HLA. In specific embodiments, T cell receptor negative and/or HLA negative T cells are generated using zinc finger nucleases, for example. CAR+ T cells from allogeneic healthy donors can be administered to any patient without causing graft-versus-host disease (GVHD), and act as universal reagents for the mass treatment of medical conditions such as cancer, autoimmune diseases and infection.

PCT publication WO 2011/041093, assigned to the US Department of Health and Human Services, describes chimeric anti-vascular endothelial growth factor receptor 2 antigen receptors containing the antigen binding domain of the KDR-1121 or DC101 antibody, an extracellular hinge domain, a T cell transmembrane domain receptor and intracellular signaling domain of the T cell receptor, and their use in the treatment of cancer.

PCT publications WO 2012/079000 and WO 2013/040557, each of which are incorporated herein by reference in their entirety, assigned to the University of Pennsylvania, share a common inventor with Carl H. June; these publications describe a CAR containing an antigen binding domain, a transmembrane domain, a co-stimulatory signaling region, and a CD3-zeta signaling domain, as well as methods for generating T cells transfected with CAR RNA, respectively.

PCT publication WO2013/126712, also assigned to the University of Pennsylvania and shared by Carl H. June, describes compositions and methods for producing a persistent population of T cells characterized by long-term exponential expansion in culture, ligand-independent and adding exogenous cytokines or feeder cells that are beneficial for cancer treatment. In some embodiments, the antigen binding domain is a cMet binding domain. In some embodiments, the antigen binding domain is a mesothelin binding domain. In some embodiments, the antigen binding domain is a CD19 binding domain. The hinge domain is the IgG4 domain, the transmembrane domain is the CD28 transmembrane domain. In some embodiments, the co-stimulatory signaling region is a CD28 signaling region. Also provided is a vector containing a nucleotide sequence encoding a chimeric antigen receptor (CAR), and a CAR containing an antigen binding domain, a hinge domain, a transmembrane domain, a co-stimulatory signaling region and a CD3-zeta signaling domain.

PCT publication WO 2014/039513, assigned to the University of Pennsylvania, describes compositions and methods for inhibiting one or more isoforms of diacylglycerol kinase (DGK) in a cell to enhance the cytolytic activity of the cell. The cells can be used for adoptive transfer of T cells, where the cells are modified to express a chimeric antigen receptor (CAR). Inhibition of DGK in T cells used for T cell adoptive transfer results in increased cytolytic activity of T cells and thus can be used in the treatment of various conditions, including cancer, infection and immune disorders.

PCT publication WO 2014/055771, assigned to the University of Pennsylvania, describes compositions and methods for the treatment of ovarian cancer. In particular, the invention relates to the administration of genetically modified T cells having a folate receptor alpha (FR-alpha) binding domain and a CD27 co-stimulatory domain for the treatment of ovarian cancer. In one embodiment, the FR-alpha binding domain is claimed to be fully human, thereby preventing a host immune response.

In some embodiments, the CARs of the invention may be designed to target tumors. Such CARs may have specificity for one or more TACAs. In some cases, the ectodomains of such CARs may contain one or more of the antibody variable domains provided herein, or fragments thereof. In some embodiments, the CARs of the invention are expressed in T cells, herein referred to as CAR-modified T cells or CAR-T. CAR-Ts can be engineered with CAR ectodomains having one or more of the antibody variable domains provided herein.

Polyspecific antibodies.

In some embodiments, the antibodies of the present invention may bind more than one epitope. As used herein, the terms multibody or multispecific antibody mean an antibody in which two or more variable regions bind different epitopes. Epitopes can be on the same or different targets. In specific embodiments, a polyspecific antibody is a bispecific antibody that recognizes two different epitopes on the same or different antigens.

Bispecific antibodies.

Bispecific antibodies are capable of binding two different antigens. Such antibodies typically contain antigen binding regions from at least two different antibodies. For example, bispezi

- 43 045483 physical monoclonal antibody (bsmAb, bsAb) is an artificial protein consisting of fragments of two different monoclonal antibodies, which allows the bsAb to bind antigens of two different types. One common application of this technology is anticancer immunotherapy, where bsmAbs are designed to simultaneously bind to a cytotoxic cell (via a CD3-like receptor) and to a target, such as a tumor cell, that is to be killed.

Bispecific antibodies may include any of those described in Riethmuller, G., 2012. Cancer Immunity. 12:12-18; Marvin, J.S. et al., 2005. Acta Pharmacologica Sinica. 26(6):64958; and Schaefer, W. et al., 2011. PNAS. 108(27): 11187-92, the contents of each publication are incorporated herein by reference in their entirety.

A new generation of bsmAbs, called trifunctional bispecific antibodies, have been developed. They consist of two heavy and two light chains, one each from two different antibodies, with two Fab regions (arms) directed against two antigens, and an Fc region (leg) containing two heavy chains and forming a third binding site.

Of the two paratopes that form the upper portions of the variable domains of a bispecific antibody, one may be directed against a target antigen and the other against a T-lymphocyte antigen such as CD3. In the case of trifunctional antibodies, the Fc region may further bind to a cell expressing Fc receptors, such as a macrophage, natural killer (NK) cell, or dendritic cell. In total, the target cell becomes associated with one or two cells of the immune system, which subsequently leads to its death.

Other types of bispecific antibodies have been developed to overcome certain problems such as short half-life, immunogenicity, and side effects caused by cytokine release. These include chemically linked Fabs, consisting only of Fab regions, and various types of divalent and trivalent single chain variable fragments (scFv), fusion proteins that mimic the variable domains of two antibodies. The most developed of these new formats are bispecific T-cell recruiters (BiTE) and mAb2, antibodies engineered to contain an Fcab antigen-binding fragment instead of an Fc constant region.

Bispecific single chain fragment Fv antibody (bs-scFv) has been successfully used to kill cancer cells. Some human cancers are caused by functional defects in p53, which are rescued by gene therapy using wild-type p53. In Weisbart, el al. described the construction and expression of a bispecific single-chain antibody that enters living colon cancer cells, binds intracellular p53, and restores its wild-type function (Weisbart, et al., Int. J. Oncol. 2004 Oct; 25(4):1113-8 ; and Weisbart, et al. Jnt. J. Oncol. 2004 Dec; 25(6): 1867-73). In these studies, a bispecific single chain Fv fragment antibody (bs-scFv) was constructed from (i) a single chain Fv antibody fragment, mAb 3E10, which penetrates living cells and localizes to the nucleus, and (ii) a single chain Fv fragment antibody, mAb PAb421 , which binds the C-terminal part of p53. PAb421 binding restores wild-type functions of some p53 mutants, including mutants from human colon cancer SW480 cells. BS-scFv penetrated into SW480 cells and had a cytotoxic effect, indicating the ability to restore the activity of mutant p53. COS-7 cells (monkey kidney cells with wild-type p53) served as a control because they are insensitive to PAb421 due to the presence of SV40 large T antigen, which inhibits the binding of PAb421 to p53. BS-scFv penetrated into COS-7 cells, but did not have a cytotoxic effect, indicating the absence of nonspecific toxicity of BS-scFv not associated with p53 binding. The Fv fragments themselves were not cytotoxic, indicating that killing was due to p53 transduction. A single mutation in CDR1 of PAb421 VH abolished bs-scFv binding to p53 and abolished cytotoxicity to SW480 cells, without affecting cell entry, further suggesting that PAb421 binding to p53 is required for cytotoxicity (Weisbart, et al., Int. J Oncol 2004 Oct;25(4):1113-8; and Weisbart, et al., Int J Oncol 2004 Dec;25(6):1867-73).

In some embodiments, the antibodies of the present invention may be diabodies. Diabodies are functional bispecific single chain antibodies (bots-Abs). These divalent antigen-binding molecules consist of non-covalently linked scFv dimers and can be produced in mammalian cells by recombinant methods (see, for example, Mack et al., Proc. Natl. Acad. Sci., 92: 7021-7025, 1995). Several diabodies have advanced to clinical development. An iodine-123-labeled variant of the anti-CEA chimeric antibody diabody cT84.66 was evaluated as a tool for the pre-surgical immunoscintigraphic detection of colorectal cancer in a study sponsored by the Beckman Research Institute of City of Hope (Clinicaltrials.gov NCT00647153) (Nelson, A. L., MAbs 2010 Jan-Feb;2(1):77-83).

Using molecular genetic techniques, two scFvs can be assembled into a single polypeptide, separated by a linker domain, into a tandem called a tandem scFv (ta-scFv). It was established

- 44 045483 It is noted that ta-scFvs are poorly soluble and require refolding when produced in bacteria, or they can be produced in mammalian cell culture systems, in which case the need for refolding is eliminated, but the result may be low product yield. Construction of tascFv with genes for two different scFvs results in bispecific single chain variable fragments (bis-scFv). Only two ta-scFvs have been clinically developed by commercial companies; both are bispecific reagents in active early phase development by Micromet for the treatment of oncologic conditions, and are described as bispecific T cell recruiters (BiTEs). Blinatumomab is an anti-CD19/anti-CD3 bispecific ta-scFv that potentiates T cell responses to B-cell non-Hodgkin lymphoma in phase 2 studies. MT110 is an anti-EP-CAM/anti-CD3 bispecific ta-scFv that potentiates T -cellular responses to solid tumors in phase 1 studies. Bispecific tetravalent tan-Abs are also the subject of research by Affimed (Nelson, A.L., MAbs. 2010. Jan-Feb; 2(1):77-83).

Maxbodies (divalent scFVs fused to the amino terminus of the Fc (CH2-CH3 domains) of IgG are also covered.

Bispecific T cell recruiter antibodies (BiTE) are designed to temporarily recruit cytotoxic T cells to lyse selected target cells. These typically include two scFvs (one that binds to CD3 on T cells and one that binds to a target antigen on the surface of the cell being targeted for destruction). In some embodiments, two scFvs are connected by a linker. In other embodiments, the two scFvs are different regions on the antibody. The clinical activity of BiTE antibodies confirms that ex vivo expanded autologous T cells derived from tumor tissue or transfected with specific T cell receptors have therapeutic potential in the treatment of solid tumors. Although these personalized approaches confirm that T cells themselves can have significant therapeutic activity even in advanced cancers, they are too cumbersome to use on a large scale. This is not the case with cytotoxic T-lymphocyte antigen 4 (CTLA-4) antibodies, which facilitate the generation of tumor-specific T-cell clones, and with bi- and trispecific antibodies, which directly recruit the majority of patients' T cells to lyse cancer cells. The potential for global recruitment of T cells by T cell recruiter antibodies for human cancer therapy is being actively studied (Baeuerle PA, et al., Current Opinion in Molecular Therapeutics. 2009, 11(1):22-30 and Baeuerle PA and Reinhardt C, Cancer Res 2009, 69(12): 4941-4, the contents of each publication are incorporated herein by reference in their entirety).

Third generation molecules include miniaturized antibodies. Some of the best examples of mAb miniaturization include Small Modular Immunopharmaceuticals (SMIPs) from Trubion Pharmaceuticals. These molecules, which may be monovalent or divalent, are recombinant single-chain molecules containing one VL, one VH antigen-binding domain, and one or two constant effector domains, all connected by linker domains. Conceivably, such a molecule could have the benefits of enhanced tissue or tumor penetration associated with fragments while retaining the immune effector functions conferred by constant domains. At least three miniaturized SMIPs have advanced to clinical development. TRU-015, an anti-CD20 SMIP developed in collaboration with Wyeth, is the closest project to reach phase 2 trials for the treatment of rheumatoid arthritis (RA). Earlier efforts in systemic lupus erythematosus (SLE) and B-cell lymphomas were eventually abandoned. Trubion and Facet Biotechnology are collaborating on the development of TRU-016, an anti-CD37 SMIP, for the treatment of CLL and other lymphoid neoplasms, the project has reached phase 2. Wyeth has licensed the anti-CD20 SMIP SBI-087 for the treatment of autoimmune diseases, including RA, SLE and, possibly multiple sclerosis, although these projects are still in the very early stages of clinical testing. (Nelson, A.L., MAbs. 2010. Jan-Feb; 2(1):77-83).

Genmab is conducting research into the use of its unitel technology, in which the hinge region is removed from IgG4 molecules. Although IgG4 molecules are unstable and can exchange light-heavy chain heterodimers with each other, deletion of the hinge region completely prevents heavy chain pairing with each other, leaving highly specific monovalent light/heavy chain heterodimers while retaining the Fc region to provide stability and increase half-life in vivo. This configuration may minimize the risk of immune activation or oncogenic growth, since IgG4 interacts weakly with FcR and monovalent units are not able to stimulate the formation of an intracellular signaling complex. These claims, however, are supported more by laboratory than clinical evidence. Biotecnol is also developing a miniaturized mAb, CAB051, which is a compact 100 kDa anti-HER2 antibody being studied in preclinical studies (Nelson, A.L., MAbs. 2010. Jan-Feb; 2(1):77-83).

Recombinant therapeutic agents consisting of single antigens have also been developed.

- 45 045483 binding domains, although they currently represent only 4% of the pipeline of drugs in development. These molecules are extremely small, with a molecular weight that is approximately one-tenth that of full-length mAbs. Arana and Domantis are making molecules composed of the antigen-binding domains of human immunoglobulin light or heavy chains, although only Agap has a candidate for clinical testing, ART-621, an anti-TNFa molecule in a phase 2 study for the treatment of psoriasis and rheumatoid arthritis. Ablynx produces nanobodies derived from the antigen-binding variable heavy chain (VHH) regions of antibodies found in camels and llamas, which are composed of heavy chains and lack light chains. Two anti-von Willebrand factor nanobodies from Ablynx have advanced to clinical development, including ALX-0081, in Phase 2 development, as an intravenous therapy to prevent thrombosis in patients undergoing percutaneous coronary intervention for acute coronary syndrome, and ALX- 0681, in Phase 1, a subcutaneous molecule designed for both patients with acute coronary syndrome and patients with thrombotic thrombocytopenic purpura (Nelson, A. L. MAbs. 2010. Jan-Feb; 2(1):77-83).

Obtaining polyspecific antibodies.

In some embodiments, antibody sequences of the invention can be used to produce polyspecific antibodies (eg, bispecific, trispecific, or more polyspecific). Polyspecific antibodies may be specific for different epitopes of a target antigen of the present invention, or may be specific for both a target antigen of the present invention and a heterologous epitope, such as a heterologous glycan, peptide, or solid support material. (See, for example, WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tub, A. et al., Trispecific F(ab)3 derivatives that use cooperative signaling via the TCR/CD3 complex and CD2 to activate and redirect resting cytotoxic Tcells. J. Immunol. 1991 Jul 1; 147(1):60-9; US Patent Nos. 4474893; 4714681; 4925648; 5573920; 5601819; and Kostelny, S.A. et al., Formation of a bispecific antibody by the use of leucine zippers. J. Immunol. 1992 Mar 1; 148(5): 1547-53); US Patent No. 5932448.

PCT publication WO2014144573, licensed to Memorial Sloan-Kettering Cancer Center, describes and claims multimerization technologies for creating dimeric polyspecific binders (eg, fusion proteins containing antibody components) with improved properties compared to non-dimerizable multispecific binders.

PCT publication WO2014144357, rights granted to Merck Patent GMBH, describes and claims tetravalent bispecific antibodies (tetby-Abs), as well as methods for the preparation and methods of using tetby-Abs for the diagnosis and treatment of cancer or immune diseases. Tetbi-Abs have a second pair of Fab fragments with a second antigen specificity attached to the C-terminus of the antibody, resulting in a molecule that is bivalent for each of the two antigen specificities. A tetravalent antibody is produced by genetic engineering by covalently linking the antibody heavy chain to a light chain Fab, which binds to its corresponding co-expressed heavy chain Fab.

PCT publication WO2014028560, rights issued to IBC Pharmaceuticals, Inc., describes and claims T cell redirecting bispecific antibodies (bs-Abs) with at least one binding site for a T cell antigen and at least one binding site for an antigen on disease-affected cell or pathogen, to treat the disease. Preferably, the bs-Ab is an anti-CD3 x anti-CD19 bispecific antibody, although antibodies against other T cell antigens and/or disease-associated antigens can be used. The complex can be targeted to effector T cells to induce T cell-mediated cytotoxicity against cells associated with a disease, such as cancer, an autoimmune disease, or an infectious disease. The cytotoxic immune response is enhanced by co-administration of interferon-based agents, which include interferon-a, interferon-bgr; interferon-λ 1. interferon-λ2 or interferon-λ3.

PCT publication WO2013092001, rights issued to Synimmune GMBH, describes and claims a bispecific antibody molecule, a method for its preparation, its use, and a nucleic acid molecule encoding the bispecific antibody molecule. In particular, an antibody molecule is provided that is capable of mediating target cell-limited activation of immune cells.

PCT publication WO2012007167 describes and claims a polyspecific modular antibody that specifically binds at least a glycoepitope and an erbB class receptor on the surface of a tumor cell, due to which cross-linking of the glycoepitope and receptor occurs, this antibody has apoptotic activity, causing cytolysis independent of NK cells .

PCT publications WO2012048332 and WO2013055404 describe and claim meditopes, meditope binding antibodies, meditope delivery systems, as well as a monoclonal antibody framework binding surface for meditopes, and methods of using them. In particular, it was shown that two anti-

- 46 045483 lo-binding peptides, C-QFDLSTRRLK-C (cQFD; sequence number 1 herein; SEQ ID NO: 17 herein) and C-QYNLSSRALK-C (cQYN; sequence number 2 herein; SEQ ID NO: 18 herein) have novel mAb binding properties. cQFD and cQYN, also called meditopes, have been shown to bind the Fab framework region of the anti-EGFR mAb cetuximab and do not bind the antigen-binding complementarity determining regions (CDRs). The binding region on the Fab scaffold differs from those for other scaffold-binding antigens, such as the superantigens Staphylococcus protein A (SpA) (Graille el al. 2000) and Peptostreptococcus magnus protein L (PpL) (Graille et al. 2001). Accordingly, one disclosed embodiment is a framework binding interface comprising a unique mouse-human antibody framework region, or a functional fragment thereof, that binds a cyclic meditope.

Illustrative patents and patent applications of interest include: US Patent No. 5,585,089; Nos. 5,693,761 and 5,693,762, all filed June 7, 1995, and U.S. Patent No. 6,180,370, all assigned to Protein Design Labs, Inc., disclose methods for the preparation and composition of humanized immunoglobulins having one or more complementarity determining regions (CDRs) and possible additional amino acids from the donor immunoglobulin and the framework region of the human immunoglobulin acceptor. It is stated that each humanized immunoglobulin chain typically contains, in addition to the CDR, amino acids from the donor immunoglobulin backbone which, for example, are capable of interacting with the CDR to affect binding affinity, for example one or more amino acids located in close proximity to the CDR in the immunoglobulin donor, or amino acids located within approximately 3 A, as predicted by molecular modeling. Each of the heavy and light chains can be designed using any one, or all, of the different position criteria. When combined into an intact antibody, the humanized immunoglobulins of the present invention are described to be substantially non-immunogenic in humans and retain substantially the same affinity as the immunoglobulin donor for an antigen, such as a protein or other epitope-containing compound.

US Patent No. 5,951,983, assigned to De Louvain Catholic University and Bio Transplant, Inc., discloses a humanized anti-T cell antibody. It utilizes framework regions from the human kappa V gene designated HUM5400 (EMBL accession no. X55400) and from the human antibody clone Amu 5-3 (GenBank accession no. U00562).

US Patent No. 5,091,513 to Creative Biomolecules, Inc. describes a family of synthetic proteins having an affinity for a preselected antigen. Proteins are characterized by one or more amino acid sequences constituting a region that acts as a biosynthetic antibody binding site (BABS). The sites include 1) non-covalently linked or disulfide-linked synthetic dimers of VH and VL, 2) single chains of VH-VL or VLVH, where VH and VL are linked by a polypeptide linker, or 3) individual VH or VL domains. Binding domains contain linked CDR and FR regions, which can be derived from individual immunoglobulins. Proteins may also include other polypeptide sequences that act, for example, as an enzyme, a toxin, a binding site, or a site for attachment to an immobilized medium or radioactive atom. Methods for obtaining proteins for constructing BABS having any specificity that can be achieved during in vivo production of antibodies, and methods for obtaining their analogues are disclosed.

US Patent No. 8,399,625 to ESBATech, Alcon Biomedical Research Unit, LLC, describes antibody acceptor scaffolds and methods for grafting non-human antibodies, such as rabbit antibodies, using particularly well-suited antibody acceptor scaffolds.

Intrabodies.

In some embodiments, the antibodies of the present invention may be intrabodies. Intrabodies are a form of antibody that is not secreted from the cell in which it is produced, but instead targets one or more intracellular proteins. Intrabodies are expressed and function intracellularly, and can be used to influence many cellular processes, including, but not limited to, intracellular directed migration, transcription, translation, metabolic processes, proliferative signaling and cell division. In some embodiments, the methods described herein include intrabody-based therapy. In some such embodiments, the variable domain sequences and/or CDR sequences disclosed herein are included in one or more intrabody therapy constructs. For example, intrabodies can target one or more glycated intracellular proteins or can modulate the interaction between one or more glycated intracellular proteins and an alternative protein.

Intracellular antibodies against intracellular targets were first described more than two decades ago (Biocca, Neuberger and Cattaneo EMBO J. 9: 101-108, 1990). Intracellular expression of intrabodies in different compartments of mammalian cells makes it possible to block or modulate

- 47 045483 function of endogenous molecules (Biocca, et al., EMBO J. 9: 101-108, 1990; Colby et al., Proc. Natl. Acad. Sci. U.S.A. 101: 17616-21, 2004). Intrabodies can alter protein folding, protein-protein, protein-DNA, protein-RNA interactions, and protein modification. They can perform phenotypic knockout and work as neutralizing agents by directly binding to the target antigen, by diverting its intracellular directional migration, or by inhibiting its binding to binding partners. They have already been used as research tools and are beginning to be used as therapeutic molecules to treat human diseases such as viral pathologies, cancer and diseases caused by misfolded proteins. The rapidly growing biological market for recombinant antibodies is introducing intrabodies with increased binding specificity, stability and solubility, along with reduced immunogenicity, for therapeutic applications (Biocca, abstract in: Antibody Expression and Production Cell Engineering Volume 7, 2011, pp. 179- 195).

In some embodiments, intrabodies have advantages over interfering RNA (mRNA); for example, mRNAs have been shown to produce many nonspecific effects, whereas intrabodies have higher specificity and affinity for target antigens. Moreover, as proteins, intrabodies have a much longer active half-life than mRNAs. Thus, if the active half-life of an intracellular target molecule is long, gene silencing by mRNA may be slow to produce an effect, whereas the effects of intrabody expression may be almost immediate. Finally, intrabodies can be designed to block some binding interactions of a particular target molecule without affecting others.

Receiving intrabody.

Intrabodies are often single-stranded fragment variable (scFv) expressed from a recombinant nucleic acid molecule and designed to remain within the cell (eg, to remain in the cytoplasm, endoplasmic reticulum, or periplasm). Intrabodies can be used, for example, to turn off the function of a protein to which the intrabody binds. Expression of the intrabody can also be controlled through the use of inducible promoters in a nucleotide expression vector containing the sequence for the intrabody. Intrabodies can be prepared using methods known in the art, such as those described and discussed in: (Marasco et al., 1993 Proc. Natl. Acad. Sci. USA, 90: 7889-7893; Chen et al. , 1994, Hum. Gene Ther. 5:595-601; Chen et al., 1994, Proc. Natl. Acad. Sci. USA, 91: 5932-5936; Maciejewski et al., 1995, Nature Med., 1: 667-673; Marasco, 1995, Immunotech, 1: 1-19; Mhashilkar, et al., 1995, EMBO J. 14: 1542-51; Chen et al., 1996, Hum. Gene Therap., 7: 1515- 1525; Marasco, Gene Ther. 4:11-15, 1997; Rondon and Marasco, 1997, Annu. Rev. Microbiol. 51:257-283; Cohen, et al., 1998, Oncogene 17:244556; Proba et al. , 1998, J. Mol. Biol. 275:245-253; Cohen et al., 1998, Oncogene 17:2445-2456; Hassanzadeh, et al., 1998, FEBS Lett. 437:81-6; Richardson et al. , 1998, Gene Ther. 5:635-44; Ohage and Steipe, 1999, J. Mol. Biol. 291:1119-1128; Ohage et al., 1999, J. Mol. Biol. 291:1129-1134; Wirtz and Steipe, 1999, Protein Sci. 8:2245-2250; Zhu et al., 1999, J. Immunol. Methods 231:207-222; Arafat et al., 2000, Cancer Gene Ther. 7:1250-6; der Maur et al., 2002, J. Biol. Chem. 277:45075-85; Mhashilkar et al., 2002, Gene Ther. 9:307-19; and Wheeler et al., 2003, FASEB J. 17: 1733-5; as well as in the references provided therein). In particular, the preparation of the CCR5 intrabody is described in Steinberger et al., 2000, Proc. Natl. Acad. Sci. USA 97:805-810). See mainly Marasco, W.A., 1998, Intrabodies: Basic Research and Clinical Gene Therapy Applications, Springer: New York; and for a review of scFv see Pluckthun in The Pharmacology of Monoclonal Antibodies, 1994, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315.

In some embodiments, antibody sequences are used to construct intrabodies. Intrabodies are often recombinantly expressed as single-domain fragments, such as isolated VH and VL domains, or as single-chain variable fragments (scFv) of an antibody in a cell. For example, intrabodies are often expressed as a single polypeptide forming a single chain antibody containing heavy and light chain variable domains connected by a flexible linker polypeptide. Intrabodies generally do not contain disulfide bonds and are capable of modulating the expression or activity of target genes through their specific binding activity. Single chain antibodies can also be expressed as a single chain variable region fragment connected to a light chain constant region.

As is known in the art, an intrabody sequence can be incorporated into recombinant polynucleotide vectors encoding subcellular targeting signals at its N or C terminus to allow expression at high concentrations in the subcellular compartments where the target protein is located. For example, endoplasmic reticulum (ER)-targeted intrabodies are designed to include a leader peptide and, optionally, a C-terminal ER retention signal, such as the KDEL amino acid motif (SEQ ID NO: 23). Intrabodies designed to exhibit activity in the nucleus are designed to include a nuclear localization signal. Lipid moieties are coupled to intrabodies to tether the intrabodies to the cytosolic side of the plasma membrane. Intrabodies can also be directed to perform functions in the cytosol. For example, cytosolic intrabodies are used to sequester factors in the cytosol, thereby preventing their transport to their natural destination in the cell.

There are some technical difficulties with intrabody expression. In particular, the conformational folding of the protein and the structural stability of the newly synthesized intrabody in the cell are influenced by the reducing conditions of the intracellular environment. In human clinical therapy, there are concerns about the safety of transfected recombinant DNA used to achieve intrabody expression in a cell. Of particular concern are the various viral vectors commonly used in genetic manipulation. Thus, one approach to avoid such problems is to fuse protein transduction domains (PTDs) with scFv antibodies to create a cell-penetrating antibody or transbody. Transbodies are cell-penetrating antibodies in which a protein transduction domain (PTD) is fused to antibodies in the form of single chain variable fragments (scFv) (Heng and Cao, 2005, Med Hypotheses. 64:11058).

Upon interaction with a target gene, the intrabody modulates the function of the target protein and/or carries out phenotypic/functional knockout through mechanisms such as accelerated degradation of the target protein and sequestration of the target protein in a non-physiological subcellular compartment. Other mechanisms of intrabody-mediated gene inactivation may depend on the epitope targeted by the intrabody, such as binding to a catalytic site on a target protein or epitopes that are involved in protein-protein, protein-DNA, or protein-RNA interactions.

In one embodiment, intrabodies are used to trap a target in the nucleus, thereby preventing its activity in the nucleus. Such intrabodies include directional signals into the nucleus to achieve the desired targeting. Such intrabodies are designed to specifically bind to a specific target domain. In another embodiment, cytosolic intrabodies that specifically bind a target protein are used to prevent the target from gaining access to the nucleus, thereby preventing it from exhibiting any biological activity in the nucleus (e.g., preventing the target from forming transcriptional complexes with other factors) .

To specifically target expression of such intrabodies to specific cells, intrabody transcription is placed under the regulatory control of an appropriate tumor-specific promoter and/or enhancer. To direct intrabody expression specifically to the prostate gland, for example, the PSA promoter and/or promoter/enhancer can be used (See, for example, US Pat. No. 5,919,652, issued July 6, 1999).

Protein transduction domains (PTDs) are short peptide sequences that allow proteins to pass through the cell membrane and be internalized into the cytosol through atypical secretion and internalization pathways. There are a number of specific advantages that a transbody may have over conventional intrabodies expressed in the cell. To begin with, proper conformational folding and disulfide bond formation can occur prior to introduction into the target cell. More importantly, the use of cell-penetrating antibodies or transbodies would avoid the excess safety or ethical concerns associated with the direct use of recombinant DNA technology in humans, which is required for expression of the intrabody in the cell. Transbodies introduced into a cell will have only a short active half-life, without causing any permanent genetic change. This would reduce safety concerns for their use in humans (Heng and Cao 2005, Med Hypotheses. 64:1105-8).

Intrabodies are promising therapeutics for the treatment of protein misfolding diseases, including Alzheimer's, Parkinson's, Huntington's and prion diseases, due to their virtually unlimited ability to specifically recognize different protein conformations, including pathological isoforms, and because they can be targeted potential areas of aggregation (both intra- and extracellular areas). These molecules may act as neutralizing agents against amyloidogenic proteins by preventing their aggregation and/or as molecular switches in intracellular transport by redirecting the protein away from its potential site of aggregation (Cardinale and Biocca, Curr. Mol. Med. 2008, 8: 2-11).

Illustrative patent publications that disclose intracellular antibodies, or intrabodies, are set forth below herein, all of which are incorporated herein by reference in their entirety.

PCT Publication WO03014960 and US Patent No. 7,608,453 to Cattaneo, et al. describe an intracellular antibody capture technology and a method for identifying at least one intracellular antibody consensus sequence (ICS), comprising the steps of: creating a database of validated intracellular antibody sequences (a database VIDA data) and sequence alignment of validated intracellular antibodies according to Kabat; determining the frequency with which a particular amino acid is present at each position of the aligned antibody sequences; selecting the frequency threshold (LP or consensus threshold)

- 49 045483 in the range from 70% to 100%; identifying positions in aligned sequences in which the frequency of occurrence of a particular amino acid is greater than or equal to the LP value; and identifying the most frequently occurring amino acid at the position of said aligned sequences.

In PCT publications WO0054057; WO03077945; WO2004046185; WO2004046186; WO2004046187; WO2004046188; WO2004046189; US patent application publications US2005272107; US2005276800; US2005288492; US2010143939; U.S. Patents 7,569,390 and 7,897,347 and European Patents EP1560853 and EP1166121, all of which are assigned to the Medical Research Council, and whose authors include Cattaneo, el al., describe intracellular single domain immunoglobulins and a method for determining the ability of a single immunoglobulin domain to bind a target in the intracellular environment, as well as methods for obtaining intracellular antibodies.

In PCT publication WO0235237; publication of US patent application 2003235850 and issued European patent EP1328814, which lists Catteneo as the inventor, and the rights to which are assigned to S.I.S.S.A. Scuola Intemazionale Superiore, a method for in vivo identification of intracellular antigen epitopes is described.

PCT publication WO2004046192 and European patent EP1565558, assigned to Lay Line Genomics SPA and listing Catteneo as the inventor, describe a method for isolating intracellular antibodies that disrupt and neutralize the interaction between protein ligand x and protein ligand y within a cell. Also disclosed is a method for identifying a protein ligand x capable of binding a known ligand y using intracellular antibodies capable of interacting between x and y; as well as a method for isolating a set of antibody fragments against a significant part of the protein-protein interactions of a particular cell (interactome) or against protein interactions that make up intracellular pathways or networks.

US Patent Application Publication 2006034834 and PCT Publication WO9914353, entitled Intrabody-mediated control of immune reactions, assigned to Dana Farber Cancer Institute, Inc., and crediting Marasco and Mhashilkar, describe methods for altering the regulation of the immune system. for example, by selectively targeting individual, or classes, immunomodulatory receptor molecules (IRM) on cells, including transducing the cells with an intracellularly expressed antibody, or intrabody, against the IRM. In a preferred embodiment, the intrabody is a single chain antibody against an IRM, for example, MHC-1 molecules.

In PCT publication WO2013033420, the rights to which are assigned to Dana Farber Cancer Institute Inc. and the Whitehead Institute for Biomedical Research, and which lists Bradner, Rahl and Young as inventors, describe methods and compositions useful for inhibiting the interaction between a bromodomain protein and an immunoglobulin (Ig) regulatory element and for down-regulating the expression of an oncogene displaced from Ig locus, as well as for the treatment of cancers (eg, hematological malignancies) characterized by increased expression of an oncogene that is intermingled with the Ig locus. In general, a description of the intrabody is given.

PCT Publication WO02086096 and US Patent Application Publication 2003104402, entitled Methods of producing or identifying intrabodies in eukaryotic cells, assigned to the University of Rochester Medical Center and crediting Zauderer, Wei and Smith, describe a highly efficient method for expressing molecules intracellular immunoglobulins and libraries of intracellular immunoglobulins in eukaryotic cells using the trimolecular recombination method. Methods for selection and screening for molecules of intracellular immunoglobulins and their fragments, kits for obtaining, screening and selection of molecules of intracellular immunoglobulins, as well as molecules of intracellular immunoglobulins and fragments obtained using these methods are also proposed.

PCT publication WO2013023251, assigned to Affinity Biosciences PTY LTD and listing Beasley, Niven and Kiefel as inventors, describes polypeptides, such as antibody molecules, and polynucleotides encoding such polypeptides, as well as libraries thereof, in which polypeptides are expressed that demonstrate high stability and solubility. In particular, polypeptides containing paired VL and VH domains that are expressed in a soluble form and folded under reducing or intracellular conditions have been described, and a library of human scFvs was screened, which led to the isolation of soluble scFv genes having framework regions identical to the germline sequences. line of human antibodies, as well as remarkable thermostability and tolerance to grafting of CDR3 onto the scFv scaffold.

European patent application EP2314622 and PCT publications WO03008451 and WO03097697, assigned to Esbatech AG and the University of Zurich, and which list Ewert, Huber, Honneger and Plueckthun as inventors, describe the modification of human variable domains and propose compositions , useful as scaffolds for the generation of highly stable and soluble single chain Fv (scFv) antibody fragments. These scaffolds were selected for intracellular use and are thus ideal for generating scFv antibody fragments or scFv antibody libraries for applications where stability and solubility are limiting factors when using antibody fragments, such as in the reducing environment of a cell. Such scaffolds can also be used to identify highly conserved residues and consensus sequences that exhibit increased solubility and stability.

PCT Publication WO02067849 and US Patent Application Publication 2004047891, entitled Systems devices and methods for intrabody targeted delivery and reloading of therapeutic agents, describe systems, devices and methods for using intrabody for targeted delivery of molecules. More specifically, some embodiments relate to reloadable drug delivery systems that provide targeted delivery of drugs to a tissue region of a subject in a precise and timely manner.

PCT Publication WO2005063817 and US Patent 7884054, assigned to Amgen, Inc. and crediting Zhou, Shen and Martin as inventors, describe methods for identifying functional antibodies, including intrabodies. In particular, a homodimeric intrabody is described, wherein each polypeptide chain of the homodimer contains an Fc region, a scFv and a subcellular localization sequence. The subcellular localization sequence may result in intrabody localization to the ER or Golgi apparatus. Optionally, each polypeptide chain contains at most one scFv.

PCT publication WO2013138795 by Vogan, et al., assigned to Permeon Biologies Inc., describes cell-penetrating compositions for the delivery of intracellular antibodies and antibody-like fragments, as well as methods for delivering them (as used herein, AAM fragments or fragment AAM) into the cage. Without being bound by theory, the present invention is based, at least in part, on the discovery that an AAM fragment can be delivered into a cell by complexing the AAM fragment with a cell penetrating polypeptide having a positive surface charge (herein referred to as Surf+ penetrating polypeptide). . Examples of some applications of intraphilin technology are also provided.

PCT publication WO2010004432, assigned to the Institut Pasteur, describes immunoglobulins from camelids (dromedary camels, Bactrian camels, llamas and alpacas), approximately 50% of which are antibodies lacking light chains. These heavy chain antibodies interact with antigen through only one variable domain, called VHH, VHH domain(s), or VHH antibody(s). Despite the absence of light chains, these homodimeric antibodies exhibit a broad antigen-binding repertoire due to the expansion of their hypervariable regions, and can act as a transbody and/or intrabody in vitro, as well as in vivo when the VHH domain is directed against an intracellular target.

PCT publication WO2014106639 describes a method for identifying a cellular target involved in a cell phenotype by identifying an intrabody that is capable of modifying the cellular phenotype and identifying a direct or indirect cellular target of the intrabody. In particular, intrabodies 3H2-1, 3H2-VH and 5H4 are able to inhibit the degranulation reaction in mast cells triggered by the allergen; moreover, intrabodies 3H2-1 and 5H4 directly or indirectly target ABCF1 family and C120RF4 family proteins, respectively. These ABCF1 and C120RF4 inhibitors are reported to be useful in therapy, particularly for the treatment of allergic and/or inflammatory conditions.

PCT publication WO0140276, assigned to Urogenesis, Inc., generally describes the possibility of inhibiting STEAP proteins (six transmembrane domain prostate epithelial antigen) using intracellular antibodies (intrabodies).

PCT publication WO02086505, assigned to the University of Manchester, and US patent application publication US2004115740, which lists Simon and Benton as inventors, describe a method for intracellular analysis of a target molecule for which intrabodies are preferred. In one embodiment, a vector (designated pScFv-ECFP) capable of expressing an anti-MUC1 intrabody associated with CFP is described.

PCT publications WO03095641 and WO0143778, assigned to Gene Therapy Systems, Inc., describe compositions and methods for intracellular protein delivery and generally describe intrabodies.

PCT publication WO03086276, assigned to Selective Genetics, Inc., describes a platform technology for the treatment of intracellular infection. The compositions and methods described herein include non-target-specific vectors that are directed to cells to be infected by bound ligands that bind and are internalized through cell surface receptors/fragments associated with infection. Vectors contain exogenous nucleotide sequences that are expressed after internalization into the target cell. Ligands and nucleic acid molecules associated with the vector can be modified to target other infectious agents. In addition, the invention relates to methods for identifying epitopes and ligands capable of controlling vector internalization and capable of blocking the entry of viruses.

PCT publication WO03062415, the rights to which are assigned to Erasmus University Rotterdam, describes a transgenic organism containing a polynucleotide construct encoding an intracellular antibody that disrupts the catalytic reaction of the formation of the galactose xenoantigen α1,3-galactose, and/or a polynucleotide construct encoding an intracellular antibody that specifically binds a retroviral protein such as the PERV particle protein. Cells, tissues and organs of the transgenic organism can be used for xenotransplantation.

PCT publication WO2004099775, entitled Means for detecting protein conformation and applications thereof, describes the use of scFv fragments as conformation-specific antibodies to specifically detect the conformational state of a protein, for use as sensors in living cells to control, after intracellular expression, behavior endogenous proteins.

PCT Publication WO2008070363, assigned to Imclone Systems, Inc., describes a single-domain intrabody that binds an intracellular protein or intracellular domain of an intracellular protein, such as Etk, an endothelial and epithelial tyrosine kinase, which is a member of the Tec family of non-receptor tyrosine kinases. Also proposed is a method of inhibiting an intracellular enzyme and treating a tumor in a patient by administering an intrabody or a nucleic acid expressing an intrabody.

PCT publication WO2009018438, assigned to the Cornell Research Foundation, Inc., describes a method for identifying a protein that binds a target molecule and has intracellular functionality by using a construct containing a DNA molecule encoding a protein that binds the target molecule with the molecule DNA connected to a stop sequence. The host cell is transformed with the construct and then cultured under conditions effective to form a complex of the translation-arrested protein, protein-encoding mRNA, and ribosomes within the host cell. The protein in the complex is in the correctly folded active form, and the complex is removed from the cell. This method can be carried out with a cell-free extract preparation containing ribosomes instead of a host cell. The present invention also provides a construct comprising a DNA molecule encoding a protein that binds a target molecule and a SecM stop sequence linked to the DNA molecule. The DNA molecule and the SecM stop sequence are associated with sufficient distance between them to permit expression in the cell of the protein they encode in the correctly folded active form. In general, a description of the intrabody is given.

PCT publication WO2014030780, assigned to Mogam Biotechnology Research Institute, describes a method called Tat-associated protein engineering (TARE) for screening a target protein having increased solubility and excellent thermostability, in particular the variable domain (VH or VL) immunoglobulin derived from human germ cells by creating a gene construct in which the target protein and the antibiotic resistance protein are linked to a Tat signal sequence, followed by expression of the construct in E. coli. Also described are human or recombinant VH and VL domain antibodies, and human or recombinant VH and VL domain antibody frameworks having excellent solubility and thermostability that are screened by the TARE method. A library is also proposed, including random CDR sequences in the framework of a human or recombinant VH or VL domain antibody, subject to screening by the TARE method, a method for its preparation, a VH or VL domain antibody having the ability to bind the target protein screened using the library, and a pharmaceutical composition , containing a domain antibody.

European patent application EP2422811 describes an antibody that binds an intracellular epitope; such intrabodies contain at least a portion of the antibody that is capable of specifically binding the antigen and, preferably, does not contain functional sequences causing its secretion, and thus remains in the cell. In one embodiment, the intrabody is a scFv. The scFv polypeptide further contains a polypeptide linker between the VH and VL domains, which allows the scFv to form the desired structure for antigen binding. A specific embodiment is also described in which the intrabody binds the cytoplasmic domain of the Eph receptor and prevents its signaling (eg, autophosphorylation). In another specific embodiment, the intrabody binds the cytoplasmic domain of ephrin type B (eg, ephrin B1, ephrin B2, or ephrin B3).

PCT publication WO2011003896 and European patent application EP2275442 describe intracellular functional PCNA chromobodies obtained using a nucleic acid molecule encoding a polypeptide that specifically binds proliferating cell nuclear antigen (PCNA). Examples of such polypeptides having conservative substitutions of one or more amino acids in one or two framework regions include MANVQLNESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSDISPSGAVKAYSDSVKGRFTISRDNAKNRLYLQMNSLTPEDTGEYFCTKVQSPRTRIPAPSS QGTQVTVSS (SEQ ID NO: 19) and MANVQLNESGGGLVQPGGSLRLSCA ASGFTFSSYAMSWVRQAPGKGLEWVSEI SPSGAVKAYSDSVKGRFTISRDNAKNRLYLQMNSLTPEDTGEYFCTKVQSPRTRIPAPSS QGTQVTVSS (SEQ ID NO: 20), including framework regions of polypeptides. The examples identify framework regions as well as CDR regions involved in PCNA binding.

European patent application EP2703485 describes a method for selecting plasma cells, or plasmablasts, as well as a method for producing target antigen-specific antibodies and new monoclonal antibodies. In one embodiment, cells expressing intracellular immunoglobulin are described.

Antibody coated agents.

In some embodiments, antibodies, or antibody fragments, described herein can be used to prepare a composition comprising an antibody-coated agent. As used herein, the term antibody-coated agent means any particle, nanoparticle, molecule, protein, fusion protein, lipid, liposome, cell membrane, cell or other structure that contains one or more surface-bound antibodies or antibody fragments. Antibody-coated agents can be targeted to one or more glycans, proteins, cells, tissues, and/or organs depending on the specificity of the antibody or antibody fragments used for coating.

Antibody-coated agents may include a bound, encapsulated, or embedded payload. The load may be a detectable mark. Some loads may include one or more therapeutic agents. Such therapeutic agents may include, but are not limited to, drugs, chemotherapeutic agents, and cytotoxic agents. Cytotoxic agents can be used to kill or otherwise inactivate a cell. Cytotoxic agents may include, but are not limited to, inhibitors of the cytoskeletal system [eg, inhibitors of tubulin polymerization such as maytansines or auristatins (eg, monomethyl auristatin E [MMAE] and monomethyl auristatin F [MMAF]), and inhibitors of the spindle-critical protein kinesin (KSP )], DNA damaging agents (e.g., calicheamicins, duocarmycins, and pyrrolobenzodiazepine dimers such as thalirine and tesirine), topoisomerase inhibitors [e.g., camptothecin compounds or derivatives such as 7-ethyl-10-hydroxycamptothecin (SN-38), and derivative exatecan DXd], transcription inhibitors (eg, RNA polymerase inhibitors such as amanitin) and kinase inhibitors [eg, phosphoinositide 3-kinase (PI3K) inhibitors or mitogen-activated protein kinase (MEK) inhibitors].

In some embodiments, the antibody-coated agents may include nanoparticles coated with one or more antibodies or antibody fragments described herein. Such antibody-coated agents can be directed to one or more glycans, including, but not limited to, cell-associated glycans. Some such antibody-coated agents include one or more cytotoxic agents.

Proteins and options.

The glycan-interacting antibodies of the present invention may exist as a whole polypeptide, multiple polypeptides, or polypeptide fragments that may independently be encoded by one or more nucleic acids, multiple nucleic acids, nucleic acid fragments, or variants of any of the above. As used herein, the term polypeptide means a polymer of amino acid residues (natural or non-natural) linked together, most commonly by peptide bonds. As used herein, the term refers to proteins, polypeptides and peptides of any size, structure or function. In some cases, the encoded polypeptide is less than about 50 amino acids in size, in which case the polypeptide is called a peptide. If the polypeptide is a peptide, it will be at least about 2, 3, 4, or at least 5 amino acid residues in length. Thus, polypeptides include gene products, natural polypeptides, synthetic polypeptides, homologues, orthologues, paralogs, fragments and other equivalents, variants and analogues of the foregoing. The polypeptide may be a single molecule or may be a multimolecular complex such as a dimer, trimer, or tetramer. They may also include single-chain or multi-chain polypeptides, and may be associated or linked. The term polypeptide can also refer to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding natural amino acids.

The term polypeptide variant refers to molecules that differ in their amino acid sequence from the natural or reference sequence. Amino acid sequence variants may have substitutions, deletions and/or insertions at certain positions of the amino acid sequence when compared to the natural or reference sequence. Typically, variants have at least about 50% identity (homology) with the natural or reference sequence and, preferably, they will be at least about 80%, more preferably at least about 90% identical (homologous) to the natural or reference sequence .

In some embodiments, simulator variants are provided. As used herein, the term mimic variant means a variant containing one or more amino acids that mimic the activated sequence. For example, glutamate can serve as a mimic for phospho-threonine and/or phospho-serine. Alternatively, mock variants may result in inactivation or an inactivated mimic-containing product, for example, phenylalanine may act as an inactivating replacement for tyrosine; or alanine may act as an inactivating replacement for serine. The amino acid sequences of the glycan-interacting antibodies of the invention may contain naturally occurring amino acids and, therefore, may be considered proteins, peptides, polypeptides, or fragments thereof.

Alternatively, glycan-reactive antibodies may contain both natural and unnatural amino acids.

The term amino acid sequence variant refers to molecules having some differences in their amino acid sequences compared to the natural or original sequence. Amino acid sequence variants may have substitutions, deletions and/or insertions at certain positions in the amino acid sequence. The natural or original sequence should not be confused with the wild type sequence. As used herein, natural or original sequence is a relative term indicating the original molecule to which comparison is made. Natural or original sequences or molecules may be wild-type sequences (naturally occurring sequences), but need not be wild-type sequences.

Typically, the variants are at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% sequence identity compared to the natural sequence.

The term sequence identity, as applied to amino acid sequences or nucleotide sequences, is defined as the percentage of residues in a candidate sequence that are identical to residues in a second sequence after sequence alignment, taking into account gaps and fragments as necessary, to achieve the maximum percentage of sequence identity. Calculation of the percent identity of two polymer sequences, for example, can be done by aligning the two sequences for the purpose of optimal comparison (for example, gaps can be introduced in one or both of the first and second polymer sequences for optimal alignment, and non-identical sequences can be omitted for comparison purposes) . In specific embodiments, the length of the sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the length of the reference sequence. The residues at the corresponding positions are then compared. If a position in the first sequence is occupied by the same residue as the corresponding position in the second sequence, the molecules are identical at that position. The percentage identity between two sequences depends on the number of identical positions shared by the two sequences, taking into account the number of gaps and the length of each gap that must be introduced for optimal alignment of the two sequences. Comparing sequences and determining the percent identity between two sequences can be done using a mathematical algorithm. For example, the percentage identity between two nucleotide sequences can be determined using methods such as those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; the contents of each of which are incorporated herein by reference. For example, the percent identity between two nucleotide sequences can be determined using the Myers-Miller algorithm (CABIOS, 1989, 4:11-17), which is included in the ALIGN program (version 2.0), using the PAM120 residue substitution weight table, gap length penalty 12 and omission penalty 4. Alternatively, the percentage identity between two nucleotide sequences can be determined using the GAP program in the package

- 54 045483 GCG programs using the NWSgapdna.CMP matrix. Methods commonly used to determine percent identity between sequences include, but are not limited to, those described in Carillo, H., and Lipman, D., SIAM J Applied Math., 48:1073 (1988); the contents of which are incorporated herein by reference. Methods for determining identity are included in publicly available computer programs. Exemplary computer programs for determining homology between two sequences include, but are not limited to, the GCG software package, Devereux, J., el al., Nucleic Acids Research, 12(1), 387 (1984)), BLASTP, BLASTN and FASTA Altschul, S. F. et al., J. Molec. Biol., 215, 403 (1990).

The term homologue, as applied to amino acid sequences, means a corresponding sequence in one biological species that has substantial identity with a second sequence in a second biological species.

The term analogs is intended to include polypeptide variants that differ in one or more amino acid changes, such as substitutions, additions or deletions of amino acid residues, but still retain the properties of the parent polypeptide.

The present invention covers several types of glycan-interacting antibodies with amino acid differences, including variants and derivatives. These include variants with substitutions, insertions, deletions, and covalent modifications. Therefore, glycan-interacting antibody molecules containing substitutions, insertions and/or additions, deletions and covalent modifications are included within the scope of the present invention. For example, tag sequences or amino acids, such as one or more lysine residues, can be added to the peptide sequences of the invention (eg, at the N-terminus or C-terminus). Tag sequences can be used to purify or localize the peptide. Lysine residues can be used to increase the solubility of the peptide or to enable biotinylation. Alternatively, amino acid residues located in the carboxy- and amino-terminal regions of the amino acid sequence of a peptide or protein can optionally be removed to produce truncated sequences. Alternatively, certain amino acids (eg, C-terminal or N-terminal residues) may be removed depending on the use of the sequence, for example, to express the sequence as part of a larger sequence that is soluble or bound to a solid support.

The term substitution variants, as applied to proteins, means variants resulting from the removal of at least one amino acid residue in the natural or original sequence and the insertion in its place at the same position of another amino acid. Substitutions can be single, when only one amino acid in a molecule is replaced, or they can be multiple, when two or more amino acids are replaced in the same molecule.

As used herein, the term conservative amino acid substitution means the replacement of an amino acid normally present in a sequence with another amino acid of similar size, charge, or polarity. Examples of conservative substitutions include replacing a non-polar (hydrophobic) residue such as isoleucine, valine and leucine with another non-polar residue. Likewise, examples of conservative substitutions include the substitution of one polar (hydrophilic) residue for another, for example, reciprocal exchanges between arginine and lysine, between glutamine and asparagine, and between glycine and serine. In addition, replacing a basic residue, such as lysine, arginine, or histidine, with another, or replacing one acidic residue, such as aspartic acid or glutamic acid, with another acidic residue are further examples of conservative substitutions. Examples of non-conservative substitutions include replacing a non-polar (hydrophobic) amino acid residue such as isoleucine, valine, leucine, alanine, methionine with a polar (hydrophilic) residue such as cysteine, glutamine, glutamic acid or lysine, and/or replacing a polar residue with a non-polar residue .

The term insertion variants, as applied to proteins, means variants in which one or more amino acids are inserted immediately adjacent to an amino acid at a particular position in the natural or original sequence. Immediately adjacent to an amino acid means to form a bond with either the alpha-carboxy or alpha-amino functional group of the amino acid.

The term deletion variants, when applied to proteins, means variants in which one or more amino acids in the natural or original amino acid sequence have been deleted. Typically, deletion variants will have one or more amino acids removed from a specific region of the molecule.

As used herein, the term derivative is synonymous with the term variant and means a molecule that has been modified or altered in any way relative to the reference molecule or parent molecule. In some embodiments, the derivatives include natural or parent proteins that have been modified by an organic protein or non-protein derivatizing agent and post-translational modifications. Covalent modifications are typically introduced by reacting target amino acid residues of a protein with an organic derivatizing agent that is capable of reacting with selected side chains.

- 55 045483 chains or terminal residues, or through the use of post-translational modification mechanisms that operate in selected recombinant host cells. The resulting covalent derivatives are useful in programs aimed at identifying residues important for biological activity, for immunoassays, or for producing antibodies against a protein for the purpose of immunoaffinity purification of a recombinant glycoprotein. Such modifications are within the skill of the ordinary person skilled in the art and are carried out without undue experimentation.

Some post-translational modifications result from the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are often posttranslationally deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under slightly acidic conditions. Any form of these residues may be present in the proteins used in the present invention.

Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of alpha-amino groups of lysine, arginine and histidine side chains (I.E. Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco , pp. 79-86 (1983)).

Covalent derivatives particularly include fusion molecules in which the proteins of the invention are covalently linked to a non-protein polymer. The non-protein polymer is typically a hydrophilic synthetic polymer, that is, a polymer not found in nature. However, polymers that exist naturally and are produced recombinantly or in vitro are also useful, as are polymers that are isolated from a natural source. Hydrophilic polyvinyl polymers are included within the scope of the present invention, for example polyvinyl alcohol and polyvinylpyrrolidone. Particularly useful are polyvinylalkylene ethers such as polyethylene glycol and polypropylene glycol. Proteins can be associated with various non-protein polymers, such as polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in the manner described in US patent No. 4640835; 4496689; 4301144; 4670417; 4791192 or 4179337.

The term features, when applied to proteins, refers to specific components of a molecule based on amino acid sequence. Features of the proteins of the present invention include surface features, local conformational shape, folding, loops, half-loops, domains, half-domains, sites, ends of the molecule, or any combination thereof.

As used herein in relation to proteins, the term surface element means the polypeptide component of a protein appearing on the outer surface.

As used herein in relation to proteins, the term local conformational form means a polypeptide structural element of a protein located in a specific protein space.

As used herein in relation to proteins, the term fold means the conformation of an amino acid sequence obtained by minimizing energy. Laying may take place at the secondary or tertiary level of the laying process. Examples of secondary level folds include beta sheets and alpha helices. Examples of tertiary level arrangements include domains and regions formed due to the aggregation or separation of energetic forces. The regions formed in this way include hydrophobic and hydrophilic pockets, and the like.

As used herein, the term turn in relation to protein conformation means a bend that changes the direction of the peptide or polypeptide backbone and may involve one, two, three or more amino acid residues.

As used herein in relation to proteins, the term loop means a structural feature of a peptide or polypeptide that reverses the backbone of the peptide or polypeptide and includes four or more amino acid residues. Oliva et al. have identified at least 5 classes of protein loops (J. Mol Biol 266 (4): 814-830; 1997).

As used herein in relation to proteins, the term half-loop means a portion of a particular loop containing at least half the number of amino acid residues of the loop from which it originates. It is clear that loops may not always contain an even number of amino acid residues. As a consequence, in cases where a loop contains or is determined to include an odd number of amino acids, a half-loop of a loop with an odd number of residues will include an integer portion or a portion with the nearest integer number of residues in the loop (number of loop amino acids/2+/-0 .5 amino acids). For example, a loop defined as having 7 amino acids can produce half-loops with 3 amino acids or 4 amino acids (7/2=3.5+/-0.5 is 3 or 4).

As used herein in relation to proteins, the term domain means a fragment of a polypeptide having one or more readily distinguishable structural or functional characteristics or properties (eg, having binding ability, serving as a site for protein-protein interactions).

As used herein in relation to proteins, the term half-domain means a portion of a specific domain having at least half the number of amino acid residues of the domain from which it originates. It is clear that domains may not always contain an even number

- 56045483 amino acid residues. As a consequence, in cases where a domain contains or is determined to include an odd number of amino acids, a half-domain of a domain with an odd number of residues will include an integer portion or a portion with the nearest integer number of residues of the domain (number of domain amino acids/2+/-0 .5 amino acids). For example, a domain defined as having 7 amino acids can result in half-domains with 3 amino acids or 4 amino acids (7/2=3.5+/-0.5 is 3 or 4). It is also understood that subdomains may be identified within domains or half-domains, these sub-domains having less than all of the structural or functional properties identified in the domains or half-domains from which they originate. It is also understood that amino acid domains of any of the types described herein do not necessarily need to be continuous along the backbone of the polypeptide (ie, non-adjacent amino acids may stack structurally to form a domain, half-domain, or subdomain).

As used herein in relation to proteins, the term site, when referring to amino acid-based embodiments, is synonymous with the terms amino acid residue and amino acid side chain. A site is a position in a peptide or polypeptide that can be modified, manipulated, altered, derivatized, or replaced in the polypeptide molecules of the present invention.

As used herein, the terms ends or end in relation to proteins mean the extreme points of a peptide or polypeptide. Such endpoints are not limited to only the first and last sites of the peptide or polypeptide, but may also include additional amino acids in the terminal regions. The polypeptide molecules of the present invention can be characterized as having both an N-terminus (ending with an amino acid with a free amino group (NH2)) and a C-terminus (ending with an amino acid with a free carboxyl group (COOH)). In some cases, the proteins of the invention consist of several polypeptide chains linked together by disulfide bonds or non-covalent bonds (multimers, oligomers). Proteins of this type will have several N- and C-termini. Alternatively, the ends of the polypeptides may be modified so that they begin or end, as appropriate, with a non-polypeptide moiety, such as an organic conjugate.

Once any features have been identified or defined as components of the molecule of the invention, various manipulations and/or modifications to these features can be made by movement, exchange, inversion, deletion, randomization or duplication. Moreover, it is clear that manipulation of features can lead to the same results as modification of the molecules of the invention. For example, a manipulation involving the removal of a domain would result in a change in the length of the molecule in the same way that modifying a nucleic acid to encode a less than full-length molecule would.

Modifications and manipulations can be accomplished by methods known in the art, such as site-directed mutagenesis. The resulting modified molecules can then be tested for activity using in vitro or in vivo assays, such as those described herein, or any other suitable screening assays known in the art.

Isotopic options.

The glycan-reactive antibodies of the present invention may contain one or more atoms that are isotopes. As used herein, the term isotope means a chemical element that has one or more additional neutrons. In one embodiment, the compounds of the present invention may be deuterated. As used herein, the term deuterated refers to a substance in which one or more hydrogen atoms have been replaced by the isotope deuterium. The isotope deuterium is an isotope of hydrogen. The hydrogen nucleus contains one proton, while the deuterium nucleus contains both a proton and a neutron. Glycan-reactive antibodies can be deuterated to change a physical property of the compound, such as stability, or to allow the compounds to be used in diagnostic or experimental applications.

Conjugates and combinations.

The present invention provides that the glycan-interacting antibodies of the present invention can be complexed, conjugated or combined with one or more homologous or heterologous molecules. As used herein, the term homologous molecule means a molecule that is similar in at least one of structure or function to the parent molecule, while a heterologous molecule is a molecule that differs in at least one of structure or function from the original molecule. Thus, structural homologs are molecules that have substantially similar structures. They may be identical. Functional homologues are molecules that have substantially similar functions. They may be identical.

The glycan-reactive antibodies of the invention may include conjugates. Such

- 57 045483 the conjugates of the invention may contain a natural substance or ligand such as a protein (eg human serum albumin (HSA), low density lipoprotein (LDL), high density lipoprotein (HDL) or globulin); carbohydrate (eg dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid) or lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, eg, a synthetic polyamino acid, oligonucleotide (eg, an aptamer). Examples of polymers include polyamino acids such as polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolide) copolymer, divinyl ether-maleic anhydride copolymer , N-(2-hydroxypropyl) methacrylamide (HMPA) copolymer, polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethyl acrylic acid), Nisopropylacrylamide polymers or polyphosphazine. Examples of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, polyamine quaternary salt, or alpha-helical peptide.

Conjugates may also include targeting groups, for example, targeting a cell or tissue with an agent or group, such as a lectin, glycoprotein, lipid or protein, such as an antibody, that binds to a particular cell type, such as kidney cells. The guide group can be thyroidropin, melanotropin, lectin, glycoprotein, surfaktant protein a, carbon mucin, irrigation lactose, polyvalent galactose, n-acetylgalactosamine, n-acetylhlugosamin, irrigation mannose, watervalent fascosis, glycosylated polyiamin acids, polyvalent galax , transferrin, bisphosphonat, polyglutamate, polyaspartate, lipid, cholesterol, steroid, bile acid, folate, vitamin B12, biotin, RGD peptide, RGD peptidomimetic or aptamer.

The targeting groups may be proteins, such as glycoproteins, or peptides, such as molecules having specific affinity for a co-ligand, or antibodies, such as an antibody that binds a specific cell type, such as a cancer cell, endothelial cell, or bone cell. Guiding groups may also include hormones and hormone receptors. They may also include non-peptide molecules such as lipids, lectins, carbohydrates, vitamins, cofactors, polyvalent lactose, polyvalent galactose, N-acetylgalactosamine, Nacetylglucosamine, polyvalent mannose, polyvalent fucose or aptamers.

The targeting group can be any ligand capable of targeting to a particular receptor. Examples include, but are not limited to, folate, GalNAc, galactose, mannose, mannose-6P, aptamers, integrin receptor ligands, chemokine receptor ligands, transferrin, biotin, serotonin receptor ligands, PSMA receptor ligands, endothelin, GCPII, somatostatin, LDL and HDL. In specific embodiments, the targeting group is an aptamer. The aptamer may be unmodified or may have any combination of modifications disclosed herein.

In other embodiments, the glycan-interacting antibodies are covalently conjugated to a cell-penetrating polypeptide. The cell-penetrating peptide may also contain a signal sequence. The conjugates of the invention can be designed to have increased stability; increased ability to transfect cells; and/or altered biodistribution patterns (eg, being targeted to specific tissue or cell types).

Conjugated moieties can be added to glycan-reactive antibodies to allow labeling or targeting of targets for clearance. Such tagging/tagging molecules include, but are not limited to, ubiquitin, fluorescent molecules, human influenza virus hemagglutinin (HA), c-myc [a 10 amino acid segment of the human proto-oncogene myc with the sequence EQKLISEEDL (SEQ ID NO: 21)], histidine (His ), FLAG marker [short peptide with the sequence DYKDDDDK (SEQ ID NO: 22)], glutathione-S-transferase (GST), V5 (simian paramyxovirus 5 epitope), biotin, avidin, streptavidin, horseradish peroxidase (HRP) and digoxigenin .

In some embodiments, glycan-interacting antibodies may be combined with each other or with another molecule in the treatment of a disease or condition.

Nucleic acids.

The present invention relates to nucleic acid molecules. In some embodiments, the nucleic acids encode antibodies of the invention (including, but not limited to, antibodies, antibody fragments, intrabodies, and chimeric antigen receptors). Such nucleic acid molecules include, but are not limited to, DNA molecules, RNA molecules, polynucleotides, oligonucleotides, mRNA molecules, vectors, plasmids and other constructs. As used herein, the term construct means any recombinant nucleic acid molecule, including, but not limited to, plasmids, cosmids, autonomously replicating polynucleotide molecules, or linear or circular single-stranded or double-stranded DNA or RNA polynucleotide molecules. The present invention also relates to cells programmed or engineered to express nucleic acid molecules encoding glycan-interacting antibodies. Such cells

- 58 045483 can be obtained using transfection, electroporation, viral delivery and the like. Recombinant viruses with constructs of the invention may include, but are not limited to, lentiviruses, adenoviruses, adeno-associated viruses and phages. In some cases, the nucleic acids of the invention include codon-optimized nucleic acids. Methods for producing codon-optimized nucleic acids are known in the art and may include, but are not limited to, those described in US Pat. Nos. 5,786,464 and 6,114,148, each of which is incorporated herein by reference in its entirety. In some embodiments, the nucleotide sequence is codon-optimized to improve protein expression or to remove cryptic splice sites.

II. Methods and applications.

Therapeutic uses.

The methods of the present invention include, but are not limited to, methods of using one or more glycan-interacting antibodies for therapeutic, diagnostic, quantitation, biological processing, and experimental and/or research purposes. Such glycan-interacting antibodies may include anti-STn antibodies.

Uses related to cancer treatment.

Aberrant glycosylation is a hallmark of malignant cell transformation. Multiple aberrant forms of glycosylation have been described in various forms of human cancer, leading to the identification of certain tumor-associated carbohydrate antigens (TACAs) as a class of cell surface molecules suitable for the specific targeting of drugs to tumors (Cheever, M.A. et al., Clin Cancer Res. 2009 Sep 1;15(17):5323-37). Expression of TACA antigens has been detected in epithelial cancers, including, but not limited to, breast, colon, lung, bladder, cervical, ovarian, gastric, prostate, and liver cancers. Expression of TACA antigens has been detected in embryonal cancers, including, but not limited to, yolk sac tumors and seminomas. In addition, expression of TACA antigens has been found in many forms of melanoma, carcinoma and leukemia of various tissues (Heimburg-Molinaro et al., Vaccine. 2011 Nov 8: 29(48): 8802-8826). Antibodies of the present invention directed to one or more TACAs are referred to herein as anti-TACA antibodies.

It is estimated that cells in approximately 80% of all carcinomas express a truncated glycan, the Tn antigen. With some exceptions, Tn and the sialylated form, sialyl-Tn (STn), are not expressed in normal healthy tissues. In addition, an immunogenic sialic acid absent in humans, Nglycolylneuraminic acid (Neu5Gc), appears to be differentially expressed on carcinomas such as breast cancer in the form Neu5Gc-STn (GcSTn).

Multiple aberrant forms of glycosylation have been described in various forms of human cancer, allowing the identification of certain glycans as a class of cell surface molecules suitable for specific tumor targeting of drugs (Cheever, M.A. et al., Clin Cancer Res. 2009 Sep 1; 15(17 ):5323-37). For example, various human cancers (such as bladder, breast, cervical, colon, lung, and ovarian cancers, among others) show high expression of the STn antigen, which is rarely found in normal human tissues (Karlen, P. et al ., Gastroenterology. 1998 Dec; 11 5(6): 1395-404; Ohno, S. et al., Anticancer Res. 2006 NovDec; 26(6A):4047-53). In addition, the presence of STn on tumor-associated mucins is associated with poor disease prognosis and, as a consequence, it is considered a promising epitope for cancer detection and targeted therapy (Cao, Y. et al., Virchows Arch. 1997 Sep; 431(3): 159-66; Julien, S. et al., Br J Cancer. 2009 Jun 2; 100(11): 1746-54; Itzkowitz, S. H. et al., Cancer. 1990 Nov 1; 66(9): 1960-6 ; Motoo, Y. et al., Oncology. 1991; 48(4):321-6; Kobayashi, H. et al., J Clin Oncol. 1992 Jan; 10(1):95-101). The formation of Tn and STn is associated with somatic mutations in the Cosmc gene, which encodes a molecular chaperone necessary for the formation of active T synthase (Ju, T. et al., Nature. 2005 Oct 27; 437(7063):1252; Ju, T. et al., Cancer Res. 2008 Mar 15;68(6):1636-46). It may also result from increased expression of the sialyl transferase, ST6GalNAc I (Ikehara, Y. et al., Glycobiology. 1999 Nov; 9(11): 1213-24; Brockhausen, I. et al., Biol Chem. 2001 Feb; 382(2):219-32). De novo expression of STn can lead to modulation of carcinoma cells, changes in the malignant phenotype and lead to more aggressive cell behavior (Pinho, S. et al., Cancer Lett. 2007 May 8; 249(2): 157-70). Although STn is expressed at high levels in malignant tissues, low levels of expression have also been found in healthy human cells (Jass, J.R. et al., J Pathol. 1995 Jun; 176(2): 143-9; Kirkeby, S. et al. , Arch Oral Biol 2010 Nov;55(11):830-41). STn itself has attracted attention as a target for cancer detection and therapy (Cheever, M.A. et al., Clin Cancer Res. 2009 Sep 1; 15(17):5323-37).

In addition to the presence of STn, other glycosylation changes have been noted in cancer. One of them includes Neu5Gc. N-acetylneuraminic acid (Neu5Ac) and Neu5Gc are the two major sialic acids on mammalian cell surfaces. Neu5Ac and Neu5Gc differ only in that Neu5Gc contains an additional oxygen atom associated with a chemical group attached to carbon 5. Due to the loss of a functional gene, sialic acid in the human body can only be synthesized in the form of Neu5Ac, but not Neu5Gc. However, Neu5Gc can be metabolically absorbed into the human body from animal foods such as red meat (Tangvoranuntakul, P. et al., Proc Natl Acad Sci USA. 2003 Oct 14; 100(21): 12045-50; Nguyen, D.H. et al., J Immunol. 2005 Jul 1; 175(1):228-36; US7682794, US8084219, US2012/0142903, WO2010030666 and WO2010030666, the entire contents of which are incorporated herein by reference). Neu5Gc is present in significant excess in human tumors (Higashi, H. et al., Cancer Res. 1985 Aug; 45(8):3796-802; Miyoshi I. et al., Mol Immunol. 1986. 23: 631-638; Hirabayashi, Y. et al., Jpn J Cancer Res. 1987. 78: 614-620; Kawachi. S., et al., Int Arch Allergy Appl Immunol. 1988. 85: 381-383; Devine, P. L. et al., Cancer Res. 1991. 51: 5826-5836; Malykh, Y. N. et al., Biochimie. 2001. 83: 623-634 and Inoue, S. et al., 2010. Glycobiology. 20(6): 752-762) and is at very low levels in normal human tissue, which has been overlooked for several decades (Diaz, S.L. et al., PLoS One. 2009. 4: e4241; Tangvoranuntakul, P. et al., Proc Natl Acad Sci U S A. 2003. 100: 12045-12050; Varki, A. et al., Glycoconj J. 2009. 26: 231-245). The increased metabolic accumulation of dietary Neu5Gc in cancer tissues compared to healthy human tissues can most likely be explained by at least three factors: rapid growth with insufficient production of competing endogenous Neu5Ac, increased macropinocytosis induced by growth factors (Dharmawardhane, S. et al., Mol Biol Cell. 2000 Oct; 11(10):3341-52; Simonsen, A. et al., Curr Opin Cell Biol. 2001 Aug; 13(4):485-92; Johannes, L. et al., Curr Opin Cell Biol. 2001 Aug; 13(4):485-92; Johannes, L. et al. al., Traffic. 2002 Jul; 3(7):443-51; Amyere, M. et al., Int J Med Microbiol. 2002 Feb; 291(6-7):487-94), and stimulation of lysosomal gene expression the sialic acid transporter sialin as a result of hypoxia (Yin, J. et al., Cancer Res. 2006 Mar 15; 66(6):2937-45). Additionally, all humans tested to date have a reservoir of polyclonal antibodies against the non-human Neu5Gc, making it the first example of a xeno-autoantigen (Padler-Karavani, V. et al., Glycobiology. 2008 Oct; 18(10): 818-30; Varki, N.M. et al., Annu Rev Pathol. 2011;6:365-93). The accumulation of dietary Neu5Gc in malignant tumors, despite the anti-Neu5Gc response, has been shown to promote tumor progression by inducing low-grade chronic inflammation (Hedlund, M. et al., Proc Natl Acad Sci US A. 2008 Dec 2; 105(48) :18936-41). Thus, Neu5Gc containing glycan epitopes on human tumors provides a valuable drug targeting opportunity. A recent study found antibodies against Neu5Gc-containing STn (GcSTn) but not Neu5Ac-STn (AcSTn) in cancer patients and examined their potential as a specific biomarker for cancer detection (Padler-Karavani, V. et al ., Cancer Res. 2011 May 1;71(9):3352-63).

MUC1 is a key cell surface glycoprotein that is typically highly glycosylated but is underglycosylated in tumor cells. Weak glycosylation of MUC1 results in exposure of immunogenic antigens. They may be located on the MUC1 core peptide sequence or on core carbohydrate residues. These TASAs include, but are not limited to, N-acetylgalactosamine (Tn), sialyl(α2,6)N-acetylgalactosamine (STn), and galactose((31-3)N-acetylgalactosamine (also known as Thomson-Friedenreich antigen or TF). Cells from approximately 80% of all carcinomas are estimated to express Tn among the MUC1 core carbohydrates, with STn being abundantly expressed on human carcinoma cells and associated with cancer progression and metastasis. With some exceptions, Tn and STn are not expressed in normal healthy tissues. Sialic acid forms prominent epitope on STn The invention takes advantage of the fact that aberrant expression of the Neu5Gc-STn glycan (GcSTn) appears to be highly specific for various carcinomas.

In the case of MUC1, incorporation of Neu5Gc into STn results in the formation of a tumor-specific target, a site that is a promising target for antibody-based therapy of tumor tissues. In some embodiments of the present invention, the glycan-interacting antibodies are directed to MUC1-expressing cancer cells containing Neu5Gc. To date, Neu5Gc has been detected in glycoconjugates from a variety of human cancer tissues, including, but not limited to, colon cancer, retinoblastoma tissue, melanoma, breast cancer, and yolk sac tumor tissue. Some embodiments of the present invention provide methods for treating these forms of cancer with glycan-interacting antibodies, as well as other forms of cancer not specifically mentioned herein that are characterized by the presence of Neu5Gc-containing cancer cells.

Additional antigens have been identified, including glycans, whose expression correlates with cancer (Heimburg-Molinaro, J. et al., Cancer vaccines and carbohydrate epitopes. Vaccine. 2011 Nov 8; 29(48):8802-26). These tumor-associated carbohydrate antigens include, but are not limited to, Lewis blood group-associated antigens [including, but not limited to, LewisY (Le ^Y ), Lewisx (Lex), sialyl-Lewis ^x (SLe ^x ) and sialyl-Lewis ^A ( SLe ^A )], glycosphingolipid-associated antigens [including, but not limited to, Globo H, stage-specific embryonic antigen 3 (SSEA-3) and glycosphingolipids, which include sialic acid], ganglioside-associated antigens [including, but not limited to, GD2 gangliosides , GD3, GM2, fucosyl GM1 and Neu5GcGM3] and polysialic acid-related antigens.

- 60 045483

In some embodiments, the therapeutic agents of the present invention may be directed against Lewis blood group-related antigens. Lewis blood group-related antigens contain a fucose residue linked to a GlcNAc a1-3 linkage or an a1-4 linkage. They can be found on both glycolipids and glycoproteins. Lewis blood group-related antigens can be found in the body fluids of individuals who secrete these antigens. Their presence on red blood cells is a consequence of the absorption of Lewis antigens from serum by red blood cells.

In some embodiments, the therapeutic agents of the present invention may be directed against Le ^Y. Le ^Y (also known as CD174) consists of Gale1,4GlcNAC having α1,2- as well as α1,3-linked fucose residues to form the epitope Fuca(1,2)Gale(1,4)Fuca(1,3 )GlcNAc. It is synthesized from the H antigen by α1,3-fucosyltransferases, which add α1,3-fucose to the GlcNAc residue of the parent chain. Le may be expressed in a variety of cancers, including, but not limited to, ovarian, breast, prostate, colon, lung, and epithelial cancers. Due to its low expression level in normal tissues and increased expression levels in many cancers, the Le ^Y antigen is a promising target for therapeutic antibodies.

In some embodiments, the therapeutic agents of the present invention may be directed against Lex. Lex includes the Gale1-4(Fuca1-3)GlcNAce-R epitope. It is also known as CD15 and stage-specific embryonal antigen 1 (SSEA-1). This antigen was first identified as immunoreactive with serum obtained from mice immunized with F9 teratocarcinoma cells. Lex has also been correlated with embryonic development at certain stages. It is also expressed in various tissues, both in the presence and absence of cancer, but can also be found in breast and ovarian cancer cells, where it is expressed only by malignant cells.

In some embodiments, the therapeutic agents of the present invention may be directed against SLe ^A and/or SLex. SLe ^A and SLex contain the structures Neu5Aca2-3Gale13(Fuca1-4)GlcNAce-R and Neu5Aca2-3Gale1-4(Fuca1-3)GlcNAce-R, respectively. Their expression is increased in cancer cells. The presence of these antigens in serum correlates with malignancy and poor prognosis. SLex is primarily found as a terminal epitope of mucin. It is expressed in a number of different cancers, including breast, ovarian, melanoma, colon, liver, lung and prostate cancers. In some embodiments of the present invention, the targets SLe ^A and SLex contain Neu5Gc (herein referred to as GcSLeA and GcSLex, respectively).

In some embodiments, the therapeutic agents of the present invention may be directed against glycolipids and/or epitopes found on glycolipids, including, but not limited to, glycosphingolipids. Glycosphingolipids contain the lipid ceramide linked to a glycan by the hydroxyl group of the ceramide. On the cell membrane, glycosphingolipids form clusters called lipid rafts.

In some embodiments, the therapeutic agents of the present invention may be directed against Globo H. Globo H is a cancer-associated glycosphingolipid first identified in breast cancer cells. The glycan moiety of Globo H includes Fuca(1-2)Gale(1-3)GalNAce(1-3)Gala(1-4)Gale(1-4)Glce(1). Although it can be found in a variety of normal epithelial tissues, Globo H has been found to be associated with many tumor tissues, including, but not limited to, small cell lung cancer, breast, prostate, lung, pancreatic, gastric, ovarian, and endometrial tumors.

In some embodiments, the therapeutic agents of the present invention may be directed against gangliosides. Gangliosides are glycosphingolipids containing one or more sialic acids. According to ganglioside nomenclature, G is used as an abbreviation for ganglioside. This abbreviation is followed by the letters M, D, or T, indicating the number of sialic acid residues attached (1, 2, or 3, respectively). Finally, the numbers 1, 2 or 3 are used to indicate the order of the distance each travels when analyzed by thin layer chromatography (with 3 traveling the longest distance, followed by 2, and then 1). Gangliosides are known to be involved in cancer growth and metastasis and can be expressed on the cell surface of tumor cells. Gangliosides expressed on tumor cells may include, but are not limited to, GD2, GD3, GM2, and Fucosyl-GMI (also referred to herein as Fuc-GM1). In some embodiments of the present invention, the glycan-interacting antibodies are directed against GD3. GD3 is a cell growth regulator. In some embodiments, GD3-directed antibodies are used to modulate cell growth and/or angiogenesis. In some embodiments, GD3-directed antibodies are used to modulate cell attachment. In some embodiments of the present invention, the glycan-interacting antibodies are directed against GM2. In some variants

- 61 045483 implementation of antibodies directed at GM2 are used to modulate cell-cell contacts. In some embodiments, the ganglioside targets of the present invention contain Neu5Gc. In some embodiments, such targets may include a GM3 variant containing Neu5Gc (herein referred to as GcGM3). The glycan component of GcGM3 is Neu5Gca2-3Gale1-4Glc. GcGM3 is a known component of tumor cells.

In some embodiments, the TACAs targeted by the anti-TACA antibodies of the present invention may include, but are not limited to, any of those listed in US Patent Publication No. US2013/0236486A1, US2013/0108624A1, US2010/0178292A1, US2010/0104572A1 , US2012/0039984A1, US2009/0196916A1 and US2009/0041836A1, the contents of each publication are incorporated herein by reference in their entirety.

The method of the present invention includes methods of treating cancer with one or more antibodies described herein. Such antibodies may contain one or more variable domains listed in table. 2. Antibodies may also contain one or more IgG constant domains, listed in table. 3. The antibodies may be humanized antibodies. Antibodies may be antibody-drug conjugates that contain a drug, including, but not limited to, any of the agents listed herein. The drug may be a cytotoxic agent, including, but not limited to, any of the agents listed herein. The cytotoxic agent may be MMAE. The cytotoxic agent may be linked to the antibody via a linker.

STn in cancer.

The immune system has multiple mechanisms to stimulate immune activity against tumor cells, including both innate and adaptive immunity. As used herein, the term immune activity against tumor cells means any activity of the immune system that results in the death or prevention of growth and/or proliferation of tumor cells. In some cases, antitumor immune activity involves recognition and killing of tumor cells by natural killer (NK) cells and phagocytosis by macrophages. Adaptive antitumor immune responses involve the uptake and presentation of tumor antigen by antigen presenting cells (APCs), such as dendritic cells (DCs), leading to modulation of antitumor activity of T cells and/or expansion of B cells with the secretion of tumor-specific antibodies. Binding of tumor-specific antibodies to tumors can lead to tumor cell death through the mechanisms of antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

As used herein, the term immunoresistant tumor cell means a tumor cell that is capable of attenuating, or avoiding, immune activity against tumor cells. Some studies indicate that expression of STn (known as TACA) on the surface of tumor cells or secretion into the microenvironment of tumor cells may contribute to tumor cell evasion of antitumor immune activity. As used herein, the term tumor cell microenvironment means any area adjacent to, or around, a tumor cell. Such areas include, but are not limited to, areas between tumor cells, between tumor and non-tumor cells, surrounding fluids, and surrounding extracellular matrix components.

Stalled mucins containing STn have been shown by Ogata et al. to reduce NK cell targeting of tumor cells (Ogata, S. et al., 1992. Cane. Res. 52:4741-6, incorporated herein by full references). This study found that the presence of ovine, bovine, or porcine submandibular gland mucin (OSM, BSM, and PSM, respectively) resulted in nearly 100% inhibition of cytotoxicity (see Table 2 in Ogata et al.). Further studies by Jandus et al. demonstrated that some tumor cells can evade NK killing by expressing sialoglycan ligands that can interact with SIGLEC receptors on NK cells, resulting in NK inhibition (Jandus, S. et al., 2014, JCI. pii: 65899, the contents of the publication are incorporated herein by reference in their entirety).

In studies by Toda et al. it has been shown that STn can bind to CD22 receptors on B cells, resulting in decreased signaling and decreased B cell activation (Toda, M. et al., 2008. Biochem Biophys Res Commun. 372(1):45-50, the contents of the publication are incorporated herein by reference in their entirety). Dendritic cells (DCs) can influence adaptive immune activity by modulating T cell activity. Studies by Carrascal et al showed that STn expression by bladder cancer cells resulted in DC tolerance, reducing their ability to induce anti-tumor cell immune activity in T cells (Carrascal, MA et al., 2014. Mol Oncol, pii: S1574-7891( 14)00047-7, the contents of the publication are incorporated herein by reference in their entirety). The results of these studies showed that DCs coming into contact with STn-positive bladder cancer cells exhibited a tolerogenic expression profile with low expression of CD80, CD86, IL-12 and TNF-α. In addition, DCs are found to modulate regulatory T cells such that the T cells have low IFNy expression and high FoxP3 expression. Other studies by van Vliet and co-workers indicate that surface expression of macrophage galactose-type lectin (MGL) on DCs may lead to the targeting of these cells to tumor tissues (van Vliet, SJ., 2007. Amsterdam: Vrije Universiteit. P1- 232 and van Vliet, SJ et al., 2008. J Immunol. 181(5):3148-55, Nollau, P. et al., 2013. J Histochem Cytochem. 61(3): 199-205, content of each publications are incorporated herein by reference in their entirety). DCs arriving in tissues due to MGL interactions can influence T helper (Th) cells in one of three ways. DCs can induce T cell tolerance, T cell immune activity, or suppression of effector T cells. MGL has been shown to bind both AcSTn and GcSTn, and the affinity has been analyzed in detail (Mortezai, N. et al., 2013. Glycobiology. 23(7): 844-52, incorporated herein by reference in its entirety ). Interestingly, expression of MUC1 on tumors has been shown to lead to T cell tolerance, protecting tumor cells from destruction by the immune system.

In some embodiments, the glycan-interacting antibodies (including, but not limited to, anti-STn antibodies) of the present invention can be used to treat subjects having one or more tumor cells expressing one or more TACAs. In some cases, glycan-interacting antibodies (including, but not limited to, anti-STn antibodies) of the invention can be used to enhance immune antitumor activity against STn-expressing tumor cells. Such antibodies may lead to an enhancement of the adaptive immune response and/or the innate immune response against immune-resistant tumor cells. Some glycan-interacting antibodies can be used to enhance the antitumor activity of NK cells. In some cases, such glycan-interacting antibodies can block the interaction between glycan receptors expressed on NK cells and STn glycans on cancer cells or in surrounding tissues.

In some embodiments, glycan-interacting antibodies (including, but not limited to, anti-STn antibodies) of the invention can be used to enhance the antitumor activity of B cells. Such antibodies may reduce the interaction between CD22 receptors on B cells and STn glycans on cancer cells or in surrounding tissues. A study by Sjoberg et al. showed that 9-O-acetylation of α2,6-linked sialic acids on glycoproteins also results in decreased interaction between the B cell receptor CD22 and such glycoproteins (Sjoberg, E.R. et al. 1994. JCB. 126(2): 549-562). Another study by Shi et al found that higher levels of 9-O-acetylated sialic acid residues on murine erythroleukemia cells made the cells more susceptible to complement-mediated lysis (Shi, W-X. et al., 1996. J of Biol Chem. 271(49): 31526-32, the contents of the publication are incorporated herein by reference in their entirety). In some embodiments, anti-STn antibodies of the invention are capable of selectively binding non-9-O-acetylated STn, which reduces overall STn binding but results in decreased tumor cell growth and/or proliferation (e.g., by increasing B cell antitumor activity and increasing complement-mediated killing of tumor cells). In some embodiments, glycan-interacting antibodies (including, but not limited to, anti-STn antibodies) of the invention can be used to enhance the antitumor activity of DCs. Such antibodies can be used to reduce DC tolerance to tumor cells. Decreased DC tolerance may involve increased DC expression of CD80, CD86, IL-12, and/or TNF-α. In some cases, the antitumor activity of DCs may involve stimulation of T cell antitumor activity. Such antibodies can prevent binding between MGL DCs and glycans expressed on or near cancer cells.

Findings from a study by Ibrahim et al suggest that high levels of anti-STn antibodies along with endocrine therapy may improve overall survival and time to disease progression (TTP) in women with metastatic breast cancer (Ibrahim, N.K. et al., 2013. 4(7): 577-584, the contents of the publication are incorporated herein by reference in their entirety). In this study, levels of anti-STn antibodies were increased after vaccination with STn bound to keyhole limpet hemocyanin (KLH). In some embodiments, the anti-STn antibodies of the invention may be used in combination with endocrine therapy (eg, administration of tamoxifen and/or an aromatase inhibitor).

STn expression is thought to contribute to the metastatic potential of ovarian tumor cells. In accordance with some methods of the invention, anti-STn antibodies can be used to reduce metastasis of ovarian tumor cells. Such methods may result in a reduction in metastasis of from about 1% to about 15%, from about 5% to

- 63 045483 approximately 25%, from approximately 10% to approximately 50%, from approximately 20% to approximately 60%, from approximately

30% to about 70%, from about 40% to about 80%, from about 50% to about 90%, from about 75% to about 95%, or at least 95%.

Some methods of the present invention include methods of treating cancer in a subject with one or more of the antibodies described herein, wherein the subject has at least one cancer cell expressing STn. Antibodies can bind STn. Such antibodies may contain one or more variable domains listed in table. 2. Some antibodies, or antigen binding fragments, may contain different combinations of antibody sequences described herein. Such antibodies may also contain one or more IgG constant domains, listed in table. 3. The antibodies may be humanized antibodies. Antibodies may be antibody-drug conjugates that contain a drug, including, but not limited to, any of the agents listed herein. The drug may be a cytotoxic agent, including, but not limited to, any of the agents listed herein. The cytotoxic agent may be MMAE. The cytotoxic agent may be linked to the antibody via a linker.

In some embodiments, the present invention provides methods for treating cancer in a subject, wherein the subject has at least one cancer cell expressing STn, and wherein the subject has a disease that is resistant to treatment with platinum compounds. Platinum-resistant disease is resistance to treatment with platinum compounds that is observed in a certain percentage of the entire population of subjects receiving treatment for cancer. The subject may be treated by administering an anti-STn antibody to the subject. The at least one cancer cell may be an ovarian cancer cell. At least one cancer cell may be resistant to cisplatin. At least one cancer cell may be part of the tumor.

Anti-STn antibodies used to treat cancer in subjects with platinum-resistant disease may be an ADC. ADCs may be cytotoxic agent conjugates, including any of the formats described herein. Anti-STn antibodies can be administered at a dose of from about 0.1 mg/kg to about 25 mg/kg. Administration can be done by intravenous injection. Administration may include, but is not limited to, daily administration, weekly administration, or monthly administration.

In some embodiments, treatment with anti-STn antibodies may result in a decrease in detectable levels of STn in body fluids and/or tissues of the subject. STn may be bound to proteins or other carriers. In some cases, serum STn levels decrease.

Cancer stem cells as therapeutic targets.

Cancer stem cells or CSCs (also called tumor-initiating cells) are a subset of cells within a heterogeneous cancer tissue or population of tumor cells that drive the initiation, growth, dissemination and recurrence of primary and metastatic tumors (Karsten and Goletz, SpringerPlus, 2013, 2, 301), and may occur in different proportions of the general population depending on the type of tumor. CSCs differ from terminally differentiated cells in their ability to self-renew and their ability to produce non-CSC differentiated progeny (Gupta et al., Nature medicine, 2009, 15, 1010-1012). These properties are similar to those of normal stem cells. Such differences between normal stem cells and CSCs have important implications for therapy.

An increasing number of cell surface biomarkers have been identified that are claimed to distinguish CSCs from their non-CSC counterparts (Medema et al., Nature cell biology, 2013, 15, 338-344; Zoller, Cancer, 2011, 11, 254-344). 267). These may include, but are not limited to, CD44, CD133, CD117, and aldehyde dehydrogenase isoform 1 (ALDH1). While some of these have been identified in studies of mouse tumors and human cell lines, others have been confirmed using primary human tumor samples. One of these, the membrane spanning glycoprotein CD44, or hyaluronan receptor, which is a well-known component of tumors of various types, has also recently been accepted as a reliable marker of CSC in human cancers and, indeed, is the most common (Lobo et al., 2007 , 23, 675-699).

CD44 exists in several variant isoforms resulting from alternative splicing events occurring among the 20 exons and 19 introns of the full-length CD44 gene (Williams et al., Experimental biology and medicine, 2013, 238, 324-338). Growing evidence supports a role for CD44 and its variants in shaping the CSC phenotype with characteristic metastatic potential and drug resistance (Negi et al., Journal of drug targeting, 2012, 20, 561-573), in part due to modulation of intracellular transmission pathways signals (Williams et al., Experimental biology and medicine, 2013, 238, 324-338). In addition, it is known that patients with triple-negative breast cancer, as well as some other types of cancer, who have high numbers of CD44 cells, have a poor prognosis and high

- 64 045483 mortality rate (Negi et al., Journal of drug targeting, 2012, 20, 561-573). These observations support the fact that targeting CD44 is a way to treat cancer by inhibiting or eliminating CSCs, in addition to mature cancer cells. Indeed, many experimental approaches to targeting CD44 have been tried with varying degrees of success. A wide range of technologies have been used, including the use of conjugated and non-conjugated antibodies, nanodrug carrier systems and hyaluronan-conjugated drugs (Negi et al., Journal of drug targeting, 2012, 20, 561-573). However, toxic effects have been observed in several cases in in vivo studies; these adverse side effects could be explained by the widespread distribution of CD44 and its variants on the membranes of most vertebrate cells (Naor et al., Seminars in cancer biology, 2008, 18, 260-267), in addition to its presence on the surface of targeted CSCs and mature tumor cells . Targeting the CD44 protein, which is also a component of normal human stem cells (Williams et al., Experimental biology and medicine, 2013, 238, 324-338), can also impair the function of normal stem cells (Leth-Larsen et al., Molecular medicine, 2012, 18, 1109-1121). Although a large number of studies indicate the desirability of targeting CD44 protein to CSCs as well as mature tumor cells, an inherent challenge with this approach currently remains the difficulty of developing inhibitors that will spare normal tissues as well as normal stem cells.

Another well-known tumor antigen associated with CSC biology is the epithelial mucin MUC1, a membrane-associated glycoprotein that is differentially expressed at high levels on most adenocarcinomas but at low levels, or not at all, on normal epithelial cells. MUC1 has recently been identified as a CSC biomarker in various neoplasms, including breast (Engelmann et al., Cancer research, 2008, 68, 2419-2426) and pancreatic cancer, where its expression correlates with high rates of metastasis and poor prognosis. As a component of the CSC, MUC1 has been shown to be involved in cell adhesion, proliferation, survival and signaling (Engelmann et al., Cancer research, 2008, 68, 2419-2426) and can also be co-expressed with CD44 (Leth-Larsen et al. , Molecular medicine, 2012, 18, 1109-1121). Immunotherapeutic approaches to targeting MUC1 in cancer include the use of a vaccine as well as other approaches, but mainly in the context of therapy against mature cancer cells (Julien et al., Biomolecules, 2012, 2, 435-466; Acres et al., Expert review of vaccines, 2005, 4, 493-502).

There is a hypothesis that cancer stem cells are formed through epithelial-mesenchymal transition (EMT) (Gupta et al., Nature medicine, 2009, 15, 1010-1012) and/or vice versa, through mesenchymal-epithelial transition (MET) occurring in the area of metastasis ( Leth-Larsen et al., Molecular medicine, 2012, 18, 1109-1121) (also use the term CSC plasticity when non-CSCs give rise to CSCs). This finding further highlights the need to eliminate both CSCs and non-CSCs from cancer tissue or tumor cell populations.

Recent studies with enriched CSC populations have shown that these cells, unlike most tumors, are relatively inactive and largely resistant to many modern therapies, including chemotherapy and radiation (Leth-Larsen et al., Molecular medicine, 2012, 18, 1109-1121). Thus, current therapeutic strategies target non-CSC components of the tumor, leaving CSCs largely unaffected so that they can reawaken after appropriate stimuli, causing recurrence of primary tumors at the original site or spread to distant sites, colonization and metastasis, a leading cause of cancer mortality.

Modern understanding of the properties of cancer stem cells clearly indicates the need not only to specifically target the bulk of tumor cells, as is currently the case, but also the CSC compartment in order to achieve a potentially complete cure.

As described above, strategies developed based on tumor-associated (including CSC) biomarkers face the challenge that most cancer biomarkers are also present on normal cells, including normal stem cells. Therapy targeting a protein biomarker to eliminate CSCs can also target normal stem cells, causing elimination of normal cells.

Tumor-specific glycans in CSCs.

Aberrant forms of glycosylation, including the appearance of the Thomsen-nouveau (Tn) antigen (GalNAc-O-Ser/Thr), have been described in various forms of human cancer, allowing the identification of glycans as an entirely new class of tumor-associated carbohydrate antigens suitable for specific targeting tumors (Rabu et al., Future oncology, 2012, 8, 943-960). The formation of the sialyl derivative of Tn (STn) is mediated by the sialyl transferase ST6GalNAc I, which adds an α2,6-linked sialic acid to the Tn antigen. Tn sialylation prevents further addition of sugars, thus reducing further glycan elongation (Schultz et al., Cancer metastasis reviews, 2012, 31, 501-518).

Although the presence of STn in normal adult tissues is rare, STn is present

- 65 045483 in various human cancer cells, including ovarian, bladder, breast, cervical, colon and lung cancers, among others (Ferreira et al., Molecular oncology, 2013, 7, 719731; Kinney et al. , Cancer, 1997, 80, 2240-2249). Additionally, the presence of STn in tumors is associated with disease metastasis, poor prognosis, and decreased overall survival (Ferreira et al., Molecular oncology, 2013, 7, 719-731; Kinney et al., Cancer, 1997, 80, 2240-2249) ; thus, STn is considered a very attractive target for cancer detection and therapy. There are two different forms of sialic acid - Neu5Ac and Neu5Gc - located at the end of the STn. The Neu5Ac-sialylated form is predominant in humans, since Neu5Gc cannot be synthesized in the human body due to the inactive CMP-Neu5Ac hydroxylase (CMAN) gene. However, consumption of foods rich in Neu5Gc leads to the incorporation of foreign Neu5Gc into human cells, in particular, into carcinoma cells. Previous studies have shown that solid tumors uptake and express the Neu5Gc form of sialic acid (Inoue et al., Glycobiology, 2010, 20, 752-762; Malykh et al., Biochimie, 2001, 83, 623-634; Padler- Karavani et al., Cancer research, 2011, 71, 3352-3363). Mats that bind both STn glycoisoforms [Neu5Ac-STn (AcSTn) and Neu5Gc-STn (GcSTn)], which are potential targets in cancer, have been developed as pan-STn antibodies.

STn accumulation is associated with certain somatic mutations regularly observed in solid tumors and with inactivation of the gene encoding the molecular chaperone core-specific 1 beta3-galactosyltransferase (COSMC), which is required for the formation of active T synthase (Ju et al., Nature, 2005, 437, 125). T synthase competes with ST6GalNAc I for the GalNAc substrate and, thus, its inactivation due to mutation leads to an increase in STn synthesis. In addition, the accumulation of STn may be a consequence of increased expression of ST6GalNAc I, which is observed quite often (Brockhausen et al., Biological chemistry, 2001, 382, 219-232; Ikehara et al., Glycobiology, 1999, 9, 12131224). De novo expression of STn can modulate carcinoma cells, alter the malignant phenotype and lead to more aggressive cell behavior (Pinho et al., Cancer letters, 2007, 249, 157170). Therefore, not only is STn an interesting cancer biomarker and therapeutic target, but also interfering with STn function has the potential to provide significant functional antimetastatic therapeutic benefits.

Although glycosylation of cellular glycoproteins is well known to be altered in cancer, aberrant glycosylation appears to be selective for both the glycoproteins and glycans in question. In fact, in human tumor CSCs, only CD44 and MUC1 are the main carriers of the STn antigen (Cazet et al., Breast cancer research: BCR, 2010, 12, 204; Julien et al., Glycobiology, 2006, 16, 54-64), which immediately suggests a selective approach to targeting not only mature tumor cells, but also CSCs. While MUC1 is a normal surface component of some epithelial cells where it has a barrier function, tumor-associated MUC1 is characterized by hypoglycosylation and increased sialylation on CSCs in a manner similar to that observed in mature cancer cells, with STn apparently being a specific marker as CSCs and mature tumor cells (Curry et al., Journal of surgical oncology, 2013, 107, 713-722). The aberrant oligosaccharide profile of MUC1 leads to the expression of neomarkers such as sialyl-Lea (used in the test for CA19-9), sialyl-Le ^x and sialyl-Tn (TAG-72), as well as cryptic epitopes such as Tn in cancer cells (eg CSC). In addition, due to insufficient glycosylation, the mucin peptide core becomes exposed, so that epitopes in the core (not available in the MUC1 core in normal tissues) can serve as potential antigens.

Clinical approaches to target STn to date have consisted exclusively of obtaining STn vaccines. The most promising candidate for clinical use is teratope, a therapeutic vaccine consisting of STn bound to cochlear limpet hemocyanin. In in vivo studies in mice, immunization with teratope induced a strong antibody response that was shown to mediate growth retardation of injected STn-expressing breast carcinoma cells (Julien et al., British journal of cancer, 2009, 100, 1746- 1751). However, teratope failed to provide the primary outcome measure of treatment efficacy in a phase III clinical trial in metastatic breast cancer. The main hypothesis explaining why the teratope trials were unsuccessful on the primary outcome measure of treatment is that the patient population was not assessed for STn expression before enrollment in the study. Because there is strong heterogeneity in STn expression in breast cancer among patients, with a range of 25%–80% depending on the study and method of determination, failure to correlate STn expression with response may have masked any benefit provided by the teratope. Importantly, in the subgroup of patients receiving hormonal therapy, there was a significant 7.5-month increase in median overall survival with teratope compared with hormonal therapy alone (Ibrahim et al., Journal of clinical oncology: official journal of the American Society of Clinical Oncology, 2004, 22, 2547; and Miles et al., The oncologist, 2011, 16, 1092-1100), This supports the therapeutic potential of targeting STn in certain patient populations. In addition, because the immune response often varies significantly among vaccinated patients, vaccine approaches do not control or modulate antibody titers, resulting in wide variation in therapeutic antibody exposure among patients. However, teratope was well tolerated with minimal toxicity, demonstrating the safety of STn targeting in cancer therapy.

Increasing understanding of the molecular basis of STn expression in cancer cells strongly suggests that cells expressing STn on any cell surface protein will also express STn on many (if not all) other O-glycosylated cell surface proteins, making its excellent widespread cancer-related therapeutic target. Thus, STn-positive cancer cell populations may be enriched for CSCs. In addition, recent evidence indicates that loss of STn expression makes the cancer less aggressive with significantly less metastasis (Gill et al., Proceedings of the National Academy of Sciences of the United States of America 2013, 110, E3152-3161).

Anti-STn antibodies targeting CSCs as anticancer therapy.

Several anti-STn antibodies have been described in the field, however some of them exhibit low specificity for the STn antigen or stalled isoforms. For example, the commercial anti-STn antibody B72.3 has been shown to bind not only to STn, but also to the Tn antigen (Bapat, S. A. (2010) Human ovarian cancer stem cells. Reproduction 140, 33-41). The availability of monoclonal antibodies (mAbs) targeting STn, engineered to induce antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC), or conjugated to a cytotoxic payload [eg, antibody-drug conjugate (ADC)], provides the opportunity obtaining significant therapeutic benefit for cancer patients with STn-expressing tumors. In addition, such antibodies would also allow the development of companion diagnostics to pre-select patients most likely to respond to therapy.

STn is often present on one or more of the surface antigens of CSCs, and these together mediate the stemness and chemotherapy resistance properties characteristic of CSCs. Thus, anti-STn antibodies are a cancer cell targeting agent directed to CSCs, with the potential not only to directly kill CSCs through direct interaction and/or ADCC, but also with the unique ability to bind a wide panel of cell surface proteins and interfere with their functions important for CSC viability, self-renewal and replication.

As described herein, the rationale and benefits of targeting STn to CSCs may include the following: (1) many tumor-specific truncated glycoproteins carry STn in cancer; (2) STn is a unique glycan target expressed predominantly on CD44, MUC1, and potentially other important cell surface markers on both CSCs and mature tumor cells, regardless of proliferative status, allowing both of these tumor components to be targeted with a single medicine; (3) STn is also a component of CA-125, a biomarker for ovarian and other cancers; (4) STn is a component of the ovarian cancer CSC marker CD44. Thus, use of murine anti-pan-STn mAbs targeting an epitope covering both Neu5Ac and Neu5Gc, the Tn-bound form of sialic acid, will result in binding and destruction or impairment of CSC function and, by virtue of the presence of a common epitope, non-CSC tumor cells.

In some embodiments, the present invention provides an anti-pan-STn mAb for specifically eliminating human CSCs as well as mature tumor cells. In one aspect of the present invention, the anti-STn antibody will be directed to the validated STn glycan itself - not to a specific glycopeptide or carrier protein, which opens up the potential for binding to CD44, MUC1 or other STn glycosylated markers on both CSCs and non-CSCs. -CSC populations of tumor cells.

Given the excellent specificity of targeting tumor-associated STn, the present invention avoids damage to normal tissues, including normal adult stem cells, thereby providing an excellent therapeutic window.

Provided herein is a unique immunotherapy solution designed to kill human tumors by eliminating both cancer stem cells (CSCs) and mature cancer cells contained within cancer tissues and/or tumor cell populations. Elimination specifically occurs by targeting cell surface sialylated Tn (STn) antigen structures that are uniquely present in cancer tissues and/or cancer cell populations, including such structures associated with cancer stem cells.

Colorectal cancer.

Colorectal cancer (CRC) is the 4th most common disease and is currently the third leading cause of cancer death in the United States. Currently, 20% of patients are diagnosed with metastatic disease and approximately 50% of patients with CRC will eventually develop metastases. For people diagnosed with metastatic disease, the 5-year survival rate is 13.1%. In patients with metastatic colon cancer (mCRC), there is precedent for the use of therapeutic antibodies (eg, monoclonal antibodies) such as

- 67 045483 monoclonal antibodies against epidermal growth factor receptor (EGFR) (for example, cetuximab and panitumumab) and monoclonal antibodies against vascular endothelial growth factor (VEGF) (for example, bevacizumab and ramucirumab).

STn expression is reported to occur in 83.4% of samples from CRC patients and is correlated with increased malignancy and poor prognosis. The STn antigen is present in normal adult colon cells, but is only detectable after removal of the Oacetyl groups by saponification, a process that does not occur naturally in vivo (Juben et al., Biomolecules, 2012, 2, 435-466). Thus, STn can be used as a therapeutic target in the treatment of CRC.

In some embodiments, the glycan-interacting antibodies of the present invention can be used to treat CRC and/or mCRC. In some cases, such glycan-interacting antibodies are anti-STn antibodies, including, but not limited to, any of those described herein. Glycan-interacting antibodies used to treat CRC and/or mCRC can be conjugated to a cytotoxic agent (eg, MMAE and MMAF). Glycan-interacting antibodies can be used in combination with other therapies, such as chemotherapeutic agents (eg, fluoropyrimidine, oxaliplatin and/or irinotecan) and/or with a therapeutic antibody (eg, cetuximab, panitumumab, bevacizumab and/or ramucirumab) . In some cases, glycan-interacting antibodies can be used to treat forms of colorectal cancer that are resistant to one or more other therapeutic treatments.

In some embodiments, glycan-interacting antibodies used to treat colorectal cancer can be administered at a dose of from about 0.5 mg/kg to about 20 mg/kg. For example, antibodies can be administered in doses of about 0.5 mg/kg to about 2 mg/kg, from about 1 mg/kg to about 5 mg/kg, from about 2.5 mg/kg to about 10 mg/kg, or from about 5 mg/kg to about 20 mg/kg.

Ovarian cancer.

In some embodiments, the methods of the present invention include methods for treating ovarian cancer. Ovarian cancer is the leading type of female reproductive system cancer affecting women in the United States. In 2013, an estimated 22,240 women will be diagnosed and 14,030 will die from the disease, making it the fifth leading cause of cancer death in women and the most lethal gynecologic malignancy in the United States (Siegel et al., Cancer statistics, 2013. CA: a cancer journal for clinicians 63, 11-30). This high mortality rate may be explained by the asymptomatic onset of the disease, initial diagnosis at a late stage, the aggressiveness of this type of cancer and, in general, the lack of genetic changes that can serve as targets for therapy. The current standard of treatment is tumor debulking followed by chemotherapy with taxanes and platinum compounds. While this initial treatment results in ~70% of patients experiencing an initial complete clinical response, the majority of these patients will unfortunately relapse with chemotherapy-resistant disease (Foster et al., Cancer letters, 2013, 338, 147-157; and McCann et al., PloS one, 2011,6, e28077). In part, disease relapse is explained, as in the case of other types of cancer, by the presence of CSCs in the general population of tumor cells. Indeed, ovarian cancer CSCs have been identified and shown to be resistant to chemotherapy and radiation therapy (Burgos-Ojeda et al., Cancer letters, 2012, 322, 1-7). Thus, again, as in other forms of cancer, elimination of CSCs along with mature cells in cancer tissues and/or tumor cell populations is the best chance for controlling disease relapse and, ideally, cure.

In some embodiments, the present invention provides methods for treating ovarian cancer using anti-STn antibodies. The methods include administering anti-STn antibodies to subjects having or suspected of having ovarian cancer. In some embodiments, ovarian cancer CSCs, including, but not limited to, those present in cancer tissues and/or tumor cell populations, may be targeted for treatment of ovarian cancer. Although CD133 is the most extensively studied of the putative ovarian cancer CSC markers, CD44, a known STn carrier described above, is recognized to be associated with ovarian cancer and is included in the set of markers that identify ovarian cancer CSCs (Zhang et al., Cancer research, 2008, 68, 4311-4320; Foster et al., Cancer letters, 2013, 338, 147-157; and Zoller, Cancer, 2011, 11, 254-267). In addition, STn is expressed on the well-known ovarian cancer biomarker CA-125 (MUC16) as well as MUC1, and serum levels of these STn-associated mucins have recently been used as additional features distinguishing malignant from benign ovarian disease. Elevated serum STn levels occur in ~50% of patients with ovarian cancer and correlate with poor 5-year survival (Kobayashi et al., Journal of clinical oncology: official journal of the American Society of Clinical Oncology, 1991, 9, 983- 987; Kobayashi et al., Journal of clinical oncology: official journal of the American Society of Clinical Oncology, 1992, 10, 95-101; and Chen et al., Journal of proteome research, 2013, 12, 1408-1418). Finally, Vathipadiekal et al. in a differential expression study

- 68 045483 genes in CSC primary human ovarian carcinoma and non-CSC cell populations revealed that the expression of the STn-creating sialyl transferase ST6GalNAc I does not differ in cells of these two groups.

In some embodiments, administration of anti-STn antibodies to a subject having or suspected of having ovarian cancer, in accordance with the methods described herein, results in a decrease in the number of STn-positive cells in such subjects and/or a decrease in the number of STn-positive cells in one or more ovarian cancer tissues or tumor cell populations present in such subjects. In some embodiments, the reduction in number may include a reduction in STn-positive cells by an amount from about 10% to more than about 90% (e.g., at least 10%, at least 20%, at least 30%, at least 40% , at least 50%, at least 60%, at least 70%, at least 80%, at least 85% or at least 90%).

In some embodiments, the present invention provides antibodies for targeting CSCs for the purpose of preventing, controlling, or treating CSC-associated cancer. Such antibodies may include anti-STn antibodies, including, but not limited to, any of the antibodies described herein. In addition, anti-STn antibodies may include antibody 3F1 (SBH Sciences, Natick, MA) or derivatives thereof, including recombinant antibodies with CDR regions from 3F1 and/or humanized derivatives.

In some embodiments, the anti-STn antibodies of the invention can be used to target ovarian cancer stem cells that are resistant to other treatments. Such treatments may include chemotherapy. As used herein, chemotherapy refers to a method of treatment using chemicals. Such chemicals are referred to herein as chemotherapeutic agents. In the treatment of cancer, chemotherapeutic agents are agents that slow or stop the proliferation of cancer cells. As used herein, the terms chemotherapy-resistant or chemoresistant refer to cells that are resistant to, or have limited responsiveness to, chemotherapeutic treatments. Such chemotherapy treatments may include treatment with olaparib, carboplatin and/or paclitaxel. Methods of targeting chemotherapy-resistant ovarian cancer stem cells may benefit from changes in STn expression in ovarian cancer stem cells that occur following chemotherapy treatment. In some cases, chemotherapy-resistant ovarian cancer stem cells express STn before and/or after chemotherapy treatment. In some cases, cell surface expression of STn in chemotherapy-resistant ovarian cancer stem cells may be increased after chemotherapy treatment. Following chemotherapy treatment with olaparib, carboplatin, and/or paclitaxel, some ovarian cancer stem cells may proliferate, resulting in a population of STn-expressing cancer cells that are resistant to olaparib, carboplatin, and/or paclitaxel. In some embodiments, anti-STn antibodies can be used to target olaparib, carboplatin, and/or paclitaxel-resistant cells. In some cases, these resistant cells are cancer stem cells. In some embodiments, treatment of a subject with anti-STn antibodies can be performed after treatment of the subject with olaparib, carboplatin, and/or paclitaxel.

Accordingly, the methods of the invention may include methods of treating cancer by administering an anti-STn antibody to a subject with ovarian cancer. Anti-STn antibodies may be administered before, during, or after treatment with chemotherapeutic agents (eg, olaparib, carboplatin, and/or paclitaxel). Anti-STn antibodies can be directed to STn-expressing ovarian cancer stem cells present before, during, or after administration of chemotherapeutic agents (eg, olaparib, carboplatin and/or paclitaxel). Anti-STn antibodies may contain a variable domain with an amino acid sequence selected from one or more of SEQ ID NOs: 1-12. In some embodiments, anti-STn antibodies are antibody-drug conjugates. Such antibody-drug conjugates may contain a cytotoxic agent (eg, monomethyl auristatin E). A decrease in the number of STn-positive cells may occur in cancer tissues from subjects treated with anti-STn antibodies. In some embodiments, reducing the number of cells may include reducing the number of STn-positive cells by an amount from about 10% to about more than 90% (e.g., at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, or at least 90%). Antibodies can be directed to CSC.

In some embodiments, subjects having one or more chemotherapy-resistant ovarian cancer stem cells may be treated with anti-STn antibodies of the invention following treatment with one or more chemotherapeutic agents (eg, olaparib, carboplatin and/or paclitaxel). Treatment with anti-STn antibodies after chemotherapy treatment may prevent tumor recurrence. Tumor resurgence is the development of one or more tumor cells or tumors following a reduction in the number of one or more tumor cells or tumors (eg, due to previous or ongoing therapy).

In some methods of treating ovarian cancer, anti-STn antibodies of the present invention are administered in combination with modulators of cell signaling associated with stemness and/or differentiation. Such modulators may include modulators of Notch and/or Hedgehog signaling.

The methods of the present invention include methods for treating ovarian cancer by obtaining a sample from a subject having or suspected of having ovarian cancer and detecting STn in the sample, wherein if STn is detected, administering an anti-STn antibody to the subject. In some embodiments, the sample is a cellular sample (eg, a cancer tissue sample or a tumor sample). Cellular samples may include BRCA1 mutant cells or non-BRCA1 mutant cells.

Detection of STn in subject samples can be performed by any methods known in the art for detecting molecular compounds. Such methods may include the use of one or more STn detecting antibodies. STn detecting antibodies may include any antibody capable of binding STn. Some methods for detecting STn may include, but are not limited to, mass spectrometry, Western blotting, flow cytometry, immunoprecipitation, and enzyme-linked immunosorbent assay (ELISA). In some embodiments, protein-bound STn is detected.

In some embodiments, the detected STn may be associated with proteins associated with ovarian cancer stem cells. As used herein, the term ovarian cancer stem cell-associated protein refers to any protein that is associated with one or more ovarian cancer stem cells. Such proteins may include, but are not limited to, cell surface proteins, markers, intracellular proteins, transcription factors and proteins involved in cell signaling that influence the survival, growth, replication and/or maintenance of ovarian cancer stem cells. Proteins associated with ovarian cancer stem cells may include, but are not limited to, Notch, Hedgehog, MUC1, CD44, CD117, CD133 and integrin.

In some embodiments, the present invention provides methods for treating ovarian cancer including administering a combination treatment of olaparib and an anti-STn antibody. Such methods may include treating a subject with olaparib, followed by treating the subject with an anti-STn antibody. In some cases, methods of treating ovarian cancer include identifying a subject as not responding completely to treatment with olaparib and administering an anti-STn antibody to the subject.

The methods of the present invention may include methods for consolidated anticancer treatment. Consolidated treatment is treatment given after chemotherapy to achieve sustained remission. Typically, consolidated treatment involves the use of lower doses of chemotherapy to prevent tumor recurrence while maintaining low levels of toxicity. Methods of the present invention for consolidated treatment of cancer may include reducing the number of cancer cells in a subject by administering at least one chemotherapeutic agent and maintaining a reduced number (or further reducing the number) of cancer cells in the subject, or in one or more cancer tissues of the subject, by administering anti-STn antibodies. In some embodiments, the cancer is ovarian cancer. The chemotherapy agent may be olaparib, carboplatin and/or paclitaxel. In some embodiments, the anti-STn antibody is an antibody-drug conjugate (ADC). The ADC may contain monomethyl auristatin E (MMAE).

In some embodiments, the methods of the invention include completely killing ovarian tumor cells to induce sustained initial remission by administering one or more anti-STn antibodies. Other methods include inhibiting ovarian tumor recurrence over a period of time by administering one or more anti-STn antibodies, in some cases without causing excess toxicity. Such time periods may range from about 1 month to about 18 months, from about 1 year to about 5 years, from about 2 years to about 10 years, or more than 10 years.

In some embodiments, the present invention provides methods for treating cancer, comprising obtaining one or more ovarian tumor cells from a subject, forming a xenograft tumor in a host (e.g., mouse, rat, rabbit, pig, or primate) from one or more ovarian tumor cells, administering one or more anti-STn antibodies to the host and selecting at least one of the one or more tested anti-STn antibodies for use as a therapeutic antibody for treating the subject. An anti-STn antibody may be selected based on the ability of the antibody to reduce tumor volume in the host.

Immunological targets.

In some embodiments, the glycan-interacting antibodies of the invention may be immunomodulatory antibodies. As used herein, the term immunomodulatory antibody means an antibody that enhances or suppresses one or more immune functions or pathways.

Many bacterial glycans are known to contain sialic acid. In some cases

- 70 045483 such glycans allow bacteria to evade the innate immune system of hosts, including, but not limited to, humans. In one example, bacterial glycans inhibit the alternative pathway of complement activation through recognition of factor H. In another example, bacterial glycans mask underlying residues that may be antigenic. Some bacterial glycans are involved in cell signaling events by activating the binding of inhibitory sialic acid to Ig-like lectins (Siglec), which leads to a decrease in the immune response to elements containing some stylated fragments (Chen, X. et al., Advances in the biology and biology chemistry of sialic acids. ACS Chem Biol. 2010 Feb 19; 5(2): 163-76). In some embodiments, the glycan-interacting antibodies of the present invention can be used to treat immune complications associated with bacterial glycans.

Due to the foreign nature of Neu5Gc, as described herein, some Neu5Gc glycans are immunogenic, resulting in immune-associated destruction of cells and other elements on which these glycans may be expressed. This autoimmune destruction may be pathogenic. In some embodiments, glycan-interacting antibodies can be used to treat patients suffering from autoimmune diseases associated with Neu5Gc glycans.

In some embodiments, the immunomodulatory antibodies of the invention can be used to stimulate or suppress T cell-mediated immunity. Such antibodies may interact with one or more glycans present on T cells, T cell-associated proteins, and/or one or more other cell types that interact with T cells. Immunomodulatory antibodies that enhance T-cell-mediated immunity can be used to promote T-cell-mediated targeting of cancer cells.

In some tumors, infiltration by tumor-associated macrophages (TAMs) can lead to immunosuppression by promoting tumor cell survival and growth. This is thought to occur due to immunosuppressive cellular signaling that occurs through interactions between myeloid C-type lectin receptors (CLRs) present on TAMs and tumor-associated mucins (Allavena, P. et al., Clin Dev Immunol. 2010; 2010 :547179). In some embodiments, binding of immunomodulatory antibodies of the invention to one or more tumor-associated mucins or TACA prevents immunosuppressive cellular signaling in the TAM.

Application in veterinary medicine.

It is envisaged that the glycan-reactive antibodies of the invention will find use in the field of veterinary medicine, including the care and treatment of non-human vertebrate animals. As used herein, the term non-human vertebrate includes all vertebrates other than Homo sapiens, including wild and domesticated animals such as companion animals and livestock. Non-human vertebrates include mammals such as alpaca, banteng, bison, camel, cat, cattle, deer, dog, donkey, guyal, goat, guinea pig, horse, llama, mule, pig, rabbit, reindeer, sheep, buffalo and yak. Livestock includes domestic animals raised on agricultural farms for inputs such as food, draft power, and derived products such as fibers and chemicals. In general, livestock includes all mammals, birds and fish of potential agricultural importance. Specifically, four-legged animals for slaughter include oxen, heifers, cows, calves, bulls, cattle, pigs and sheep.

Bioprocessing.

In some embodiments, the invention provides methods for producing biological products in host cells by contacting the cells with one or more glycan-interacting antibodies (such as an antibody or fusion protein) capable of modulating gene expression or altering the levels and/or types of glycans produced, where In this case, such modeling or change leads to increased production of biological products. The bioprocessing methods of the present invention can be improved by using one or more glycan-interacting antibodies of the present invention. They can also be improved by providing, replacing or adding one or more glycan-interacting antibodies.

Diagnostics.

In some embodiments, the compounds and compositions of the invention may be used as diagnostic agents. In some cases, the antibodies of the invention can be used to identify, label or stain cells, tissues, organs, etc., expressing target antigens. In further embodiments, the antibodies of the invention can be used to identify STn present in tissue sections (ie, histological tissue sections), including tissues containing or suspected of containing malignant cells. Such methods of using the antibodies of the invention may in some cases

- 71 045483 be used to identify malignant cells or tumors in tissue sections. Tissue sections may be obtained from any tissue or organ, including, but not limited to, breast, colon, pancreas, ovary, brain, liver, kidney, spleen, lung, skin, stomach, intestine, esophagus, or bone.

In some embodiments, the diagnostic methods of the invention may include analyzing one or more cells or tissues using immunohistochemical methods. Such methods may include the use of one or more of any of the glycan-interacting antibodies described herein. The immunohistochemical methods of the invention may include staining tissue sections to determine the presence and/or level of one or more glycosylated proteins or other markers. Tissue sections can be obtained from tumors of subjects (eg, patient tumors and animal tumors, such as animal tumor models). Tissue sections can be obtained from formalin-fixed or unfixed fresh frozen tissue. In some cases, tissue sections are obtained from formalin-fixed paraffin-embedded (FFPE) tissues. The glycan-reactive antibodies described herein can be used as primary antibodies. Primary antibodies are used to directly contact tissue sections and bind to target epitopes. Primary antibodies can be directly conjugated to a detectable label or can be detected through the use of a detection agent such as a secondary antibody. In some embodiments, the primary antibody or detection agent includes an enzyme that can be used in reaction with a substrate to produce a visible product (eg, a precipitate). Such enzymes may include, but are not limited to, horseradish peroxidase, alkaline phosphatase, beta-galactosidase and catalase.

The anti-STn antibodies described herein can be used in the immunohistochemical methods of the present invention to detect STn-glycosylated proteins in tissues or cells. In some cases, these antibodies are used to detect and/or determine the level of STn in tumor tissues. Such tumor tissues may include tumor tissues included in tumor micropanels. Suitable types of tumors include, but are not limited to, tumors of the breast, colon, ovary, pancreas, skin, intestine, lung, and brain. The levels of anti-STn antibodies used in immunohistochemical staining techniques can be varied to increase visible staining or to reduce background staining levels. In some embodiments, antibody concentrations of from about 0.01 μg/ml to about 50 μg/ml are used. For example, antibody concentrations of from about 0.01 μg/ml to about 1 μg/ml, from about 0.05 μg/ml to about 5 μg/ml, from about 0.1 μg/ml to about 3 μg/ml can be used. ml, from about 1 μg/ml to about 10 μg/ml, from about 2 μg/ml to about 20 μg/ml, from about 3 μg/ml to about 25 μg/ml, from about 4 μg/ml to about 30 µg/ml or from about 5 µg/ml to about 50 µg/ml.

In some embodiments, the diagnostic methods of the invention include methods for obtaining a profile of STn-related glycoproteins. As used herein, the term STn-linked glycoprotein profile means a collection of information indicating the level and/or specific type of STn-linked glycoproteins in a sample or body of a subject. Methods for obtaining a profile of STn-related glycoproteins can be performed on a sample obtained from a subject. Such samples may be biological samples, including, but not limited to, any of those described herein. Biological samples may be cellular samples. In some cases, the cellular samples may include at least one tumor cell. In some embodiments, the tumor cell samples may include BRCA1 mutant or non-BRCA1 mutant tumor cells.

Glycoproteins included in the STn-related glycoprotein profiles may include, but are not limited to, cancer cell markers, stem cell markers, cancer stem cell markers, and stem cell-related proteins. In some embodiments, the glycoproteins identified and/or quantified as part of the STn-related glycoprotein profile may include, but are not limited to, CD44, CD133, CD117, integrin, Notch, and Hedgehog.

The levels and/or specific species of STn-linked glycoproteins in STn-linked glycoprotein profiles can be determined by any methods known in the art for identifying proteins and/or quantifying protein levels. In some embodiments, such methods may include, but are not limited to, mass spectrometry, array analysis (eg, antibody array or protein array), Western blotting, flow cytometry, immunoprecipitation, and ELISA. In some cases, STn-bound glycoproteins may be immunoprecipitated from the sample prior to analysis. Such immunoprecipitation can be performed using an anti-STn antibody. Anti-STn antibodies used to immunoprecipitate STn-bound glycoproteins may include any of those known in the art or described herein. In some embodiments, STn glycoproteins are immunoprecipitated from biological samples using an anti-STn antibody and then identified and/or quantified by mass spectrometry.

In some embodiments, information obtained from profiling STn-associated glycoproteins is used in the treatment of cancer. Accordingly, the present invention provides methods for treating cancer, comprising obtaining a sample from a subject in need of cancer treatment, determining a profile of STn-linked glycoproteins in the sample, selecting a glycan-interacting antibody that binds an STn-glycosylated protein from the profile of STn-linked glycoproteins, and administering the glycan-interacting antibody to the subject. Glycan-interacting antibodies administered in accordance with such methods may contain one or more CDR regions or variable domains described herein.

In some embodiments, the methods of the present invention can be used for companion diagnostics. As used herein, the term companion diagnostic means a test whose results assist in the diagnosis or treatment of subjects. Companion diagnostics can be useful in stratifying the severity of a patient's disease, disorder, or condition, helping to adjust treatment regimens and dosages to reduce cost, shorten clinical trial duration, improve safety, and/or improve efficacy. Companion diagnostics can be used to predict the progression of a disease, disorder or condition and to assist in prescribing preventive therapy. Certain companion diagnostic options may be used to select subjects for one or more clinical trials. In some cases, companion diagnostic tests may be used in parallel with specific treatments to optimize treatment.

In some embodiments, the methods of the present invention may be useful for accompanying diagnosis of diseases, disorders and/or conditions associated with cancer. Certain companion diagnostics of the present invention may be useful for predicting and/or determining the severity of one or more forms of cancer. Certain companion diagnostics of the present invention can be used to stratify subjects according to their risk of developing one or more forms of cancer. Certain companion diagnostics of the present invention may be used to facilitate and expedite drug development for anticancer therapy.

In some embodiments, the present invention provides methods for detecting and/or quantifying STn in a sample using a capture antibody and a detection antibody. As used herein, the term capture antibody means an antibody that binds an analyte such that it can be detected. Capture antibodies can be bound to surfaces or other carriers. Detection antibodies are antibodies that help determine the presence or absence of an analyte. In some embodiments, both the capture antibody and the detection antibody bind STn. In such embodiments, the capture antibody and detection antibody may be obtained from different species. This allows the use of secondary antibodies that recognize only the detection antibody and are not affected by the presence of the capture antibody. In some embodiments, the capture antibody may bind STn, and the detection antibody may bind the protein or carrier of the bound STn. Capture antibodies and detection antibodies used to detect STn in samples can be selected from commercially available anti-STn antibodies, as well as any of the anti-STn antibodies provided herein.

Cells with modified STn expression.

In some embodiments, the present invention relates to modified cells having altered STn levels. Such cells can be used for various purposes (eg experimental, therapeutic, antibody testing, etc.). In some cases, the methods of the present invention include methods of increasing the expression of ST6GalNAc I in one or more cells or tissues. This may result in one or more cells having increased expression of cellular STn (eg, surface expressed STn). Expression of ST6GalNAc I can be increased, for example, by introducing one or more vectors carrying an ST6GalNAc I expression construct. Such expression constructs can be constructed with the natural ST6GalNAc I promoter or with a promoter designed to enhance gene expression. Promoters designed to enhance gene expression may include constitutively, or excessively, active promoter elements. In some cases, promoters can be designed for inducible gene expression. Such promoters may become active, or have increased activity, upon contact with factors that activate inducible promoter elements. STn expression constructs may include hST6GalNAcI pRc-CMV, described in Julien, S. et al., 2001. Glycoconj J, 18: 883-93, the contents of which are incorporated herein by reference in their entirety. In some embodiments, the expression constructs may encode other factors involved in synthesis and/or expression

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STn. Such factors may include, but are not limited to, T synthase and core 1 beta3galactosyltransferase specific molecular chaperone (COSMC). In some embodiments, cells with minimal STn expression are converted to STn expressing cells. Such cells may include, but are not limited to, SKOV3 cells, BRCA1 mutant cells, and non-BRCA1 mutant cells.

Also proposed are modified cells that have reduced STn expression compared to unmodified cells. Accordingly, the methods of the present invention include methods for inhibiting STn expression. Such methods may include reducing the expression of ST6GalNAc I. In some embodiments, such methods may include introducing one or more nucleic acid molecules that suppress the expression of ST6GalNAc I. Such nucleic acid molecules may include, but are not limited to, inhibitory RNA (e.g., RNAi or siRNA -silencers). In some embodiments, other factors involved in STn synthesis and/or expression may be reduced. Such factors may include, but are not limited to, T synthase and COSMC. In some embodiments, cells naturally expressing STn are converted to STn-deficient cells. Such cells may include, but are not limited to, OVCAR3 cells and OVCAR4 cells.

III. Pharmaceutical compositions.

In some embodiments, the present invention relates to pharmaceutical compositions. Such pharmaceutical compositions may contain antibodies of the present invention and/or fragments, peptides or proteins derived from such antibodies. Pharmaceutical compositions can be characterized by one or more of: bioavailability, therapeutic window and/or volume of distribution.

Bioavailability.

Glycan-reactive antibodies, when formulated in a composition with a delivery/formulation agent or carrier described herein, may exhibit increased bioavailability compared to a composition not containing the delivery vehicle described herein. As used herein, the term bioavailability means the systemic availability of a specific amount of glycan-interacting antibody administered to a mammal. Bioavailability can be assessed by measuring the area under the curve (AUC) or maximum plasma or serum concentration (C _max ) of the unchanged form of the compound after administration of the compound to a mammal. AUC is defined as the area under the curve of the concentration of a compound in serum or plasma plotted on the ordinate (Y-axis) versus time plotted on the abscissa (X-axis). In general, the AUC for a particular compound can be calculated by methods known to those skilled in the art and described in GS Banker, Modem Pharmaceutics, Drugs and the Pharmaceutical Sciences, v. 72, Marcel Dekker, New York, Inc., 1996, which is incorporated herein by reference. In some embodiments, the AUC is calculated using the linear trapezoidal method with linear/linear interpolation. AUC can be expressed in units of time multiplied by concentration (i.e. unit of time * unit of mass / unit of volume). For example, AUC may be expressed in units such as days*µg/ml. The final elimination phase of each concentration versus time curve can be determined using one or more observed final concentration values. The slope of the curve for the final elimination phase can be determined using logarithmic regression on the unweighted concentration data.

The _Cmax value represents the maximum concentration of the compound achieved in the serum or plasma of the subject after administration. The C _max value of a particular compound can be measured using methods known to those skilled in the art. As used herein, increased bioavailability or improved pharmacokinetics means that the systemic availability of a glycan-interacting antibody, measured as AUC, _Cmax , or _Cmin in a mammal, is greater when coadministered with a delivery vehicle described herein than when coadministered with a delivery vehicle described herein. absence of such co-administration. In some embodiments, the bioavailability of the glycan-interacting antibody may be increased by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, according to at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or about 100%.

In some embodiments, the bioavailability of anti-STn antibodies can be determined after administration of the pharmaceutical composition. Anti-STn antibodies can be antibody-drug conjugates containing a drug, including, but not limited to, any of those described herein. The drug may be

- 74 045483 cytotoxic agent, including, but not limited to, any of those listed herein. The cytotoxic agent may be MMAE. The cytotoxic agent may be linked to the antibody via a linker. The pharmaceutical compositions of the present invention can be used to deliver anti-STn antibodies to a subject, wherein the anti-STn antibodies have a C _max of from about 1 μg/ml to about 5 μg/ml, from about 2 μg/ml to about 10 μg/ml , from about 3 μg/ml to about 15 μg/ml, from about 4 μg/ml to about 20 μg/ml, from about 5 μg/ml to about 50 μg/ml, from about 20 μg/ml to about 100 μg /ml, from about 50 μg/ml to about 200 μg/ml, from about 75 μg/ml to about 150 μg/ml, or from about 100 μg/ml to about 500 μg/ml. The pharmaceutical compositions of the present invention can be used to deliver anti-STn antibodies to a subject, wherein the anti-STn antibodies have an AUC (from the start of administration to the last observed quantifiable concentration) of from about 1 day*μg/ml to about 5 days*μg /ml, from about 2 days*µg/ml to about 10 days*µg/ml, from about 5 days*µg/ml to about 50 days*µg/ml, from about 20 days*µg/ml to about 200 days* µg/ml, from about 100 days*µg/ml to about 500 days*µg/ml or from about 250 days*µg/ml to about 1000 days*µg/ml.

Therapeutic window.

Glycan-interacting antibodies, when formulated in a composition with a delivery vehicle described herein, may exhibit an increase in the therapeutic window of the administered glycan-interacting antibody composition compared to the therapeutic window of an administered glycan-interacting antibody composition not containing the delivery vehicle described herein. . As used herein, the term therapeutic window means the range of plasma concentrations or range of levels of a therapeutically active substance in the area of action with a high probability of producing a therapeutic effect. In some embodiments, the therapeutic window of a glycan-interacting antibody, when coadministered with a delivery vehicle described herein, can be increased by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50 %, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85% , at least about 90%, at least about 95%, or about 100%.

In some embodiments, the compound's half-life and/or clearance rate may be monitored as an indicator of the therapeutic window.

As used herein, the term half-life, or t _1/2 , means the period of time required for a particular process or compound concentration to reach half of its final value. The term terminal elimination half-life, or terminal half-life, refers to the period of time required to achieve a reduction in plasma factor concentration by half after the factor concentration has reached pseudo-equilibrium. When the decrease in concentration can be influenced by one or more factors independent of elimination (for example, the rate of absorption or the rate of distribution), the observed half-life is called the apparent half-life. As used herein, the term clearance rate means the rate at which a particular compound is cleared from a biological system or fluid. When the rate can be influenced by one or more factors independent of clearance, the clearance rate is called the apparent clearance rate.

In some embodiments, the therapeutic window of anti-STn antibodies can be determined after administration of the pharmaceutical composition. Anti-STn antibodies can be antibody-drug conjugates containing a drug, including, but not limited to, any of those described herein. The drug may be a cytotoxic agent, including, but not limited to, any of those described herein. The cytotoxic agent may be MMAE. The cytotoxic agent may be linked to the antibody via a linker. The pharmaceutical compositions of the present invention can be used to deliver an anti-STn antibody to a subject, wherein the anti-STn antibody has an apparent terminal half-life of from about 1 hour to about 10 hours, from about 2 hours to about 12 hours, from about 4 hours to about 24 hours, about 20 hours to about 30 hours, about 1 day to about 5 days, about 2 days to about 14 days, about 4 days to about 21 days, about 8 days to about 28 days, or more than 28 days. In some embodiments, the pharmaceutical compositions of the present invention can be used to deliver anti-STn antibodies to a subject, wherein the anti-STn antibodies have an apparent clearance rate of from about 1 ml/kg/day to about 10 ml/kg/day, from about 5 ml/kg/day to about 20 ml/kg/day, from about 15 ml/kg/day to about 50 ml/kg/day, or more than 50 ml/kg/day.

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Volume of distribution.

Glycan-interacting antibodies, when formulated in a composition with a delivery vehicle described herein, may have an improved volume of distribution (Vdist), e.g., reduced or targeted, compared to a composition not containing a delivery vehicle described herein. The volume of distribution ( _Vdist ) relates the amount of drug in the body and the concentration of the drug in the blood or plasma. As used herein, the term volume of distribution means the volume of fluid that would be required to maintain the total amount of drug in the body at the same concentration as in the blood or plasma: V _dist equals the amount of drug in the body/drug concentration in the blood or plasma . For example, for a dose of 10 mg and a plasma concentration of 10 mg/L, the volume of distribution would be 1 liter. The volume of distribution reflects the extent of drug presence in extravascular tissue. A larger volume of distribution reflects the tendency of the compound to bind more to tissue components than to plasma protein. In a clinical setting, V _dist can be used to determine the loading dose to achieve steady-state concentration. Vss means the apparent volume of distribution at steady state. In some embodiments, the volume of distribution of a glycan-interacting antibody, when coadministered with a delivery vehicle described herein, can be reduced by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50 %, at least about 55%, at least about 60%, at least about 65%, at least about 70%.

In some embodiments, the volume of distribution of anti-STn antibodies in the body of a subject can be determined after administration of the pharmaceutical composition. Anti-STn antibodies can be antibody-drug conjugates containing a drug, including, but not limited to, any of those described herein. The drug may be a cytotoxic agent, including, but not limited to, any of those described herein. The cytotoxic agent may be MMAE. The cytotoxic agent may be linked to the antibody via a linker. The pharmaceutical compositions of the present invention can be used to deliver anti-STn antibodies to a subject, wherein the anti-STn antibodies have an apparent V _ss of from about 1 ml/kg to about 10 ml/kg, from about 5 ml/kg to about 50 ml/ kg, from about 20 ml/kg to about 100 ml/kg, from about 75 ml/kg to about 150 ml/kg, or more than 150 ml/kg.

In some embodiments, the glycan-interacting antibodies are in compositions and/or complexes in combination with one or more pharmaceutically acceptable excipients. Pharmaceutical compositions may optionally contain one or more additional active substances, for example, therapeutically and/or prophylactically active substances. General information on the formulation and/or production of pharmaceuticals can be found, for example, in Remington: The Science and Practice of Pharmacy 21 ^st ed., Lippincott Williams & Wilkins, 2005 (the contents of which are incorporated herein by reference).

In some embodiments, the compositions are administered to humans, patients, or subjects. For purposes of the present invention, the term active ingredient generally means glycan-reactive antibodies delivered as described herein.

Although the description of pharmaceutical compositions provided herein primarily focuses on pharmaceutical compositions that are suitable for administration to humans, those skilled in the art will understand that such compositions are generally suitable for administration to any other animal, such as non-human animals, e.g. , non-human mammals. Modifications of pharmaceutical compositions suitable for administration to humans to adapt them for administration to various animals are well known, and a skilled veterinary pharmacologist will be able to plan and/or implement such modifications using routine experimentation, as appropriate. Subjects to whom the pharmaceutical compositions are administered include, but are not limited to, humans and/or other primates; mammals, including commercially valuable mammals such as cattle, pigs, horses, sheep, cats, dogs, mice and/or rats; and/or poultry, including birds of commercial value such as poultry, chickens, ducks, geese and/or turkeys.

The pharmaceutical compositions described herein can be formulated by any method known, or hereafter developed, in the art of pharmacology. Typically, such manufacturing methods involve the step of combining the active ingredient with an excipient and/or one or more other auxiliary ingredients, followed, if necessary and/or desirable, by separating, shaping and/or packaging the preparation into the desired single or multi-dose form .

The pharmaceutical composition of the invention can be manufactured, packaged and/or sold

- 76 045483 unpacked, in the form of a single standard dose and/or in the form of a plurality of single standard doses.

As used herein, the term unit dose means a discrete amount of a pharmaceutical composition containing a predetermined amount of the active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient that will be administered to the subject and/or a convenient portion of such dose, such as one-half or one-third of such dose.

The relative amounts of the active ingredient, pharmaceutically acceptable excipient and/or any additional ingredients in the pharmaceutical composition of the invention will vary depending on the characteristics, body weight and/or health status of the subject receiving treatment, and also depending on the route of administration of the composition. As an example, the composition may contain from 0.1% to 100%, for example from 0.5 to 50%, 1-30%, 5-80%, or at least 80% (by weight) of the active ingredient. In one embodiment, the active ingredients are antibodies directed against cancer cells.

A drug.

The glycan-reactive antibodies of the present invention can be formulated using one or more excipients to: (1) increase stability; (2) increasing the ability to penetrate cells; (3) providing a sustained or delayed release (eg, from a glycan-reactive antibody preparation); and/or (4) changes in biodistribution (eg, targeting glycan-interacting antibody to specific tissues or cell types). In addition to traditional excipients, such as any and all solvents, dispersion media, diluents or other liquid carriers, dispersing or suspending agents, surfactants, isotonic agents, thickeners or emulsifiers and preservatives, the preparations of the present invention may contain, without limitation, liposomes, liquid nanoparticles, polymers, lipoplexes, core/shell nanoparticles, peptides, proteins, cells transfected with glycan-reactive antibodies (eg, for transplantation into a subject), and combinations thereof.

Excipients.

As used herein, the term excipient means any substance combined with a compound and/or composition of the invention prior to use. In some embodiments, the excipients are inactive and are used primarily as a carrier, diluent, or vehicle for the compound and/or composition of the present invention. Various excipients for formulating pharmaceutical compositions and methods for preparing the compositions are known in the art (see Remington: The Science and Practice of Pharmacy, ^21st Edition, AR Gennaro, Lippincott, Williams & Wilkins, Baltimore, MD, 2006; the contents of which are incorporated herein document by reference).

The use of conventional excipients is within the scope of the present invention, except that any conventional excipient may be incompatible with the substance or its derivatives, for example, may produce any undesirable biological effect or otherwise interact adversely with any other component(s). pharmaceutical composition.

The pharmaceutical compositions described herein can be formulated by any method known, or hereafter developed, in the art of pharmacology. Typically, such manufacturing methods include the step of combining the active ingredient with an excipient and/or one or more other auxiliary ingredients.

The pharmaceutical composition of the present invention can be manufactured, packaged and/or sold in bulk, in unit unit dosage form and/or in multiple unit unit dosage form.

The relative amounts of the active ingredient, pharmaceutically acceptable excipient and/or any additional ingredients in the pharmaceutical composition of the present invention may vary depending on the characteristics, body weight and/or health status of the subject receiving treatment, and also depending on the route of administration of the composition.

In some embodiments, the pharmaceutically acceptable excipient is at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% pure. In some embodiments, the excipient is approved for human use and for veterinary use. In some embodiments, the excipient is approved by the US Food and Drug Administration. In some embodiments, the excipient is rated for pharmaceutical use. In some embodiments, the excipient conforms to United States Pharmacopoeia (USP), European Pharmacopoeia (EP), British Pharmacopoeia, and/or International Pharmacopoeia standards.

Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersants and/or granulating agents, surfactants and/or emulsifiers, disintegrants, binders, preservatives, buffering agents, lubricants and/or or oil. Such excipients

- 77 045483 may optionally be included in pharmaceutical compositions.

Exemplary diluents include, but are not limited to, calcium carbonate, sodium carbonate, calcium phosphate, dicalcium phosphate, calcium sulfate, hydrogen calcium phosphate, sodium phosphate, lactose, sucrose, cellulose, microcrystalline cellulose, kaolin, mannitol, sorbitol, inositol, sodium chloride, dry starch, corn starch, powdered sugar, etc., and/or combinations thereof.

Exemplary granulating and/or dispersing agents include, but are not limited to, potato starch, corn starch, tapioca starch, sodium starch glycolate, clays, alginic acid, guar gum, citrus pulp, agar, bentonite, cellulose and wood products, natural sponges , cation exchange resins, calcium carbonate, silicates, sodium carbonate, cross-linked poly(vinylpyrrolidone) (crospovidone), sodium carboxymethyl starch (sodium starch glycolate), carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose (croscarmellose), methyl cellulose, pregelatinized starch (starch 1500), microcrystalline starch, water-insoluble starch, calcium carboxymethylcellulose, magnesium aluminum silicate (Veegum®), sodium lauryl sulfate, quaternary ammonium compounds, etc., and/or combinations thereof.

Exemplary surfactants and/or emulsifiers include, but are not limited to, natural emulsifiers (e.g., gum arabic, agar, alginic acid, sodium alginate, tragacanth, chondrox, cholesterol, xanthan, pectin, gelatin, egg yolk, casein, lanolin, cholesterol , waxes and lecithin), colloidal clays (for example, bentonite [aluminum silicate] and Veegum® [magnesium aluminum silicate]), long-chain amino acid derivatives, high molecular weight alcohols (for example, stearyl alcohol, cetyl alcohol, oleyl alcohol, triacetin monostearate, ethylene glycol distearate, glyceryl monostearate and propylene glycol monostearate , polyvinyl alcohol), carbomers (e.g. carboxypolymethylene, polyacrylic acid, acrylic acid polymer and carboxyvinyl polymer), carrageenan, cellulose derivatives (e.g. sodium carboxymethylcellulose, cellulose powder, hydroxymethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, methylcellulose), sorbitan fatty acid esters (e.g. polyoxyethylene sorbitan monolaurate [TWEEN® 20], polyoxyethylene sorbitan [TWEEN® 60], polyoxyethylene sorbitan monooleate [TWEEN® 80], sorbitan monopalmitate [Span® 40], sorbitan monostearate [Span® 60], sorbitan tristearate [Span® 65 ], glyceryl monooleate, sorbitan monooleate [Span® 80]), polyoxyethylene esters (e.g. polyoxyethylene monostearate [MYRJ® 45], polyoxyethylene hydrogenated castor oil, polyethoxylated castor oil, polyoxymethylene stearate and SOLUTOL®), sucrose fatty acid esters, polyethylene glycol fatty acid esters (e.g. CREMOPHOR®), polyoxyethylene ethers (e.g. polyoxyethylene lauryl ether [BRIJ® 30]), poly(vinylpyrrolidone), diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, PLUORINC® F 68, POLOXAMER® 188, cetrimonium bromide, cetylpyridinium chloride, benzalkonium chloride, sodium docusate, etc., and/or combinations thereof.

Exemplary binders include, but are not limited to, starch (eg, corn starch and starch paste); gelatin; sugars (eg sucrose, glucose, dextrose, dextrin, molasses, lactose, lactitol, mannitol); natural and synthetic gums (e.g. gum arabic, sodium alginate, Irish moss extract, panvar gum, ghatti gum, flea husk gum, carboxymethylcellulose, methylcellulose, ethylcellulose, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, cellulose acetate, polyvinylpyr rolidon), aluminosilicate magnesium (Veegum®) and larch arabogalactan); alginates; polyethylene oxide; polyethylene glycol; inorganic calcium salts; silicic acid; polymethacrylates; waxes; water; alcohol and so on; as well as their combinations.

Exemplary preservatives may include, but are not limited to, antioxidants, chelating agents, antimicrobial preservatives, antifungal preservatives, alcohol preservatives, acid preservatives, and/or other preservatives. Exemplary antioxidants include, but are not limited to, alpha-tocopherol, ascorbic acid, ascorbyl palmitate, butylated hydroxyanisole, butylated hydroxytoluene, monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite and/or sodium sulfite. Exemplary chelating agents include ethylenediaminetetraacetic acid (EDTA), citric acid monohydrate, disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, and/or trisodium edetate. Exemplary antimicrobial preservatives include, but are not limited to, benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, ethyl alcohol, glycerin, hexetidine, imidomourea, phenol, phenoxyethanol, phenyleth silt alcohol , phenylmercurate nitrate, propylene glycol and/or thimerosal. Exemplary antifungal preservatives include, but are not limited to, butylparaben, methylparaben, ethylparaben, propylparaben, benzoic acid, hydroxybenzoic acid, potassium benzoate, potassium sorbate, sodium benzoate, sodium propionate and/or sorbic acid. Exemplary alcohol preservatives include, but are not limited to, ethanol, polyethylene glycol, phenol, phenolic compounds, bisphenol, chlorobutanol, hydroxybenzoate, and/or phenylethyl alcohol. Exemplary acid preservatives include, but are not limited to, vitamin A, vitamin C, vitamin E, beta-carotene, citric acid, acetic acid, dehydroacetic acid, ascorbic acid, sorbic acid and/or phytic acid. Other preservatives include, but are not limited to, tocopherol, tocopherol acetate, deteroxime mesylate, cetrimide, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BEIT), ethylenediamine, sodium lauryl sulfate (SLS), sodium lauryl ether sulfate (SLES), sodium bisulfite, metabisulfite sodium, potassium sulfite, potassium metabisulfite, GLYDANT PLUS®, PHENONIP®, methylparaben, GERMALL® 115, GERMATEN® II, NEOLONE™, KATHON™ and/or EUXYL®.

Exemplary buffer agents include, but are not limited to, citrate buffer solutions, acetate buffer solutions, phosphate buffer solutions, ammonium chloride, calcium carbonate, calcium chloride, calcium citrate, calcium glubionate, calcium gluceptate, calcium gluconate, D-gluconic acid, calcium glycerophosphate, calcium lactate, propionic acid, calcium levulinate, pentanoic acid, dibasic calcium phosphate, phosphoric acid, tribasic calcium phosphate, calcium hydroxide phosphate, potassium acetate, potassium chloride, potassium gluconate, potassium mixtures, dibasic potassium phosphate, monobasic potassium phosphate, phosphate mixtures potassium, sodium acetate, sodium bicarbonate, sodium chloride, sodium citrate, sodium lactate, dibasic sodium phosphate, monobasic sodium phosphate, sodium phosphate mixtures, tromethamine, magnesium hydroxide, aluminum hydroxide, alginic acid, pyrogen-free water, isotonic saline solution, Ringer's solution, ethyl alcohol and so on, and/or combinations thereof.

Exemplary lubricants include, but are not limited to, magnesium stearate, calcium stearate, stearic acid, silicon dioxide, talc, malt, glyceryl behenate, hydrogenated vegetable oils, polyethylene glycol, sodium benzoate, sodium acetate, sodium chloride, leucine, magnesium lauryl sulfate, sodium lauryl sulfate and so on, as well as their combinations.

Illustrative oils include, but are not limited to, almond oil, apricot kernel oil, avocado oil, babassa oil, bergamot oil, blackcurrant seed oil, borage oil, juniper oil, chamomile oil, canola oil, cumin oil, carnauba oil, castor oil, cinnamon oil, cocoa oil, coconut oil, cod liver oil , coffee, corn oil, cottonseed oil, emu oil, eucalyptus oil, evening primrose, fish oil, flaxseed oil, geranoyl, pumpkin oil, grape seed oil, hazelnut oil, hyssop, isopropyl myristate, jojoba oil, candlewood oil, lavandin, lavender oil, lemon oil , Lycea cubeba, macadamia nut, mallow, mango kernels, meadowfoam, mink oil, nutmeg oil, olive oil, orange oil, Atlantic lobster fat, palm oil, coconut kernel oil, peach kernels, peanuts, poppy seeds, pumpkin seeds , rapeseed, rice bran, rosemary, safflower, sandalwood, camellia oleifera, savory oil, sea buckthorn oil, sesame oil, shea butter, silicone, soybean oil, sunflower, tea tree, thistle, camellia, vetiver, walnut and wheat germ. Exemplary oils include, but are not limited to, butyl stearate, caprylic triglyceride, capric triglyceride, cyclomethicone, diethyl sebacate, dimethicone 360, isopropyl myristate, mineral oil, octyldodecanoyl, oleyl alcohol, silicone oil, and/or combinations thereof.

The composition may contain excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweeteners, flavoring agents and/or flavoring agents, depending on the discretion of the formulator.

In some embodiments, the antibodies of the present invention are formulated with at least one excipient. The antibodies may be anti-STn antibodies. Antibodies can be antibody-drug conjugates containing a drug, including, but not limited to, any of those described herein. The drug may be a cytotoxic agent, including, but not limited to, any of those described herein. The cytotoxic agent may be MMAE. The cytotoxic agent may be linked to the antibody via a linker.

Carriers.

Liposomes, lipoplexes and lipid nanoparticles.

The glycan-reactive antibodies of the present invention can be formulated using one or more liposomes, lipoplexes or lipid nanoparticles. In one embodiment, pharmaceutical compositions containing glycan-reactive antibodies also contain liposomes.

Liposomes are artificially produced vesicles that may advantageously contain one or more lipid bilayers and can be used as a delivery vehicle for the administration of dietary supplements and pharmaceuticals. Liposomes can come in a variety of sizes, for example, but are not limited to, multilayer vesicles (MLVs), which can be hundreds of nanometers in diameter and can contain a series of concentric bilayers separated by narrow aqueous compartments; small unilamellar vesicles (SUVs), which can have a diameter of less than 50 nm; and large unilamellar vesicles (LUVs), which can have a diameter of 50 to 500

- 79 045483 nm. Liposome constructs may include, but are not limited to, opsonins or ligands to enhance liposome attachment to unhealthy tissue or to activate events such as, but not limited to, endocytosis. Liposomes can have low or high pH to improve the delivery of pharmaceuticals.

The formation of liposomes may depend on physicochemical characteristics such as, but not limited to, the encapsulated pharmaceutical drug and liposome ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the encapsulated substance and its potential toxicity, any additional processes occurring during timing of application and/or delivery of vesicles, optimization of vesicle size, polydispersity and shelf life for intended use, as well as batch-to-batch reproducibility of product performance and the ability to produce safe and effective liposomal formulations on a large scale.

In one embodiment, such drugs can also be designed, or compositions modified, such that they are passively or actively targeted to different types of cells in vivo.

Drugs can also be selectively targeted by expressing different ligands on their surface, such as, but not limited to, folate, transferrin, N-acetylgalactosamine (GalNAc), as well as targeting antibody approaches.

Liposomes, lipoplexes or lipid nanoparticles can be used to improve the functioning of glycan-interacting antibodies, as these drugs may be able to increase the degree of transfection of cells with glycan-interacting antibodies. Liposomes, lipoplexes, or lipid nanoparticles can also be used to improve the stability of glycan-reactive antibodies.

Liposomes that are specifically formulated to be loaded with antibodies are prepared by methods known in the art, such as those described in Eppstein et al. (Eppstein, D.A. et al., Biological activity of liposome-encapsulated murine interferon gamma is mediated by a cell membrane receptor. Proc Natl Acad Sci USA. 1985 Jun; 82(11):3688-92); Hwang et al. (Hwang, K.J. et al., Hepatic uptake and degradation of unilamellar sphingomyelin/cholesterol liposomes: a kinetic study. Proc Natl Acad Sci USA. 1980 Jul; 77(7):4030-4); US 4485045 and US 4544545. The preparation of liposomes with extended circulation period is also described in US 5013556.

Liposomes containing the glycan-reactive antibodies of the present invention can be prepared by reverse phase evaporation using lipids such as phosphatidylcholine, cholesterol, and phosphatidylethanolamine that has been derivatized with polyethylene glycol. To extrude liposomes of the desired diameter, filters with a certain pore size are used. In another embodiment, the glycan-interacting antibodies of the present invention can be conjugated to the outer surface of liposomes through a disulfide exchange reaction described in Martin et al. (Martin, F.J. et al., Irreversible coupling of immunoglobulin fragments to preformed vesicles. An improved method for liposome targeting. J Biol Chem. 1982 Jan 10; 257(1):286-8).

Polymers and nanoparticles.

The glycan-reactive antibodies of the invention can be formulated using natural and/or synthetic polymers. Non-limiting examples of polymers that can be used for delivery include, but are not limited to, DMRI/DOPE, poloxamer, chitosan, cyclodextrin and poly(lactic-co-glycolic acid) (PLGA) polymers. They can be biodegradable.

The polymeric formulation may provide sustained or delayed release of glycan-reactive antibodies (eg, after intramuscular or subcutaneous injection). An altered release profile of glycan-interacting antibodies may result, for example, in the release of glycan-interacting antibodies over a longer period of time. The polymer preparation can also be used to increase the stability of glycan-interacting antibodies.

Polymeric drugs can also be selectively targeted through the expression of various ligands such as, but not limited to, folate, transferrin, N-acetylgalactosamine (GalNAc) (Benoit et al., Biomacromolecules. 2011 12:2708-2714; Rozema et al. ., Proc Natl Acad Sci USA 2007 104:12982-12887; Davis, Mol Pharm 2009 6:659-668; Davis, Nature 2010 464:1067-1070; full contents of publications are incorporated herein by reference).

The glycan-reactive antibodies of the invention may also be formulated as nanoparticles using a combination of polymers, lipids and/or other biodegradable compounds such as, but not limited to, calcium phosphate. The components can be combined in a core/shell, hybrid, and/or layer-by-layer structure allowing the nanoparticles to be finely tuned to increase the efficiency of delivery of glycan-reactive antibodies. For glycan-interacting antibodies, systems based on poly(2(methacryloyloxy)ethylphosphorylcholine)-block-(2-(diisopropylamino)ethyl methacrylate), (PMPC-PDPA), can be used.

- 80 045483 pH-sensitive diblock copolymer, which during the process of self-assembly forms nanometer-sized vesicles, also known as polymerosomes, at physiological pH values. Such polymerosomes have been shown to successfully deliver relatively high antibody payloads into living cells. (Massignani, et al., Cellular delivery of antibodies: effective targeted subcellular imaging and new therapeutic tool. Nature Proceedings, May, 2010).

In one embodiment, a charge-variable PEG polymer (Pitella et al., Biomaterials. 2011 32:3106-3114) can be used to produce nanoparticles delivering the glycan-reactive antibodies of the present invention. Charge-variable PEG polymer is superior to polyanionic PEG block copolymers in that it converts to a polycationic polymer at acidic pH, which increases endosomal release.

In the field of core/shell nanoparticle applications, the focus is on a high-throughput method for synthesizing cationic cores from cross-linked nanogels and different shells (Siegwart et al., Proc Natl Acad Sci USA. 2011 108:12996-13001). Complexation, delivery, and internalization of polymeric nanoparticles can be precisely controlled by varying the chemical composition of both the core and shell components of the nanoparticle.

In one embodiment, poly(ethylene-co-vinyl acetate) matrices are used to deliver the glycan-reactive antibodies of the invention. Such matrices are described in Nature Biotechnology 10, 1446-1449 (1992).

Antibody preparations.

The glycan-interacting antibodies of the invention may be formulated for intravenous or extravascular administration (Daugherty, et al., Formulation and delivery issues for monoclonal antibody therapeutics. Adv Drug Deliv Rev. 2006 Aug 7; 58(5-6):686-706 , US Patent Publication No. 2011/0135570, all of which are incorporated herein in their entirety). Methods of extravascular administration may include, but are not limited to, subcutaneous administration, intraperitoneal administration, intracerebral administration, intraocular administration, intralesional administration, topical administration, and intramuscular administration.

Antibody structures can be modified to improve their effectiveness as therapeutic agents. Improvements may include, but are not limited to, increased thermodynamic stability, reduced Fc receptor binding properties, and increased folding efficiency. Modifications may include, but are not limited to, amino acid substitutions, glycosylation, palmitoylation, and protein conjugation.

Glycan-reactive antibodies can be formulated with antioxidants to reduce antibody oxidation. Glycan-interacting antibodies can also be formulated with additives to reduce protein aggregation. Such additives may include, but are not limited to, albumin, amino acids, sugars, urea, guanidinium chloride, polyhydric alcohols, polymers (such as polyethylene glycol and dextrans), surfactants (including, but not limited to, polysorbate 20 and polysorbate 80) or even other antibodies .

The glycan-reactive antibodies of the present invention can be formulated to reduce the effect of water on antibody structure and function. Antibodies in such preparations may be lyophilized. Formulations to be lyophilized may include carbohydrate or polyol compounds to protect and stabilize the antibody structure. Such compounds include, but are not limited to, sucrose, trehalose and mannitol.

The glycan-reactive antibodies of the present invention can be formulated with polymers. In one embodiment, the polymer preparations may include hydrophobic polymers. Such polymers may be microspheres formulated with polylactide-co-glycolide by a solid-in-oil-in-water encapsulation method. Microspheres containing ethylene-vinyl acetate copolymer are also provided for the delivery of antibodies and can be used to prolong the release time of the antibody in the delivery zone. In another embodiment, the polymers may be aqueous gels. Such gels may, for example, contain carboxymethylcellulose. Aqueous gels may also contain hyaluronic acid hydrogel. Antibodies can be covalently linked to such gels by a hydrazone bond, which allows delayed delivery to tissues, including, but not limited to, tissues of the central nervous system.

Peptide and protein preparations.

The glycan-reactive antibodies of the invention may be formulated with peptides and/or proteins. In one embodiment, peptides may be used, such as, but not limited to, cell penetrating peptides, as well as proteins and peptides that provide intracellular delivery of pharmaceuticals. Non-limiting examples of cell penetrating peptides that can be used with the pharmaceutical formulations of the present invention include a cell penetrating peptide sequence attached to polycations that facilitate delivery into the intracellular space, for example, TAT peptide from HIV, penetratins, transportans or derived from hCT cell-penetrating peptides (see, for example, Caron et al., Mol. Ther. 3(3):310-8 (2001); Langel, Cell-Penetrating Peptides: Processes and Applications (CRC

- 81 045483

Press, Boca Raton FL, 2002); El-Andaloussi et al., Curr. Pharm. Des. 11(28):3597-611 (2003); and Deshayes et al., Cell. Mol. Life Sci. 62(16): 1839-49 (2005), all of which are incorporated herein by reference). The compositions can also be formulated to include a cell penetrating agent, for example, liposomes, which enhance the delivery of the compositions into the intracellular space. The glycan-interacting antibodies of the invention can be complexed with peptides and/or proteins, such as, but not limited to, peptides and/or proteins from Aileron Therapeutics (Cambridge, MA) and Permeon Biologies (Cambridge, MA), to achieve intracellular delivery (Cronican et al., ACS Chem. Biol. 2010 5:747-752; McNaughton et al., Proc. Natl. Acad. Sci. USA 2009 106:6111-6116; Sawyer, Chem Biol Drug Des. 2009 73: 3-6; Verdine and Hilinski, Methods Enzymol. 2012;503:3-33, all of which are incorporated herein by reference in their entirety).

In one embodiment, the cell penetrating polypeptide may comprise a first domain and a second domain. The first domain may contain a supercharged polypeptide. The second domain may contain a protein binding partner. As used herein, the protein binding partner includes, but is not limited to, antibodies and functional fragments, scaffold proteins, or peptides thereof. The cell-penetrating polypeptide may also contain an intracellular binding partner for the protein binding partner. The cell-penetrating polypeptide can be secreted from the cell, into which glycan-interacting antibodies can be introduced.

Peptides or proteins may be included in the formulations of the present invention to enhance the efficiency of cell transfection with glycan-interacting antibodies or to alter the biodistribution of glycan-interacting antibodies (eg, by targeting specific tissues or cell types).

Cellular preparations.

Cellular preparations of the glycan-interacting antibody compositions of the invention can be used to achieve transfection of cells (eg, in a cell carrier) or alter the biodistribution of the compositions (eg, by targeting the cellular carrier to specific tissues or cell types).

Methods of transfer into cells.

Various methods are known in the art and are suitable for introducing nucleic acids or proteins, such as glycan-interacting antibodies, into a cell, including viral and non-viral methods. Examples of typical non-viral methods include, but are not limited to, electroporation, calcium phosphate-mediated transfer, nucleofection, sonoporation, heat shock, magnetofection, liposome-mediated transfer, microinjection, microparticle (nanoparticle) bombardment-mediated transfer, cationic polymer-mediated transfer (DEAE-mediated transfer). dextran, polyethyleneimine, polyethylene glycol (PEG) and the like) or cell fusion.

Sonoporation, or cell sonication, uses sound (such as ultrasonic frequencies) to change the permeability of the cell's plasma membrane. Sonoporation techniques are known to those skilled in the art and are used to deliver nucleic acids in vivo (Yoon and Park, Expert Opin Drug Deliv. 2010 7:321-330; Postema and Gilja, Curr Pharm Biotechnol. 2007 8:355-361; Newman and Bettinger , Gene Ther. 2007 14:465-475, all of which are incorporated herein by reference in their entirety). Sonoporation methods are known in the art and are also described, for example, in relation to bacteria, in US Patent Publication 20100196983, and in relation to other types of cells, for example, in US Patent Publication 20100009424, the contents of each of which are incorporated herein by reference in their entirety. .

Electroporation techniques are well known in the art and are used to deliver nucleic acids in vivo and in clinical settings (Andre et al., Curr Gene Ther. 2010 10:267-280; Chiarella et al., Curr Gene Ther. 2010 10:281- 286; Hojman, Curr Gene Ther. 2010 10:128-138; all of which are incorporated herein by reference in their entirety). In one embodiment, glycan-interacting antibodies can be delivered by electroporation.

Introduction and delivery.

The compositions of the present invention can be administered by any of the standard methods or routes of administration known in the art.

The glycan-interacting antibodies of the present invention can be administered by any route of administration that results in a therapeutically effective result. These include, but are not limited to, enteral, enterogastric, epidural, oral, percutaneous, epidural (epidural), intracerebral (into the cerebral hemispheres), intracerebroventricular (into the ventricles of the brain), cutaneous (applied to the skin), intradermal (into the skin itself) , subcutaneous (under the skin), nasal (through the nose), intravenous (into a vein), intraarterial (into an artery), intramuscular (into a muscle), intracardiac (into the heart), intraosseous infusion (into the bone marrow), intrathecal (into the spinal brain canal), intraperitoneal (infusion or injection into the abdominal cavity), intravesical infusion, intravitreal (through the eye), intracavernous injection (into the base of the penis), intravaginal, intrauterine, extra-amniotic, percutaneous (by diffusion through the intact

- 82 045483 skin for systemic distribution), transmucosal (diffusion through the mucous membrane), insufflation (inhalation), sublingual, sublabial, using a syringe, eye drops (on the conjunctiva) or ear drops. In specific embodiments, the compositions can be administered in a manner that allows them to cross the blood-brain barrier, vascular barrier, or other epithelial barrier. Non-limiting routes of administration of the glycan-interacting antibodies of the present invention are described below.

Parenteral and injection administration.

Liquid dosage forms for oral and parenteral administration include, but are not limited to, pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and/or elixirs. In addition to the active ingredients, liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, dimethylformamide, oils (especially cottonseed, peanut, corn, seed, olive, castor and sesame oils), glycerin, tetrahydrofurfuryl alcohol, polyethylene glycols, sorbitan fatty acid esters, and their mixtures. In addition to inert diluents, oral compositions may include adjuvants such as humectants, emulsifying and suspending agents, sweeteners, flavoring agents and/or flavoring agents. In specific embodiments, parenteral compositions are mixed with solubilizing agents such as CREMOPHOR®, alcohols, oils, modified oils, glycols, polysorbates, cyclodextrins, polymers, and/or combinations thereof. In other embodiments, surfactants such as hydroxypropylcellulose are included.

Injectable preparations, for example, sterile injectable aqueous or oleaginous suspensions, can be formulated as known in the art using appropriate dispersing agents, wetting agents and/or suspending agents. Sterile injectable preparations may be sterile injectable solutions, suspensions and/or emulsions in non-toxic parenterally acceptable diluents and/or solvents, for example, as a solution in 1,3-butanediol. Acceptable media and solvents that may be used include water, Ringer's solution, U.S.? and isotonic sodium chloride solution. Sterile non-volatile oils are usually used as a solvent or suspension medium. For these purposes, any soft, fixed oil can be used, including synthetic mono- or diglycerides. Fatty acids such as oleic acid can be used to make injectables.

Injectable preparations can be sterilized, for example, by filtration through a bacteria-retaining filter and/or by incorporating sterilants in the form of sterile solid compositions that can be dissolved or dispersed in sterile water or other sterile injection media prior to use.

To prolong the effect of the active ingredient, it is often desirable to slow the absorption of the active ingredient from the site of subcutaneous or intramuscular injection. This can be accomplished by using a liquid suspension of crystalline or amorphous material with poor water solubility. The rate of absorption of the drug in this case will depend on the rate of its dissolution, which in turn may depend on the crystal size and crystalline form. Alternatively, delayed absorption of a parenterally administered dosage form is achieved by dissolving or suspending the drug in an oily medium. Injectable depot forms are created by forming microencapsulated drug matrices in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, as well as the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly(orthoesters) and poly(anhydrides). An injectable drug depot is prepared by enclosing the drug in liposomes or microemulsions that are compatible with body tissues.

In some embodiments, the compositions of the present invention can be administered intravenously. Administration can be accomplished by intravenous bolus injection.

Rectal and vaginal administration.

Compositions for rectal or vaginal administration are typically suppositories, which can be prepared by mixing the compositions with appropriate non-irritating excipients such as cocoa butter, polyethylene glycol or suppository wax, which are solid at ambient temperature but liquid at body temperature and , as a result, melt in the rectum or vaginal cavity and release the active ingredient.

Oral administration.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In such solid dosage forms, the active ingredient is mixed with at least one inert, pharmaceutically acceptable excipient such as sodium citrate

- 83 045483 or dicalcium phosphate and/or fillers or dry diluents (for example, starches, lactose, sucrose, glucose, mannitol and silicic acid), binders (for example, carboxymethylcellulose, alginates, gelatin, polyvinylpyrrolidinone, sucrose and gum arabic), humectants ( e.g. glycerin), disintegrants (e.g. agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates and sodium carbonate), solution retardants (e.g. paraffin), absorption accelerators (e.g. quaternary ammonium compounds), humectants agents (eg, cetyl alcohol and glycerol monostearate), absorbents (eg, kaolin and bentonite clay) and lubricants (eg, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate), and mixtures thereof. In the case of capsules, tablets and pills, the dosage form may contain buffering agents.

Topical or percutaneous administration.

As described herein, compositions containing the glycan-reactive antibodies of the invention can be formulated for topical administration. Skin may be an ideal target site for delivery because it is easily accessible. Gene expression can be restricted not only to the skin, with the potential to avoid nonspecific toxicity, but also to specific layers and cell types within the skin.

The site of cutaneous expression of the delivered compositions will depend on the method of delivery of the nucleic acid. Three routes are typically considered for delivering glycan-interacting antibodies to the skin: (i) topical application (eg, for local/zonal treatment and/or cosmetic applications); (ii) intradermal injection (eg, for local/zonal treatment and/or cosmetic applications); and (iii) systemic delivery (eg, for the treatment of dermatological diseases affecting both cutaneous and extracutaneous areas). Glycan-reactive antibodies can be delivered to the skin using several different approaches known in the art.

In one embodiment, the invention relates to various dressings (eg, wound dressings) or dressings (eg, adhesive bandages) for convenient and/or effective use of the methods of the present invention. Typically, dressings or dressings may contain significant amounts of pharmaceutical compositions and/or glycan-interacting antibodies described herein, allowing for multiple treatments per subject(s).

In one embodiment, the invention relates to compositions containing glycan-reactive antibodies delivered by more than one injection.

Dosage forms for topical and/or transdermal administration of the compositions may include ointments, pastes, creams, lotions, gels, powders, solutions, sprays, aerosols and/or patches. Typically, the active ingredient is mixed under sterile conditions with a pharmaceutically acceptable excipient and/or any necessary preservatives and/or buffers, as needed.

In addition, the present invention provides for the use of transdermal patches, which often have the additional advantage of providing controlled delivery of the compound to the body. Such dosage forms can be prepared, for example, by dissolving and/or dispersing the compound in an appropriate medium. Alternatively or additionally, the rate can be controlled either by the use of a rate-controlling membrane and/or by dispersing the compound in a polymer matrix and/or gel.

Formulations suitable for topical administration include, but are not limited to, liquid and/or semi-liquid preparations such as liniments, lotions, oil-in-water and/or water-in-oil emulsions, such as creams, ointments and/or pastes and/or solutions and/or suspensions.

Topically administered preparations may, for example, contain from about 1% to about 10% (by weight) of the active ingredient, although the concentration of the active ingredient can be as high as the limiting solubility of the active ingredient in the solvent allows. Topical formulations may also contain one or more additional ingredients described herein.

Introduction of the depot.

As described herein, in some embodiments, the compositions of the present invention are formulated as a sustained release depot. Typically, a specific organ or tissue (target tissue) is selected for administration.

In some aspects of the invention, glycan-reactive antibodies are retained within, or near, the target tissue. Methods are provided for delivering compositions to one or more target tissues of a mammalian subject by contacting one or more target tissues (containing one or more target cells) with the compositions under conditions under which the compositions, in particular, the component(s) of the composition comprising glycan-interacting antibody is retained to a significant extent in the target tissue, i.e. at least 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99 .9, 99.99 or greater than 99.99% of the composition is retained in the target tissue. Preferably, tissue retention is determined by measuring the level of glyca interacting

- 84 045483 by us of antibodies present in compositions that penetrate target tissues and/or target cells. For example, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.99, or more than 99, 99% of glycan-interacting antibodies administered to a subject are present within cells for some period of time after administration. For example, intramuscular injection into a mammalian subject is performed using an aqueous composition containing one or more glycan-reactive antibodies and a transfection reagent, and tissue retention of the composition is determined by measuring the level of glycan-reactive antibodies present in the muscle cells.

Some aspects of the invention relate to methods of delivering compositions to target tissues of mammalian subjects by contacting target tissues (containing one or more target cells) with compositions under conditions under which the compositions are substantially retained in the target tissue. The compositions contain an effective amount of glycan-interacting antibodies such that the effect of interest occurs in at least one target cell. The compositions typically comprise cell penetrating agents and a pharmaceutically acceptable carrier, although the administration of naked glycan-interacting antibodies (eg, glycan-interacting antibodies without cell penetrating agents or other agents) is also contemplated.

In some embodiments, the compositions contain several different glycan-interacting antibodies, wherein one or more of the glycan-interacting antibodies is directed to a glycan of interest. Optionally, the compositions also contain cell penetration agents that facilitate intracellular delivery of the compositions. Determine the dose of the composition required to target the glycans of interest in a significant percentage of cells contained in a predetermined volume of target tissue (typically without targeting glycans in tissue adjacent to the predetermined volume of tissue, or located at a distance from the target tissues). Once determined, the determined dose may be administered directly into the tissue of the mammalian subject.

In one embodiment, the invention relates to glycan-reactive antibodies delivered by more than one injection or divided dose injections.

Pulmonary administration.

Pharmaceutical compositions can be formulated, packaged and/or sold in preparations suitable for pulmonary administration via the oral cavity. Such preparations may contain dry particles containing the active ingredients and having a diameter in the range of from about 0.5 nm to about 7 nm or from about 1 nm to about 6 nm. Such compositions are conveniently in the form of dry powders administered using a device including a dry powder reservoir into which a stream of propellant may be directed to atomize the powder, and/or a container for dispensing a self-propelled solvent/powder mixture, e.g. containing the active ingredient dissolved and/or suspended in a low boiling point propellant in a sealed container. Such powders include particles of which at least 98% of the particles by weight have a diameter greater than 0.5 nm, and at least 95% of the particles by weight have a diameter of less than 7 nm. Alternatively, at least 95% of the particles by mass have a diameter greater than 1 nm, and at least 90% of the particles by number have a diameter of less than 6 nm. Dry powder compositions may contain a solid, fine powder diluent, such as sugar, and are conveniently provided in unit dosage form.

Low boiling point propellants generally include liquid propellants having a boiling point below 65°F at atmospheric pressure. Typically, the propellant may comprise from 50 to 99.9% (by weight) of the composition, and the active ingredient may comprise from 0.1 to 20% (by weight) of the composition. The propellant may also contain additional ingredients such as a liquid nonionic and/or solid anionic surfactant and/or a solid diluent (which may be of the same order of particle size as the particles containing the active ingredient).

Pharmaceutical compositions formulated for pulmonary delivery may deliver the active ingredient in the form of drops of a solution and/or suspension. Such preparations may be formulated, packaged and/or sold as aqueous and/or low alcohol solutions and/or suspensions, optionally sterile, containing the active ingredient and are conveniently administered using any nebulization and/or atomization device. Such preparations may also contain one or more additional ingredients, including, but not limited to, a flavoring agent such as sodium saccharin, a volatile oil, a buffering agent, a surfactant, and/or a preservative such as methyl hydroxybenzoate. Droplets formed by this method of administration may have an average diameter ranging from about 0.1 nm to about 200 nm.

Intranasal, nasal and buccal administration.

The formulations described herein as being suitable for pulmonary delivery are suitable for intranasal delivery of a pharmaceutical composition. Another preparation suitable for intranasal administration is a coarse powder containing the active ingredient and

- 85 045483 having an average particle size of approximately 0.2 μm to 500 μm. This drug is administered by inhalation, that is, by rapid inhalation through the nasal passages from a container of powder located close to the nose.

Formulations suitable for nasal administration may, for example, contain as little as about 0.1% (by weight) and up to 100% (by weight) of the active ingredient, and may contain one or more additional ingredients described herein. The pharmaceutical composition may be formulated, packaged and/or marketed as a preparation suitable for buccal administration. Such preparations may, for example, take the form of a tablet and/or lozenge prepared using conventional methods, and may, for example, contain from 0.1% to 20% (by weight) of the active ingredient, with the remainder comprising soluble and/or an orally disintegrating composition and, optionally, one or more of the additional ingredients described herein. Alternatively, preparations suitable for buccal administration may comprise a powder and/or aerosolized and/or nebulized solution and/or suspension containing the active ingredient. Such powdered, aerosolized and/or finely divided preparations, when nebulized, may have an average particle and/or droplet size ranging from about 0.1 nm to about 200 nm, and may further contain one or more of any of the additional ingredients described in this document.

Injection into the eye or ear.

The pharmaceutical composition may be formulated, packaged and/or sold as a preparation suitable for administration to the eye or ear. Such preparations may, for example, take the form of eye or ear drops containing, for example, a 0.1/1.0% (by weight) solution and/or suspension of the active ingredient in an aqueous or oily liquid excipient. Such drops may further contain buffering agents, salts and/or one or more of any of the additional ingredients described herein. Other useful formulations that can be administered to the eye include those that contain the active ingredient in microcrystalline form and/or in a liposomal formulation. Subretinal inlays can also be used as a route of administration.

Introducing the payload.

The glycan-interacting antibodies described herein can be used in a number of different scenarios in which delivery of a substance (payload) to a biological target is desired, for example, delivery of detectable substances for target detection, or delivery of a therapeutic or diagnostic agent. Detection methods may include, but are not limited to, both in vitro and in vivo imaging techniques, such as immunohistochemical techniques, bioluminescence imaging (BLI), magnetic resonance imaging (MRI), positron emission tomography (PET), electron microscopy , X-ray computed tomography, Raman imaging, optical coherence tomography, absorption imaging, thermal imaging, reflected fluorescence imaging, fluorescence microscopy, fluorescence molecular tomography, nuclear magnetic resonance imaging, x-ray imaging, ultrasound imaging, photoacoustic imaging, laboratory tests, or detection in any situation where marking/coloring/visualization is necessary.

Glycan-interacting antibodies can be designed to incorporate both linker and payload, in any useful orientation. For example, a linker having two ends is used to attach one end to a payload and the other end to a glycan-interacting antibody. The glycan-interacting antibodies of the invention may include more than one payload as well as a cleavable linker. In another example, a drug that can be attached to glycan-interacting antibodies via a linker and can be fluorescently labeled can be used to track the drug in vivo, for example, within a cell.

Other examples include, but are not limited to, the use of glycan-interacting antibodies for reversible drug delivery into cells.

The glycan-interacting antibodies described herein can be used to intracellularly target payloads, such as detections or drugs, to specific organelles. In addition, the glycan-interacting antibodies described herein can be used to deliver drugs to cells or tissues, for example, in living animals. For example, the glycan-interacting antibodies described herein can be used to deliver chemotherapeutic agents to kill cancer cells; Glycan-interacting antibodies linked to drugs via linkers can facilitate membrane penetration, allowing the drug to pass into the cell to reach an intracellular target.

In some embodiments, the payload may be a drug, such as a cytotoxin, radioactive ion, chemotherapeutic agent, or other drug. A cytotoxin or cytotoxic agent includes any agent that can

- 86 045483 be harmful to cells. Examples include, but are not limited to, taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, teniposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxyanthracinedione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids , procaine, tatracaine, lidocaine, propranolol, puromycin, maytansinoids, e.g. maytansinol (see US Pat. No. 5,208,020, the entire contents of which are incorporated herein), rachelmicin (CC-1065, see US Pat. Nos. 5,475,092, 5,585,499, and 5,846,545, the entire contents of of which are incorporated herein by reference), as well as their analogs or homologues. Radioactive ions include, but are not limited to, iodine (eg, iodine 125 or iodine 131), strontium 89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, samarium 153, and praseodymium. Other drugs include, but are not limited to, antimetabolites (eg, methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, decarbazine), alkylating agents (eg, mechlorethamine, thiotepa, chlorambucil, rachelmicin (CC-1065), melphalan, carmustine (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C and platinum(II) cis-dichlorodiamine (DDP) cisplatin), anthracyclines (eg daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mithramycin and anthramycin (AMC)) and antimitotics (eg, vincristine, vinblastine, taxol and maytansinoids). In the case of the anti-STn antibodies of the present invention, tumor killing can be promoted by conjugation of the toxin with such anti-STn antibodies.

In some embodiments, the payload may be a detectable agent such as various organic small molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials (e.g., luminol), bioluminescent materials (e.g., luciferase, luciferin, and aequorin ), chemiluminescent materials, radioactive materials (for example, ¹⁸ F, ⁶⁷ Ga, ^81m Kr, ⁸² Rb, ⁿ¹ In, ¹²³ I, ¹³³ Xe, ²⁰¹ Tl, ¹²⁵ I, ³⁵ S, ¹⁴ C, ³ H or ^99m Tc (for example , as pertechnetate (technetate(VII), TcO ₄ )) and contrast agents (e.g., gold (e.g., gold nanoparticles), gadolinium (e.g., chelated Gd), iron oxides (e.g., superparamagnetic iron oxide (SPIO), single crystalline nanoparticles iron oxide (MION) and ultrafine superparamagnetic iron oxide (USPIO) particles), manganese chelates (e.g. Mn-DPDP), barium sulfate, iodinated contrast media (iohexol), microbubbles or perfluorocarbons). Such optically detectable labels include, for example, but are not limited to, 4-acetamido-4'-isothiocyanate-stilbene-2,2'-disulfonic acid; acridine and its derivatives (for example, acridine and acridine isothiocyanate); 5-(2'-aminoethyl)aminonaphthalene-1sulfonic acid (EDANS); 4-amino-N-[(3-vinylsulfonyl)phenyl]naphthalimide-3,5-disulfonate; N(4-anilino-1-naphthyl)maleimide; anthranilamide; BODIPY; brilliant yellow; coumarin and its derivatives (for example, coumarin, 7-amino-4-methylcoumarin (AMC, coumarin 120) and 7-amino-4trifluoromethylcoumarin (coumarin 151)); cyanine dyes; cyanosine; 4',6-diaminidino-2phenylindole (DAPI); 5',5-dibromopyrogallolsulfonaphthalein (bromopyrogallol red); 7diethylamino-3-(4'-isothiocyanatephenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'diisothiocyanate dihydrostilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanate stilbene-2,2'disulfonic acid; 5-[dimethylamino]-naphthalene-1-sulfonyl chloride (DNS, dansyl chloride); dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC); eosin and its derivatives (for example, eosin and eosin isothiocyanate); erythrosine and its derivatives (for example, erythrosine B and erythrosine isothiocyanate); ethidium; fluorescein and its derivatives (for example, 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin2-yl)aminofluorescein (DTAF), 2',7'-dimethoxy-4'5'-dichloro-6-carboxyfluorescein, fluorescein , fluorescein isothiocyanate, X-rhodamine-5-(and-6)-isothiocyanate (QFITC or XRITC) and fluorescamine); 2-[2[3-[[1,3-dihydro-1,1-dimethyl-3-(3-sulfopropyl)-2H-benzo[o]indol-2-ylidene]ethylidene]-2-[4(ethoxycarbonyl )-1-piperazinyl]-1-cyclopenten-1-yl]ethenyl]-1,1-dimethyl-3-(3-sulfopropyl)-N-benz[o]indolium hydroxide, internal salt, compound with n,n-diethylethanamine ( 1:1) (IR144); chloro-2[2-[3-[(5-chloro-3-ethyl-2(3H)-benzothiazolilidene)ethylidene]-2-(diphenylamino)-1-cyclopenten-1yl]ethenyl]-3-ethylbenzothiazolium perchlorate (IR140 ); malachite green isothiocyanate; 4methylumbelliferone orthocresolphthadeine; nitrotyrosine; pararosaniline; phenol red; Vphycoerythrin; o-phthaldialdehyde; pyrene and its derivatives (eg pyrene, pyrene butyrate and succinimidyl-1-pyrene); butyrate quantum dots; reactive red 4 (CIBACRON™ Brilliant Red 3B-A); rhodamine and its derivatives (e.g. 6-carboxy-X-rhodamine (ROX), 6-carboxy-rhodamine (R6G), lissamine-rhodamine B, sulfonyl chloride-rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas Red), N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine and tetramethylrhodamine isothiocyanate (TRITC)); riboflavin; rosolic acid; terbium chelate derivatives; cyanine3 (Cy3); cyanine-5 (Cy5); cyanine-5.5 (Cy5.5), cyanine-7 (Cy7); IRD 700; IRD 800; Alexa 647; La Jolta Blue; phthalocyanine and naphthalocyanine.

In some embodiments, the detectable agent may be an undetectable precursor that becomes detectable upon activation (e.g., fluorogenic tetrazine fluorophore constructs (e.g., tetrazine-BODIPY FL, tetrazine-Oregon Green 488, or tetrazine-BODIPY TMR-X ) or enzyme-activated fluorogenic reagents [e.g., PROSENSE® (VisEn Medical)]. In vitro assays in which enzyme-labeled compositions may be used include, but are not limited to, enzyme-linked immunosorbent assays (ELISAs), immunoprecipitation assays, immunofluorescence assays, enzyme-linked immunosorbent assays (EIA), radioimmunoassays (RIA) and Western blot analysis.

Combinations.

Glycan-interacting antibodies can be used in combination with one or more other therapeutic, prophylactic, diagnostic or imaging agents. The expression in combination with should not imply that the agents must be administered at the same time and/or formulated for co-delivery, although such delivery methods are within the scope of the present invention. The compositions may be administered simultaneously, before or after one or more other desired therapeutic agents or medical procedures. Typically, each agent will be administered at a dose and/or schedule specified for that agent. In some embodiments, the present invention relates to the delivery of pharmaceutical, prophylactic, diagnostic and/or imaging compositions in combination with agents that are capable of increasing their bioavailability, decreasing and/or modifying their metabolism, inhibiting their excretion and/or modifying their distribution in the body.

In some embodiments, glycan-interacting antibodies may be used in combination with one or more anticancer drugs, such as a chemotherapeutic agent, a therapeutic antibody, and/or a cell cycle inhibitor. Combining glycan-interacting antibody treatment with such anticancer drugs may provide additional therapeutic benefits, such as synergistic antitumor activity, and may be used to treat cancer. In some cases, such methods can be used to target cancer cells that are resistant to chemotherapy, antibody therapy and/or treatment with cell cycle inhibitors. Methods for targeting drug-resistant cancer cells may rely on changes in STn expression in cancer cells that occur following chemotherapy, antibody therapy, and/or treatment with cell cycle inhibitors. In some cases, drug-resistant cancer cells express STn before and/or after treatment. In some cases, cell surface expression of STn in drug-resistant cancer cells may be increased after treatment. Following treatment, some cancer cells may proliferate, resulting in the formation of a population of STn-expressing cancer cells that are resistant to the anticancer drug(s) used. In some cases, drug-resistant cancer cells are cancer stem cells.

Accordingly, the methods of the invention may include methods of administering an anti-STn antibody to a subject to target STn-expressing cancer cells present following administration of one or more anticancer drugs. Such anti-STn antibodies may contain a variable domain with an amino acid sequence selected from any of SEQ ID NOs: 1-12. In subjects and/or in cancer tissues from subjects treated with anti-STn antibodies, a decrease in the number of STn-positive cells may occur. In some embodiments, reducing the number may include reducing the number of STn-positive cells by an amount from about 10% to more than about 90% (e.g., at least 10%, at least 20%, at least 30%, at least 40 %, at least 50%, at least 60%, at least 70%, at least 80%, at least 85% or at least 90%).

In some embodiments, chemotherapeutic agents used in combination with anti-STn antibodies may include, but are not limited to, taxanes (e.g., paclitaxel and docetaxel), platinum-based agents (e.g., cisplatin, oxaliplatin, and carboplatin), topoisomerase inhibitors (e.g. , irinotecan), nucleotide analogs (eg, 5-fluorouracil and gemcitabine), kinase inhibitors [eg, ponatinib (ICLUSIG®) and sorafenib (NEXAVAR®)], and PARP inhibitors [eg, olaparib (LYNPARZA™)]. Chemotherapeutic agents can cause cell death or prevent cell growth through mechanisms that include, but are not limited to, inhibition of microtubule function, enzyme function, or DNA synthesis.

In some embodiments, therapeutic antibodies used in combination with anti-STn antibodies may include antibodies that target surface receptors of cancer cells. Such surface receptors may be involved in cell signaling pathways important for cell proliferation and/or migration. In some cases, the surface receptor may be epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR), or human epidermal growth factor receptor (HER). Antibodies directed to these receptors, or related factors, can inhibit the growth and/or migration of cancer cells. Exemplary anti-EGFR antibodies include cetuximab (ERBITUX®) and panitumumab (VECTIBIX®). Exemplary anti-VEGF antibodies include bevacizumab (AVASTIN®) and ramucirumab (CYRAMZA®). Exemplary aHTu-HER2 antibodies include trastuzumab (HERCEPTIN®). Among them, cetuximab, panitumumab, bevacizumab and ramucirumab are FDA-approved antibodies for the treatment of colorectal cancer.

In some embodiments, treatment with cell cycle inhibitors may be used in combination with treatment with anti-STn antibodies. Cell cycle inhibitors may include, but are not limited to, cyclin-dependent kinase (CDK) inhibitors, checkpoint kinase inhibitors, Polo-like kinase (PLK) inhibitors, and Aurora kinase inhibitors. As used herein, the term cell cycle inhibitor means any agent that slows or stops cell cycle progression. Cell cycle inhibitors can cause cell cycle arrest at various stages, and can lead to cell death and/or inhibition of cell growth.

In some embodiments, the cell cycle inhibitors may be CDK inhibitors. Cyclin-dependent kinases (CDKs) are a group of serine/threonine kinases. CDKs and their cyclin partners are key regulatory enzymes that control phase transitions in the cell cycle. Among them, CDK4 and CDK6 are key enzymes governing the transition from GO phase or G1 phase to S phase. CDK4 and CDK6 (herein referred to as CDK4/6) are close homologues that are expressed in a tissue-specific manner. CDK4/6 forms a complex with cyclin D and phosphorylates retinoblastoma protein (Rb). Phosphorylation of Rb reduces its suppression of E2F transcription factors, which leads to transcription of genes encoding proteins required for DNA replication. This may allow cells to enter S phase. Progression through S phase is controlled by the cyclin E-CDK2 and cyclin A-CDK2 complexes. The transition from G2 phase to M phase is regulated by CDK1 through interactions with partners cyclins, cyclin A2 and cyclin B. Therapeutic targeting of CDKs (in general or specific CDKs) can impede cell cycle progression and prevent cell proliferation. See Otto and Sicinski, Nat Rev Cancer. 2017; 17(2):93-115; and Asghar et al., Nat Rev Drug Discov. 2015; 14(2): 130-146; the contents of the publications are incorporated herein by reference in their entirety.

In some cases, CDK inhibitors may be selective CDK4/6 inhibitors. Because of the role of CDK4/6 in the transition from G1 to S phase, inhibition of CDK4/6 causes cell cycle arrest in G1 phase. Exemplary CDK4/6 inhibitors include, but are not limited to, palbociclib (Pfizer), ribociclib (Novartis), and abemaciclib (Eli Lilly). Palbociclib (PD-0332991, IBRANCE®) is a drug approved for the treatment of advanced (metastatic) breast cancer (Finn et al., Breast Cancer Res. 2009; 11(5):R77; Rocca et al., Expert Opin Pharmacother 2014;15(3):407-20; US Patent Nos. 6936612; 7863278; 7208489 and 7456168, all of which are incorporated herein by reference in their entirety). Palbociclib can be prepared and characterized by the methods disclosed in US Pat. No. 7,345,171, the contents of which are incorporated herein by reference in their entirety. Similarly, ribociclib (LEE011) also selectively inhibits CDK4/6 with high potency. Ribociclib and pharmaceutically acceptable salts of ribociclib can be prepared by methods described in detail in US Pat. Nos. 8,685,980 and 9,193,732, the contents of which are incorporated herein by reference in their entirety. Abemaciclib (LY-2835219) inhibits not only CDK4 and CDK6, but also several other kinases, including PIM1 (US Patent No. 7855211, the contents of which are incorporated herein by reference in their entirety). Early activity of abemaciclib has been reported in patients with lung cancer, ovarian cancer, and melanoma (Shapiro et al., ASCO Meeting abstracts 2013;31:2500).

In some cases, CDK inhibitors may be pan-CDK inhibitors. Such inhibitors can block multiple CDKs and lead to cell cycle arrest at different stages, such as G1 arrest, G2 arrest, and/or G2/M arrest. Exemplary pan-CDK inhibitors include, but are not limited to, flavopiridol (alvocidib), dinaciclib (MK7965), R-roscovitine, AT7519, milciclib, TG02, CYC065, and RGB-286638. For example, panCDK inhibitors may be semisynthetic flavopiridol (eg, see US Pat. No. 4,900,727) or flavopiridol analogues (eg, see US Pat. No. 5,733,920 and 5,849,733, the contents of which are incorporated herein by reference in their entirety). As another example, pan-CDK inhibitors may be dinaciclib, or a pharmaceutically acceptable salt thereof, as described in US Pat. No. 7,119,200, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the cell cycle inhibitors used in combination with glycan-interacting antibodies may be checkpoint kinase inhibitors. Inhibition of checkpoint kinases, such as CHK1, CHK2, or WEE1, can lead to disruption of cell cycle checkpoints (eg, S phase checkpoint, G2/M phase checkpoint, or mitotic spindle assembly checkpoint), allowing allows the cell cycle to progress even in the event of DNA damage. This can initiate cell death during mitosis through a mechanism known as mitotic catastrophe. Exemplary checkpoint kinase inhibitors include, but are not limited to, MK-8776, prexasertib (LY2606368), AZD1775, GDC-0575, and those described in Visconti et al. J Exp Clin Cancer Res. 2016; 35:153, the contents of which are incorporated herein by reference in their entirety.

In some embodiments, the cell cycle inhibitors may be Polo-like kinase (PLK) inhibitors. Polo-like kinases (PLKs) are cell cycle regulatory serine/threonine kinases. PLK1 is the most fully characterized member of the PLK family and is a key mitotic protein kinase involved in multiple regulatory events such as G2/M phase transition, completion of spindle assembly, chromosome separation, and mitotic exit. Exemplary PLK1 inhibitors include, but are not limited to, rigosertib (ON 01910.Na), volasertib (BI 6727), BI 2536, HMN-176, TKM-080301, NMS-P937, DAP-81, cyclopaline 1, ZK-thiazolidinone (TAL ), SBE13, COM-36, LFM-A13, scytonemin, wortmannin, and GSK461364A (Kumar and Kim, Biomed Res Int. 2015; 2015:705745, incorporated herein by reference in its entirety).

In some embodiments, the cell cycle inhibitors may be Aurora kinase inhibitors. Aurora kinases are a family of highly conserved serine/threonine kinases that are important for proper progression through the phases of mitosis. Aurora A plays an important role in various events of mitosis, including centrosome maturation, relative chromosome positioning, spindle assembly, completion of meiosis, and cytokinesis. Aurora B is a component of the chromosome passenger complex and is involved in the condensation and orientation of chromosomes, as well as in the proper implementation of cytokinesis. Exemplary Aurora kinase inhibitors include barasertib (AZD1152), alisertib (MLN8237), danusertib (PHA-739358), ENMD-2076, AT9283, PF-03814735, and AMG 900 (Bavetsias and Linardopoulos, Front Oncol. 2015; 5:278, contents publications incorporated herein by reference in its entirety).

Dosage.

The present invention relates to the delivery of glycan-interacting antibodies for any of therapeutic, pharmaceutical, diagnostic, or imaging applications, in any suitable manner taking into account advances in the field of drug delivery. Naked or formulated antibodies can be delivered.

Delivery of naked antibodies.

The glycan-reactive antibodies of the present invention can be delivered to cells, tissues, organs or organisms in naked form. As used herein, the term naked refers to glycan-interacting antibodies delivered without any agents or modifications to promote transfection or cell permeabilization. Naked glycan-interacting antibodies can be delivered to cells, tissues, organs and/or organisms using routes of administration known in the art and described herein. Naked delivery may involve delivering the drug in a simple buffer such as saline or PBS.

Delivery of formulated antibodies.

The glycan-interacting antibodies of the present invention can be formulated using the methods described herein. The formulations may contain glycan-interacting antibodies, which may or may not be modified. Drugs may also include, but are not limited to, cell penetration agents, pharmaceutically acceptable carriers, delivery vehicles, biodegradable or biocompatible polymers, solvents, and sustained release depots. Formulated glycan-interacting antibodies can be delivered to cells using routes of administration known in the art and described herein.

The compositions may also be formulated for direct delivery to organs or tissues by any of several methods known in the art, including, but not limited to, direct wetting or irrigation, through catheters, using gels, powders, ointments, creams, gels, lotions and/or droplets, using substrates such as fabric or biodegradable materials coated or impregnated with compositions, and the like.

Dosing.

The present invention relates to methods comprising administering one or more glycan-interacting antibodies of the present invention to a subject in need thereof. Nucleic acids encoding glycan-interacting antibodies, proteins or complexes comprising glycan-interacting antibodies, or pharmaceutical, imaging, diagnostic or prophylactic compositions thereof, can be administered to a subject using any amount and any route of administration effective for preventing, treating, diagnosing or visualization of the disease, disorder and/or condition. The exact amount needed will vary between subjects depending on species, age and general health.

- 90 045483 the name of the subject, the severity of the disease, the specific composition, the method of its administration, the nature of its activity, and the like. The compositions of the invention are generally formulated in unit dosage form for ease of administration and uniformity of dosage. However, it should be understood that the total daily use of the compositions of the present invention will be determined by the attending physician based on sound medical judgment. The specific therapeutically effective, prophylactically effective, or appropriate imaging dose level for any given patient will depend on various factors, including the disease being treated and the severity of the disease; the activity of the particular compound used; the specific composition used; the age, body weight, general health, gender and diet of the patient; timing of administration, route of administration, and rate of excretion of the particular compound used; duration of treatment; drugs used in combination, or in parallel, with the specific compound used; and similar factors well known in the medical field.

In some embodiments, the compounds of the present invention (e.g., anti-STn antibodies) can be administered as part of a composition in which the concentration of such compounds is from about 0.01 mg/ml to about 1 mg/ml, from about 0.1 mg/ml to about 5 mg/ml, from about 0.5 mg/ml to about 10 mg/ml, from about 2 mg/ml to about 20 mg/ml, or from about 5 mg/ml to about 50 mg/ml.

In some embodiments, the compositions can be administered in a volume, based on the subject's weight, of from about 0.01 ml/kg to about 1 ml/kg, from about 0.2 ml/kg to about 1.2 ml/kg, from about 0.05 ml/kg to about 2 ml/kg, from about 0.1 ml/kg to about 3 ml/kg, from about 0.5 ml/kg to about 5 ml/kg, from about 2 ml/kg to about 10 ml/kg or from about 5 ml/kg to about 20 ml/kg. Administration may be intravenous administration (eg, intravenous bolus injection).

In specific embodiments, the compounds or compositions of the present invention can be administered at dosage levels sufficient to deliver an amount of active compound (e.g., anti-STn antibody or chemotherapeutic agent), based on the subject's body weight, of from about 0.0001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg kg, from about 1 mg/kg to about 6 mg/kg, from about 2 mg/kg to about 10 mg/kg, from about 5 mg/kg to about 20 mg/kg, or from about 10 mg/kg to about 200 mg/kg. Administration can be performed one or more times per day to achieve the desired therapeutic, diagnostic, prophylactic or imaging effect. The desired dose may be delivered according to any dosing schedule, including, but not limited to, three times daily, twice daily, once daily, every other day, every third day, every week, every two weeks, every three weeks or every four weeks, every two months, every three months, every four months, every 5 months, every 6 months or every year. In certain embodiments, the desired dose may be delivered using multiple administrations (eg, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or more administrations).

In accordance with the present invention, glycan-interacting antibodies can be administered in divided dose regimens. As used herein, the term divided dose means dividing a single unit dose or total daily dose into two or more doses, such as two or more administrations of a single unit dose. As used herein, the term unit dose unit means a dose of any therapeutic agent administered in one dose/at one time point/by one route/at one point of contact, that is, during one administration event. As used herein, the term total daily dose means the amount administered or prescribed to be administered over a 24-hour period. It may be administered as a single unit dose. In one embodiment, the glycan-interacting antibodies of the present invention are administered to a subject in divided doses. Glycan-interacting antibodies can only be formulated in a buffer, or in the preparation described herein. Pharmaceutical compositions containing the glycan-reactive antibodies described herein may be formulated in a dosage form described herein, e.g., topical, intranasal, intratracheal, or injectable (e.g., intravenous, intraocular, intravitreal, intramuscular, intracardiac, intraperitoneal, or subcutaneous). General information on the formulation and/or production of pharmaceuticals can be found, for example, in Remington: The Science and Practice of Pharmacy 21 ^st ed., Lippincott Williams & Wilkins, 2005 (the contents of which are incorporated herein by reference).

Coverings or shells.

Solid dosage forms in the form of tablets, dragees, capsules, pills and granules can be formulated with coatings and shells, such as enteric coatings and other coatings well known in the pharmaceutical field. They may optionally contain opalescent components and may be of such a composition that they release the active ingredient(s) only, or preferably, in the central part of the intestinal tract, optionally in a delayed manner. Examples of coating compositions that can be used include polymeric substances and waxes. Solid compositions of a similar type can be used as fillers in soft and hard filled gelatin capsules using excipients such as lactose or milk sugar, as well as high molecular weight polyethylene glycols and the like.

IV. Kits and devices.

Sets.

Any of the compositions described herein may be included in a kit. By way of non-limiting example, the kit includes reagents for the production of glycan-reactive antibodies, including antigen molecules. The kit may further include reagents or instructions for the production or synthesis of glycan-reactive antibodies. It may also include one or more buffers. Other kits of the invention may include components for creating matrices or libraries of proteins or nucleic acids that interact with antibody glycans and thus may include, for example, a solid support.

In some embodiments, the present invention provides kits for screening, monitoring and/or diagnosing a subject comprising one or more glycan-interacting antibodies. Such kits may be used alone or in combination with one or more other screening, monitoring and/or diagnostic methods (eg, companion diagnostics). Some kits include one or more of a buffer, a biological standard, a secondary antibody, a detection reagent, and a composition for sample pretreatment (eg, retrieval, antigen blocking, etc.).

Kit components can be packaged in either aqueous or lyophilized form. The container in the kits will typically include at least one ampoule, tube, flask, vial, syringe or other container into which the component can be placed and preferably aliquoted. In the event that there is more than one component in a kit (the labeling reagent and label may be packaged together), the kit will typically also include a second, third, and so on, additional container into which the additional components can be separately placed. The kits may also include a second container for containing a sterile, pharmaceutically acceptable buffer and/or other diluent. However, different combinations of components may be contained in the bottle. Kits of the present invention will typically also include arrangements for enclosing glycan-reactive antibodies, eg proteins, nucleic acids and any other reagent containers in secure packaging for commercial sale. Such containers may include injection molded or blow molded plastic containers that contain the desired vials.

When the kit components are provided in one and/or more liquid solutions, the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred. However, the kit components may be provided as dry powder(s). When the reagents and/or components are provided as a dry powder, the powder can be reconstituted by adding an appropriate solvent. It is contemplated that the solvent may also be provided in another container. In some embodiments, the marking dyes are provided in the form of a dry powder. The kits of the invention are intended to provide 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 120, 130, 140, 150, 160, 170, 180, 190, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mcg, or at least 1000 mcg, or not more than 10 g, of dry dye. The dye can then be resuspended in any suitable solvent such as DMSO.

The kit may include instructions for the use of kit components, as well as for the use of any other reagent not included in the kit. The instructions may include variations that may be used.

Devices.

Any of the compositions described herein may be combined with, applied to, or incorporated into the device. Devices include, but are not limited to, dental implants, stents, bone replacement materials, artificial joints, valves, pacemakers or other implantable therapeutic devices.

V. Equivalents and scope of the invention.

Those skilled in the art will recognize, or will be able to determine using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention should not be limited by the above description, but only by the contents of the appended claims.

In the claims, the singular form of nouns may mean one or more than one, unless otherwise indicated or otherwise evident from the context. Claims or descriptions that include the word or expression of one or more group members should be construed to mean that one, more than one, or all of the group members are present in, participate in, or are otherwise associated with a particular product or process if not otherwise stated or otherwise does not appear from the context.

- 92 045483

The invention includes embodiments in which exactly one member of the group is present in, participates in, or is otherwise associated with a particular product or process. The invention includes embodiments in which more than one or all members of the group are present in, participate in, or are otherwise associated with a particular product or process.

It should also be noted that the term inclusive must be open-ended, and allows, but does not require, the inclusion of additional elements or steps. When the term including is used herein, the term consisting of is therefore also intended and indicated.

In the ranges given, final values are included. Moreover, it should be understood that, unless otherwise indicated or otherwise apparent from the context and knowledge of those skilled in the art, the values that are presented as a range may be any specific value or subrange within the stated ranges in various embodiments of the invention, up to a tenth of a unit of the lower limit of the range, unless the context clearly indicates otherwise.

Moreover, it should be understood that any specific embodiment of the present invention that is part of the prior art may be expressly excluded from any one or more claims. Since such embodiments are believed to be known to those skilled in the art, they may be excluded even if such exclusion is not expressly stated herein. Any particular embodiment of the compositions of the invention (e.g., any nucleic acid or protein encoded therewith; any method of preparation; any method of administration, etc.) may be omitted from any one or more claims for any reason, related or not. associated with the existence of prior art.

All references cited, such as references, publications, databases, database values, and materials cited herein, are incorporated herein by reference, even if not specifically cited. In the event of a conflict of language in a cited source and in this application, the language in this application shall control.

Section and table headings should not be restrictive.

Examples

Example 1: Analysis with glycan matrices.

Optimized glycan arrays are used to test the affinity and specificity of antibodies against multiple glycans in a single experiment. The glycan arrays include 71 chemically synthesized and well-defined glycans, most of which are the Neu5Ac and Neu5Gc glycan pairs. Array slides are obtained commercially (ArrayIt Corp, Sunnyvale, CA) and include the glycans listed in Table 1. 4.

Table 4 Glycans of the matrix

ID no. Glycan Glycan 1 Neu5,9Ac2a2,3Gaipi,4GlcNAcPO(CH2)2CH2NH2 2 Neu5Gc9Aca2,3Gaipi,4GlcNAcPO(CH2)2CH2NH2 3 Neu5.9Ac2a2.6Gaipi,4GlcNAcPO(CH2)2CH2NH2 4 Neu5Gc9Aca2,6Gaipi,4GlcNAcPO(CH2)2CH2NH2 5 Neu5Aca2,6GalNAcaO(CH2)2CH2NH2 6 Neu5Gca2,6GalNAcaO(CH2)2CH2NH2 7 Neu5.9Ac2a2.3Gal₽l,3GlcNAcPO(CH2)2CH2NH2 8 Neu5Gc9Aca2,3Gal₽l,3GlcNAc₽O(CH2)2CH2NH2 9 Neu5,9Ac2a2,3Gaipi,3GalNAcaO(CH2)2CH2NH2 10 Neu5Gc9Aca2,3Gal₽l,3GalNAcaO(CH2)2CH2NH2 eleven Neu5Aca2,3Gal₽l,4GlcNAc₽O(CH2)2CH2NH2 12 Neu5Gca2,3Gaipi,4GlcNAcPO(CH2)2CH2NH2 13 Neu5Aca2,3Gal₽l,3GlcNAc₽O(CH2)2CH2NH2 14 Neu5Gca2,3Gal₽l,3GlcNAc₽O(CH2)2CH2NH2 15 Neu5 Aca2.3Gal₽ 1.3GalNAcaO(CH2)2CH2NH2 16 Neu5Gca2,3Gal31,3GalNAcaO(CH2)2CH2NH2 17 Neu5 Aca2.6Gal₽ 1.4GlcNAc βΟ(ΟΗ2)2ΟΗ2ΝΗ2 18 Neu5Gca2.6Gal₽ 1.4GlcNAc3O(CH2)2CH2NH2 19 Neu5Aca2,6Gaipi,4GlcPO(CH2)2CH2NH2 20 Neu5Gca2,6Gal₽l,4Glc3O(CH2)2CH2NH2 21 Neu5Aca2,3Gal₽l,4Glc₽O(CH2)2CH2NH2

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22 Neu5Gca2,3Gaipi,4GlcPO(CH2)2CH2NH2 23 Neu5.9Ac2a2.6GalNAcaO(CH2)2CH2NH2 24 Neu5Gc9Aca2.6GalNAcaO(CH2)2CH2NH2 25 Neu5Aca2,3Gal3O(CH2)2CH2NH2 26 Neu5Gca2,3Gal3O(CH2)2CH2NH2 27 Neu5Aca2,6GaipO(CH2)2CH2NH2 28 Neu5Gca2.6Gal3O(CH2)2CH2NH2 29 Neu5.9Ac2a2.3Gal3O(CH2)2CH2NH2 thirty Neu5Gc9Aca2.3Gal3O(CH2)2CH2NH2 31 Neu5.9Ac2a2.6Gal3O(CH2)2CH2NH2 32 Neu5Gc9Aca2.6Gal3O(CH2)2CH2NH2 33 Neu5Aca2,3Gal₽l,3GalNAc₽O(CH2)2CH2NH2 34 Neu5Gca2,3Gal₽l,3GalNAc₽O(CH2)2CH2NH2 35 Neu5.9Ac2a2.3Gal₽l,3GalNAc₽O(CH2)2CH2NH2 36 Neu5Gc9Aca2,3Gal₽l,3GalNAc₽O(CH2)2CH2NH2 37 Neu5.9Ac2a2.6Gaipi,4Glc3O(CH2)2CH2NH2 38 Neu5Gc9Aca2,6Gaipi,4GlcPO(CH2)2CH2NH2 39 Neu5.9Ac2a2.3Gaipi,4Glc3O(CH2)2CH2NH2 40 Neu5Gc9Aca2,3Gaipi,4GlcPO(CH2)2CH2NH2 41 Neu5Aca2,8Neu5Aca2,3Gal₽l,4Glc₽O(CH2)2CH2NH2 42 Neu5Aca2,8Neu5Aca2,8Neu5Aca2,3Gaipi,4GlcpO(CH2)2CH2 NH2 43 Gaipi,4GlcPO(CH2)2CH2NH2 45 Gaipi,4GlcNAcPO(CH2)2CH2NH2 47 GalNAcaO(CH2)2CH2NH2 51 Gal β 1.3 GalNAc3O(CH2)2CH2NH2 52 Gaipi,3GlcNAcaO(CH2)2CH2NH2 53 Gal₽l,3GlcNAc₽O(CH2)2CH2NH2 54 Gal β 1.4GlcN Ac6SP0(CH2)2CH2NH2 55 Neu5 Aca2,3 Gal β 1,4(Fucal ,3)GlcNAc3O(CH2)2CH2NH2 56 Neu5Gca2,3Gaipi,4(Fucal,3)GlcNAcPO(CH2)2CH2NH2 57 Neu5Aca2,3Gaipi,4(Fucal,3)GlcNAc6SPO(CH2)2CH2NH2 58 Neu5Gca2,3Gaipi,4(Fucal,3)GlcNAc6SpO(CH2)2CH2NH2 59 Gaipi,3GlcNAcPl,3Gaipi,4GlcPO(CH2)2CH2NH2 60 Neu5Aca2,3Gaipi,3GlcNAcPl,3Gaipi,4GlcPO(CH2)2CH2NH2 61 Neu5Gca2,3Gaipi,3GlcNAcPl,3Gaipi,4GlcPO(CH2)2CH2NH2 62 Neu5Aca2,3Gaipi,4GlcNAc6SP0(CH2)2CH2NH2 63 Neu5Gca2,3Gaipi,4GlcNAc6SP0(CH2)2CH2NH2 64 Neu5Aca2,8Neu5Aca2,3Gaipi,4GlcpO(CH2)3NHCOCH2(OCH 2CH2)6NH2 65 Neu5Aca2,8Neu5Aca2,8Neu5Aca2,3Gaipi,4GlcpO(CH2)3NHC OCH2(OCH2CH2)6NH2 66 Neu5Aca2,6(Neu5Aca2,3)Gaipi,4GlcPO(CH2)2CH2NH2 67 Neu5Aca2,6(Neu5Gca2,3)Gaipi,4GlcPO(CH2)2CH2NH2 68 Neu5Aca2,6(KDNa2,3)Gaipi,4GlcPO(CH2)2CH2NH2 69 Neu5Gca2,8Neu5Aca2,3Gaipi,4GlcPO(CH2)2CH2NH2 70 KDNa2,8Neu5Aca2,3Gaipi,4GlcPO(CH2)2CH2NH2 71 Neu5Aca2.8Kdna2.6Gaipi,4GlcPO(CH2)2CH2NH2 72 Neu5Aca2,8Neu5Gca2,3Gaipi,4GlcPO(CH2)2CH2NH2 73 Neu5Aca2,8Neu5Gca2,6Gaipi,4GlcPO(CH2)2CH2NH2 74 KDNa2,8Neu5Gca2,3Gaipi,4GlcPO(CH2)2CH2NH2 75 Neu5Gca2,8Neu5Gca2,3Gaipi,4GlcPO(CH2)2CH2NH2 76 Neu5Aca2,8Neu5Aca2,6Gaipi,4GlcPO(CH2)2CH2NH2

300 ml of epoxy blocking buffer is prepared by combining 15 ml of 2 M Tris buffer (pH 8) with 0.9 ml of 16.6 M ethanolamine and 284.1 ml of distilled water. The solution is adjusted to a final pH of 9.0 with HCl. The solution is filtered using a nitrocellulose membrane with 0.2 μm pores. The epoxy buffer solution, as well as 1 liter of distilled water, are preheated to 50°C. Glass slides are assembled in a slide holder and quickly immersed in a staining bath containing heated epoxy blocking buffer. The slides are incubated in epoxy blocking buffer for 1 hour at 50°C with occasional shaking to deactivate the epoxy binding sites. The slides are then washed and blocked with PBS with 1% OVA at 25°C for one hour. Serum samples with polyclonal antibodies (1:1000) or purified monoclonal antibodies (1 μg/ml) were diluted in PBS with 1% OVA and added to the glycan matrices for one hour at 25°C. After extensive washing, antibody binding was detected by incubating glycan array slides with Cy3-conjugated anti-mouse IgG antibodies (Jackson Immunoresearch, West Grove, PA) for one hour. The slides are then extensively washed, dried, and scanned on a Genepix 4000B scanner (laser at 100%; gain at 350; 10-μm pixels). Raw data from scanned images is obtained using Genepix software and raw data analysis is performed. Antibodies are considered highly specific for AcSTn and GcSTn if they demonstrate binding to both molecules, but not to Tn or any other glycans on the matrix.

Based on the results of the matrix assay, antibodies are classified according to their matrix glycan binding profile. Antibodies are classified as group 1 antibodies capable of binding AcSTn and GcSTn if they bind glycans 5, 6, 23 and 24. Such antibodies are called panSTn antibodies due to their ability to bind a wide range of STn structures and the part of STn indicated by the largest ellipse on fig. 1A. Antibodies are classified as group 2 antibodies capable of binding STn, as well as some related structures containing an O-linkage to a serine or threonine residue, if they bind glycans 5, 6, 23, 24, 27 and 31. These antibodies are considered to bind the portion of STn indicated by the largest ellipse in FIG. 1B. Some group 2 antibodies bind AcSTn structures more preferentially than GcSTn structures. Antibodies are classified as group 3 antibodies (capable of binding STn, but also capable of binding a wider group of related structures) if they bind glycans 5, 6, 23, 24, 17, 3, 19, 37, 27 and 31. In contrast from group 2 antibodies, group 3 antibodies do not require such structures to have an O-bond with a serine or threonine residue. Group 3 antibodies are believed to bind the portion of STn indicated by the largest ellipse in FIG. 1C. Finally, antibodies are classified as group 4 antibodies capable of binding both AcSTn and GcSTn, as well as non-sialylated Tn antigen (i.e., having broader specificity) if they bind glycans 5, 6, 23, 24 and 47. that group 4 antibodies bind the portion of STn indicated by the largest ellipse in FIG. 1D.

Example 2. Preparation of mouse anti-STn antibodies.

Antibodies mSIA101 and mSIA102 were generated using variable domains from anti-STn monoclonal antibodies prepared as previously described in US patent publication number US2016/0130356 (the contents of the publication are incorporated herein by reference in their entirety) by immunizing mice with mucins. mSIA103 variable domain sequences were obtained from IgG1 antibody 3F1 (SBH Biosciences, Natick, MA). IgG2a expression vector constructs (plasmid H1206 for antibody heavy chains and plasmid L1206 for antibody light chains, LakePharma, Belmont, Calif.) were modified to encode the variable domains mSIA101, mSIA102, and mSIA103 upstream of the IgG2a constant regions. The sequences of the expressed domains are given in table. 5.

Table 5

Sequences used to produce IgG2a antibodies

Domain Subsequence SEQ ID NO mSIAlOl, VH domain QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQK PEQGLEWIGYFSPGNDDIKYNEKFRGKATLTADKSSSTAYM QLNSLSSDDSAVYFCKRSLSTPYWGQGTLVTVSA 1 mSIAlOl, VL domain DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNRGNHKNYLT WYRQKPGLPPKLLIYWASTRESGVPDRFTGSGSGTDFALTI SSVQAEDLAVYYCQNDYTYPYTFGGGTKLEIKR 2 mSIA102, VH domain QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQK PEQGLEWIGYFSPGNDDIKYNEKFKVKATLTADKSSSTAY MQLTSLTSEDSAVYFCKRSYYGDwgogttltvss 3 mSIA102, VL domain DIQMTQSPASLSVSVGETVTITCRASENIYSHLAWYQQKQG KSPQLLVYGATNLADGVPSRFSGSGSGTQFSLKIHSLQSEDF GSYYCQHFWGAPFTFGSGTKLEIK 4 mSIA103, VH domain QVQLQQSDAELVKPGASVKISCKASGYTFTDHAIHWVKQK PEQGLDWIGYISPGNGDIKYNEKFKDKVTLTADKSSSTACM HLNSLTSEDSAVYFCKRSLLALDywgogttltvss 5 mSIA103, VL domain DIVMTQSHKFMSTSVGDRVSITCKASQDVGTNIAWYQQKP GRSPKVLIYSASTRHTGVPDRFTGSGSGTDFTLTISNVQSED LTDYFCQQYSSFPLTFGVGTKLELK 6

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IgG2a, heavy chain constant domain AKTTAPSVYPLAPVCGDTTGSSVTLGCLVKGYFPEPVTLTW NSGSLSSGVHTFPAVLQSDLYTLSSSVTVTSSTWPSQSITCN VAHPASSTKVDKKIEPRGPTIKPCPPCKCPAPNLLGGPSVFIF PPKIKDVLMISLSPIVTCVWDVSEDDPDVQISWFVNNVEVH TAQTQTHREDYNSTLRWSALPIQHQD WMSGKEFKCKVNN KDLPAPIERTISKPKGSVRAPQVYVLPPPEEEMTKKQVTLTC MVTDFMPEDIYVEWTNNGKTELNYKNTEPVLDSDGSYFM YSKLRVEKKNWVERNSYSCSVVHEGLHNHHTTKSFSRTPG K 13 light chain constant domain RADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKW KIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYER HNSYTCEATHKTSTSSPIVKSFNRNEC 14 kappa

Plasmids encoding full-length heavy chain amino acid sequences and plasmids encoding full-length light chain amino acid sequences were transfected into cells and expressed to produce mature antibodies.

Chinese hamster ovary K1 (CHO-K1) cells were transfected to generate a stable cell line expressing IgG2a antibodies. Cells were cultured in a humidified incubator containing 5% CO2 at 37°C in a chemically defined medium (CD-CHO, Invitrogen, Carlsbad, CA) containing L-glutamine.

Approximately 80 million log-phase CHO suspension cells were transfected by electroporation (MaxCyte) with 80 μg of total plasmid encoding full-length heavy and light chains. Twenty-four hours later, transfected cells were selected for stable integration of antibody genes. During the selection process, cells were pelleted and resuspended in fresh selection medium every 2–3 days until growth rate and viability were restored in the cell pool. Cells were monitored for growth, titer, and stable integration of antibody expression constructs. The doubling period was 20 hours.

Stable cell lines were cultured for large-scale production, and 10 L of culture was obtained. Conditioned medium collected from the stable producing pool of cells was clarified by centrifugation and filtration through a 0.2-μm pore membrane. The antibody was purified by protein A affinity chromatography, then sterilized and cleared of particles by passing through a 0.2-μm pore membrane filter. After purification from endotoxin impurities and filtration, the concentration was adjusted to 5 mg/ml and 120 mg of antibody was recovered.

To prepare antibody-drug conjugates (ADCs), antibodies were conjugated to monomethyl auristatin E (MMAE). Conjugation was performed by bringing the antibodies into contact with maleimidocaproyl-valine-citrulline-p-aminobenzyloxycarbonyl-monomethyl auristatin E (MC-vc-PABMMAE, herein referred to as CL-MMAE). The resulting conjugation is based on a maleimide-cysteine bond, where the interchain disulfide bonds of the antibody have been reduced by TCEP and then linked to the maleimide moiety of the drug.

Conjugated antibodies were desalted on Sephadex G50 columns to remove residual unreacted toxins and then dialyzed in 30 mM HEPES, pH 7.7, with 150 mM NaCl. Size exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC) were used to determine drug-antibody ratio (DAR).

Example 3: Characteristics of Mouse Antibodies and ADCs.

The murine antibodies were characterized for in vitro and in vivo effectiveness in breast, colon and ovarian cancer models. Antibodies were used to identify a CRC cell line expressing STn on the surface. Five CRC cell lines were incubated in vitro and grown to confluence. STn expression, in %, was determined by flow cytometry using anti-STn antibodies. Approximately 30-70% of cultured SW403, COLO205 and LS174T cells naturally expressed STn on their surface (as determined using mSIA103), while HT29 and RKO had very little or undetectable amounts of STn (as determined using hSIA101) on its cell surface (see Table 6).

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Table 6

In addition, a tissue micropanel (TMA) of CRC (NBP2-30214; Novus Bio, Littleton, CO) was stained for STn expression. The matrix consisted of 59 samples, with and without treatment with the enzyme neuraminidase (sialidase). Neuraminidase specifically cleaves terminal sialic acids and destroys the STn antigen present in the tissue. Of the 51 neoplastic specimens on TMA, 45 had some degree of positive membrane staining, and anti-STn mSIA103 staining was absent on all 45 when treated with 250 mU/ml neuraminidase. Staining with a vimentin-specific antibody was ineffective, suggesting that binding of the anti-STn antibody mSIA103 was specific for sialic acid. Staining of normal colon samples (8 samples) was limited to the crypts and superficial mucosa, as well as the apical side of the cells, which should be protected from intravenously administered therapeutic ADC.

Preliminary xenograft studies using COLO205 cells were conducted to evaluate the in vivo efficacy of murine antibody-drug conjugates (ADCs). The COLO205 subcutaneous xenograft model was generated by injecting 5x106 cells/mouse into the right flank of athymic nude mice. Treatment began when the tumors reached an average size of 180 mm ³ . Mice were administered a once-weekly dose of 5 mg/kg for three weeks. Only mSIA103 without toxin, mSIA103-CL-MMAE, mSIA103 conjugated to monomethyl auristatin F (MMAF) via a non-cleavable linker, and a solvent control were evaluated in this study. mSIA103-CL-MMAE demonstrated significant tumor growth inhibition with a treatment-to-control (T/C) ratio of 44.5% (p=0.008) compared with the other three groups.

Example 4. Preparation of humanized anti-STn antibodies.

Humanized variants of the variable domains mSIA101, mSIA102 and mSIA103 were generated by introducing a CDR into the germline sequence of a human antibody by a method called CDR grafting. The resulting variable domains were used to produce humanized antibodies hSIA101, hSIA102 and hSIA103 by expressing constructs encoding the humanized variable domains with human IgG1 constant domains. The sequences of the variable domains and constant domains of the expressed antibodies are given in table. 7.

Table 7 Humanized variable domains

Antibody Domain Subsequence SEQ ID NO hSIAlOl VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWVR QAPGQGLEWMGYFSPGNDDIKYNEKFRGRVTMTADKS SSTAYMELRSLRSDDTAVYFCKRSLSTPYWGQGTLVTV SS 7 hSIAlOl VL DIVMTQSPDSLAVSLGERATINCKSSQSLLNRGNHKNYL TWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFT LTISSLQAEDVAVYYCQNDYTYPYTFGQGTKVEIK 8 hSIA102 VH QVQLVQSGAEVKKPGASVKISCKASGYTFTDHAIHWVR QAPGQGLEWIGYFSPGNDDIKYNEKFKVRATLTADKSSS TAYMELRSLRSDDTAVYFCKRSYYGDWGQGTLVTVSS 9 hSIA102 VL DIQMTQSPSSLSASVGDRVTITCRASENIYSHLAWYQQK PGKAPKLLVYGATNLASGVPSRFSGSGSGTQFTLTISSLQ PEDFAT YYCQHFWGAPFTFGQGTKVEIK 10 hSIA103 VH QVQLVQSGAEVKKPGASVKVSCKASGYTFTDHAIHWV RQAPGQGLEWMGYISPGNGDIKYNEKFKDRVTMTADK SSSTAYMQLRSLRSDDTAVYFCKRSLLALDYWGQGTLV TVSS eleven

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hSIA103 VL DIQMTQSPSSLSSASVGDRVTITCKASQDVGTNIAWYQQK PGKAPKVLIYSASTRHTGVPSRFSGSGSGTDFTLTISSLQP EDFAT YFCQQYS SFPLTFGQGTKVEIK 12 Human IgGl Constants domain ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVS WNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ 15 severe chains TYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELL GGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL PPSRDELTKNQVSLTCLVKGFYPSDIAVE WESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVM HEALHNHYTQKSLSLSPGK Human IgGl Light chain constant domain RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV QWKVДHKLQSGNSQESVTEQDSKDSTYSLSSTLTLSKAD YEKHKVYACEVTHQGLSSPVTKSFNRGEC 16

The preparation of humanized antibody-drug conjugates (ADCs) with MMAE was performed using the same methods as previously described for murine antibodies.

Example 5: Characteristics of Humanized Antibodies.

The characteristics of the humanized IgGl antibodies prepared as described above were obtained using a flow cytometry-based binding assay on MDA-MB-231STn cells; BSM ELISA binding assay and glycan array assay.

Flow cytometry-based binding studies screened antibodies over a concentration range from 0 to 300 nM, comparing binding to MDA-MB-231 cells with or without transfection-induced STn expression. Binding was determined using an APC-conjugated anti-human immunoglobulin secondary antibody, and only live cells were considered (by setting a negative discrimination window on propidium iodide staining). On average, 5000 events were collected for each sample. Data were analyzed using FlowJo software (Asland, OR) and mean values for APC and % APC were obtained. These data were logarithmically transformed and then a nonlinear regression model was fitted to obtain binding information to construct a dose response curve and determine the half-maximal effective concentration ( _EC5o ). Human IgGl antibody was used as a negative isotype control. Epidermal growth factor receptor (LA22, EMD Millipore, Billerica, MA) was used as a positive control.

For the BSM ELISA, antibodies were screened at concentrations ranging from 0 to 100 nM in bovine submandibular mucin (BSM)-coated wells. A set of wells was treated with a weak periodate solution before adding the antibody to remove the side chain on the terminal sialic acid residues (destruction of the STn antigen). Optical density in periodate-treated and non-periodate-treated wells was determined, logarithmically transformed, and then a nonlinear regression model was fitted to obtain a dose-response curve. Absorbance values obtained for periodate-treated wells were subtracted from those for non-periodate-treated wells to obtain a periodate-sensitive STn binding curve and corresponding EC ₅₀ values.

The glycan array assay was performed as previously described, and the glycan matrix binding profiles of the antibodies were determined based on the results obtained.

The results of flow cytometry, ELISA and glycan array analysis are shown in Table. 8.

Table 8

Antibody Characterization Results

Clone MDA-MB-231-STn Cell Binding [EC ₅₀ (nM)] BSM ELISA [ECso (nM)1 Glycan binding profile in the matrix hSIAlOl 2.0 4.2 Group 1 hSIA102 0.1 undefined Group 4 hSIA103 0.3 1.8 Group 1

All antibodies tested showed binding to cell-associated STn and BSM. Binding was absent with human IgGl isotype controls (Southern Biotech, Birmingham, AL). hSIA102 binding was not sensitive to periodate treatment in ELISA assays,

-98045483 therefore the EC50 could not be reliably determined in the BSM ELISA.

Example 6: Analysis of Humanized Antibody-Drug Conjugates.

The MMAE-conjugated ADC variants hSIA101, hSIA102 and hSIA103 were evaluated in an ADC cytotoxicity assay using MDA-MB-231 cells (parental or transfected to overexpress STn). Parental cells were grown in Eagle's minimal essential medium (EMEM) supplemented with 10% FBS, 1x penicillin/streptomycin, and 45 μg/ml gentamicin. STn-positive cells were grown in the same medium except for the addition of 1 mg/ml G418 as a selective antibiotic. Cells were seeded individually (4000 cells/well for parental cells or 2000/well for STn-positive cells) in 96-well plates using the appropriate media described above. The cells grew overnight. After 16-20 hours, cells were treated in triplicate with test antibodies at different concentrations (50 nM - 0.012 nM) for 72 hours. Cells were then analyzed using the ADC CELLTITER-GLO® Luminescent Cell Viability Assay Kit (Promega, Madison, WI) to determine the amount of ATP present, an indicator of metabolically active cells. The assay uses a single reagent that is added directly to cultured cells in serum-containing media. The reagent lyses the cells and generates a luminescent signal proportional to the amount of ATP present. Luminescent signals were analyzed and used to calculate half-maximal inhibitory concentration (IC ₅₀ ) values for each antibody used based on the concentration required to kill STn-positive cells at half the maximum level (see Table 9).

Table 9 ______IC ₅₀ values for ADC humanized antibodies______

Humanized Antibody 1C ₅₀ (nM) hSIA103 1.3 hSIA102 2.6 hSIAlOl 5.2

All antibodies tested had IC ₅₀ values in the single nanomolar range, indicating a strong ability of each to kill STn-expressing cells.

Similar studies were performed using OVCAR3 cells in culture. Doses of hSIA101-MMAE and hSIA102-MMAE from 5 pM to about 300 nM were used and an IC ₅₀ value of 29 nM was obtained for hSIA101-MMAE and an IC ₅₀ value of 15 nM for hSIA102-MMAE.

Example 7 STn Assays.

ELISA assays are being developed to identify and quantify STn levels in samples from subjects. Bovine submandibular mucin (BSM), which is highly plated, is used to generate standard curves and to optimize the assay. Initial assays use commercially available mouse anti-STn antibodies [eg, 3F1 antibody (SBH Sciences, Natick, MA), B72.3 antibody (see Colcher, D. et al., 1981. PNAS. 78(5): 3199203) and antibody CC49 (see Muraro, R. et al., 1988. Cancer Res. 48: 4588-96)] to coat assay plates and capture BSM in test samples (buffer solutions or serum samples spiked with BSM). Humanized anti-STn antibodies (eg, hSIA101, hSIA102 and hSIA103) are used to detect captured BSM. Antibodies used for capture and detection are tested in two ways. One is to test for matching pairs in sandwich ELISA assays. The second is flow cytometry testing for competitive binding using fluorescent antibody tags and alternating the order of exposure for STn-expressing cells. Antibody pairs that do not compete for binding are used for further assay development.

The following assays use anti-STn antibodies as capture antibodies coupled to assay plates and species-specific detection antibodies to detect bound proteins. For example, human anti-STn mAbs can be used as both coating and detection mAbs if the detection mAbs are directly labeled (eg, Alexa-488, HRP, etc.). For example, mouse anti-STn mAbs can be used as both coating and detection mAbs if the detection mAbs are directly labeled (eg, Alexa-488, HRP, etc.). Detection of STn in samples from mice (eg, collected in the xenograft model studies described herein) and in samples from humans (eg, serum samples obtained as described herein) is assessed with both mouse and human detection antibodies .

Example 8 STn Assay Using Humanized Anti-STn Antibodies.

An STn ELISA was performed using mSIA102 as the capture antibody and hSIA102 or hSIA103 as the detection antibody and isotype-controlled human IgG as the control detection antibody. Solutions containing five micrograms per milliliter (μg/ml) of capture antibody were used to coat the wells of ELISA plates

- 99 045483 overnight. The plates were washed and blocked, followed by treatment with serially diluted bovine submandibular mucin (BSM) at concentrations ranging from 0 nM to 6x10-8 nM. BSM bound to the analytical surface was detected using detection antibody at a concentration of 3 μg/mL. Horseradish peroxidase-conjugated anti-human immunoglobulin antibody (0.08 μg/ml) was used to bind detection antibodies, and antibody-antigen complexes were detected using HRP substrate. The results showed that humanized anti-STn antibodies were able to detect bound BSM with a half-maximal effective concentration (EC ₅₀ ) for antibody binding of 9x10-9 nM for hSIA103 and 2x10-8 nM for hSIA101. There was no binding with the isotype-control detection antibody.

Example 9 Pharmacokinetic studies.

Antibodies hSIA101 and hSIA102, both conjugated to MMAE, were administered to female nude mice at a dose of 5 mg/kg (dose determined gravimetrically) by intravenous (IV) tail vein injection to assess the terminal elimination half-life of the antibody. Each group consisted of 9 mice. Blood (serum) was collected at 1, 4, 8, 24, 48, 72, 168, 336, and 672 hours and used to calculate the half-life of the antibody.

Pharmacokinetic (PK) parameters were assessed at the end of the study using the WINNONLIN® program (Pharsight Corp., Mountain View, CA). The area under the curve (AUC) was assessed from the start of the first dose to the time after the last quantifiable observed concentration (AUC _last ) and from the start of the study to infinity (AUCNF). The maximum observed plasma concentration (C _max ), observed clearance (CL), terminal elimination half-life (HL), steady-state volume of distribution (Vss), and terminal volume of distribution (V _z ) were also determined. PK parameters were calculated using non-compartmental analysis with sparse sampling. The results are shown in table. 10.

Table 10 FC parameters

Antibody Spach (µg/ml) AUC _last (day*mcg/ml) auc _№F (day*µg/m l) CL (ml/day/kg) HL (days) v _z (ml/kg) Vss (ml/kg) hSIAlOl- MMAE 254 1050.00 1050.00 4.77 2.55 17.51 20.07 hSIA102- MMAE 285 1075.00 1079.17 4.63 3.77 25.16 23.57

In the study, hSIA101-MMAE was determined to have a terminal elimination half-life of 2.55 days, while hSIA102-MMAE was determined to have a terminal elimination half-life of 3.77 days. These values were comparable to those known for other antibody-drug conjugates (see, for example, Leal, M. et al., 2015, Bioconjugate Chem, 26: 2223-32, which reports that the terminal elimination half-life for anti-5T4 antibody-drug conjugate is 3.5 days).

Example 10. Generation of cell lines overexpressing STn.

Stabilized cancer cell lines are genetically modified to stably express STn as described by Julien et al. (Mien, S. et al., 2005. Breast Cancer Res Treat. 90(1): 77-84). Cells are transduced with lentiviral vectors delivering ST6GalNAc I expression constructs (hST6GalNAc I pRc-CMV) or a control vector (empty vector pRc-CMV). Stable transfectants are selected in medium containing geneticin 418 (G418, 1 mg/ml). Resistant cells are seeded into 96-well plates using a serial dilution strategy and subcloned three times to obtain stable clonal lines. Clonal lines exhibit different levels of ST6GalNAc I expression [as determined by quantitative polymerase chain reaction (qPCR) analysis]. OVCAR3 cell lines that stably express additional amounts of STn (compared to wild-type cells) are called OVCAR3-STn cells. Successful expression of STn was confirmed by flow cytometry analysis using anti-STn antibodies.

Example 11. Generation of SKOV3 cell lines with increased expression of ST6GalNAc I.

SKOV3 cells were transduced with lentiviral vectors delivering ST6GalNAc I expression constructs (hST6GalNAc I pRc-CMV). Stable cell pools were obtained and 6 clones were selected with varying levels of ST6GalNAc I expression [as determined by quantitative polymerase chain reaction (qPCR) analysis] (see Table 11).

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Table 11

Expression levels of ST6GalNAc I in selected clones

Clones 7, 8, and 13 exhibited the highest levels of ST6GalNAc I mRNA when compared with levels in non-transduced cell lines.

SKOV3 clones not expressing (wild type), moderately expressing (clone 15), and highly expressing (clone 13) STn were tested for sensitivity to anti-STn antibody treatment. Cells were cultured and treated with vehicle control, hSIA101, hSIA101-MMAE, hSIA102, or hSIA102-MMAE at concentrations of 2.5 nM, 5 nM, 10 nM, 50 nM, and 100 nM. Cell viability was then determined by MTT assay (EMD Millipore, Billerica, MA). Cell viability was reduced by treatment with hSIA101-MMAE and hSIA102-MMAE at all doses in the case of cells expressing high levels of STn, and at doses above 2.5 nM in the case of cells expressing moderate levels of STn. Cell viability was reduced by treatment with hSIA101-MMAE and hSIA102-MMAE at all doses for cells expressing moderate levels of STn and at doses above 5 nM for cells expressing moderate levels of STn. In cells not expressing STn, hSIA101MMAE and hSIA102-MMAE had little effect except at the highest dose used. These results indicate that sensitivity to anti-STn antibody treatment depends on the level of STn expression.

Example 12. In vivo study of xenografts obtained from OVCAR3 cells in mice.

A subcutaneous xenograft murine model in athymic nude mice was generated using human OVCAR3 ovarian cancer cells. Animals were treated with humanized anti-STn ADC (1 mg/kg, 2.5 mg/kg, 5 mg/kg), isotype control ADC (1 mg/kg, 5 mg/kg), paclitaxel (20 mg/kg), or solvent only. ADC was administered intravenously once a week for four weeks. Paclitaxel was administered intraperitoneally once a week for three weeks. Tumor volume and body weight were measured on the days indicated in the table. 12. The humanized ADC caused a significant reduction in tumor volumes in comparison with the solvent and control ADC isotype (see Table 12), although it had a minor effect on total body weight. These data suggest that antiSTn ADCs are nontoxic in vivo and are effective in reducing the volume of ovarian serous tumor xenografts. After completion of the study, tumor tissues were collected and analyzed by immunohistochemical methods for STn expression. Tumor tissues from mice treated with anti-STn ADCs showed reduced expression of STn compared to tissues from animals treated with other drugs.

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Table 12

Volume of tumors

Impact Average tumor volume (mm ⁵ ) Day 1 Day 4 Day 9 Day 12 Day 15 Day 18 Day 23 Day 26 Day thirty Day 31 Solvent 161.8 242.8 373.5 539.1 678.4 803.1 987.1 1039.3 1175.6 1224.0 hSIAlOl- MMAE, 1 mg/kg 161.7 228.9 347.7 448.2 551.5 609.0 768.4 882.7 937.1 966.8 hSIAlOl- MMAE, 2.5 mg/kg 161.9 213.2 275.8 302.6 313.5 299.7 234.0 195.9 169.7 169.1 hSIAlOl- MMAE, 5 mg/kg 161.7 203.4 211.0 193.4 165.0 131.1 77.8 59.4 39.5 39.1 hSIA102- MMAE, 1 mg/kg 161.7 219.7 335.5 465.7 567.1 707.1 871.9 897.0 957.7 984.8 hSIA102- MMAE, 2.5 mg/kg 161.8 202.9 291.8 358.5 399.5 420.8 414.3 370.2 384.0 384.7 hSIA102- MMAE, 5 mg/kg 161.7 210.7 210.6 187.4 168.7 127.2 84.0 67.3 50.5 46.8 Isotype- MMAE, 1 mg/kg 161.1 222.1 312.6 368.8 423.1 468.3 510.6 472.5 531.4 548.2 Isotype- MMAE, 5 161.4 196.5 255.9 241.8 205.9 176.9 127.1 94.3 78.0 74.0 mg/kg Paclitaxel, 20 mg/kg 161.2 210.0 370.0 482.3 620.4 767.4 971.6 1133.0 1277.8 1385.2

Example 13 STn expression in CRC patient samples.

Samples from patients with primary CRC were analyzed by flow cytometry with hSIA101 and two commercial antibodies, B72.3 and CC49. All 10 samples tested were STn positive. The results are shown in table. 13. In the table, % STn is expressed as % viable CD45-/CD34- population. STn expression was often more reliably determined using hSIA101 than using commercial antibodies. The expression-positive population ranged from 3-47%, with the metastatic sample having the highest percentage of positive cells. This indicated that anti-STn antibodies could be used to identify STn-specific populations in tumor samples.

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Table 13

STn expression in patient samples

Sample Original fabric Antibody %STn+ 160093-2 Colon SIA201a 22.9 V72.3 18.9 SS49 17.1 160101-2 Colon SIA201a 10 V72.3 5.8 SS49 5 160102-2 Colon SIA201a 12.9 V72.3 14.7 SS49 11.4 160115-2 Colon SIA201a 5.4 B72.3 13 CC49 5.7 160127-2 Colon SIA201a 4.2 B72.3 14.4 CC49 3.5 160149-1 Colon SIA201a 22.7 B72.3 10.5 CC49 9.8 160151-1 Colon SIA201a 19.6 B72.3 6.8 CC49 11.5 169068-1 Colon SIA201a 10.9 B72.3 7.4 CC49 8.4 1601666 Colon SIA201a 29.4 B72.3 17.3 CC49 20.6 160104-2 Metastasis from the colon to the liver SIA201a 47.8 B72.3 46.6 CC49 39.4

Example 14: In vitro toxin testing on CRC cell lines.

Based on preliminary flow cytometry analyzes showing that approximately 60-70% of the total cell population in the COLO205 and LS174T lines expresses STn, in vitro assays are being conducted to test anti-STn antibody therapies. Given the preliminary results for murine and humanized anti-STn-MMAE ADCs, experiments are being conducted to identify optimal toxins/linkers for anti-STn ADCs for the treatment of colorectal cancer. Resistance of 8 CRC lines (COLO205, LS174T, RKO, HT-29, LSI80 and SW403), as well as cell lines with modified STn expression [COLO205 STn-knockdown, LS174T STn-knockdown, RKO STn+ (ST6GalNAc I overexpression) and HT- 29 STn+ (ST6GalNAc I overexpression)] having different levels of STn expression are studied using a panel of 10 common ADC toxins, including auristatins (MMAE and MMAF), maytansines (DM4 and DM1), pyrrolobenzodiazepine dimers (thalirine and tesirine), and topoisomerase inhibitors (SN-38 and DXd), as well as other natural remedies (duocarmycin and amanitin). The sensitivity of CRC is determined in comparison with the standard treatment drug (irinotecan, also known as CPT-11) and vehicle alone to determine the half-maximal inhibitory (cytotoxic) concentration (IC ₅₀ ). To accomplish this, non-conjugated toxins are tested on CRC cell lines with varying levels of endogenous STn expression, as well as derivatives of these cell lines in which STn expression levels are either increased or absent.

Knockdown of STn expression in COLO205 and LS174T cells is performed using ST6GalNAc I siRNA silencer (ThermoFisher Scientific, Waltham, MA) by methods described in the literature (e.g., Gao et al., Nat Med. 2015 Nov; 21(11): 1318-25). In contrast, RKO and HT-29 cells (expressing small or undetectable amounts of STn) are transduced with lentiviral vectors delivering ST6GalNAc I expression constructs, yielding clone

- 103 045483 nal populations of cells expressing STn at elevated levels. Genetically modified cells are monitored for cell surface STn expression by flow cytometry and Western blot analysis, and ST6GalNAc I mRNA levels are monitored by qRT-PCR. Non-conjugated toxins are added to CRC cell lines, obtaining IC ₅₀ values at 24, 48, 72 and 96 hours post-incubation to determine sensitivity. The CELLTITER-GLO® Luminescent Cell Viability Assay Kit (Promega, Madison, WI) is used according to the manufacturer's instructions to assess cell viability after treatment. Two toxins with desirable ADC properties are selected for conjugation to the antibody and further characterization.

Example 15: Evaluation of anti-STn ADC in CRC models.

The two toxins are conjugated to two selected humanized anti-STn antibodies, and binding assays are used to confirm retention of STn binding specificity. Binding assays (flow cytometry, ELISA, and glycan array assays) were performed as previously described. One human IgG1 isotype antibody is separately conjugated to each of the two toxins and used as a negative control for binding. Antibodies to EGFR (clone LA22) and CEA (clone CD66e) were used as positive controls for binding to MDA-MB-231 and CRC cell lines, respectively.

The anti-STn ADC was tested against the three CRC cell lines described above for in vitro efficacy. A total of 8 conditions are tested, including: anti-STn 1+toxin 1; anti-STn 1+toxin 2; antiSTn 2+toxin 1; anti-STn 2+toxin 2; isotype+toxin 1; isotype+toxin 2; standard preparation and solvent. Anti-STn ADCs are assessed for their effect on cell viability and cell colony formation using viability assays and soft agar cell growth assays, respectively. Viability assays were performed using the CELLTITER-GLO® Luminescent Cell Viability Assay Kit (Promega, Madison, WI). The relative potency of the ADC is calculated according to the manufacturer's instructions, yielding percentage pot life and IC ₅₀ values. Colony formation assays are performed in which cells are diluted in methylcellulose-based medium and grown at 37°C for 5-14 days in the presence of ADC. The number of colonies is counted and compared. In both assays, anti-STn ADCs are tested over a range of concentrations and the corresponding IC ₅₀ values are obtained. The results are compared with those for the standard chemotherapy agent and ADC isotype controls. The two best ADCs are selected based on IC ₅₀ data and colony formation information.

TMAs for CRC xenotransplantation are evaluated with respect to STn expression to identify two STn-positive CRC models for in vivo studies. The panel consists of 20 unique models, including models for different stages, subtypes, genetic background, and treatment-responsive/resistant models. STn expression is assessed by immunohistochemical (IHC) methods. TMAs are stained with two anti-STn antibodies, an isotype control, and a secondary antibody only. IHC detection is performed using a pre-chelation strategy where the antibody is incubated with a biotin-labeled secondary anti-human IgG antibody and then incubated with tissue. Staining is scored to assess neoplastic cell membrane-specific staining. Two CRC xenograft models and one anti-STn ADC are selected for in vivo xenograft studies.

The anti-STn ADC candidate is evaluated for in vivo efficacy in a multiple-dose single-drug study using two cell lines and three dose concentrations (1, 2.5 and 5 mg/kg). Mouse xenograft models are generated by subcutaneously injecting STn-expressing human CRC cells (i.e., 5x106 cells of 1:1 (v/v) Matrigel) into the right flank of ICR SCID mice (~80 mice). When the tumors in 60 mice reach an average volume of ~200 mm ³ , the animals are randomized into 6 groups (n=10 mice per group) with approximately the same average tumor sizes in the groups. Animals in the groups received either anti-STn ADC (1, 2.5 or 5 mg/kg), isotype control ADC (5 mg/kg), standard treatment (irinotecan 40 mg/kg), or vehicle (DPBS). The drugs are administered by intravenous injection once a week for four weeks based on previously obtained PK studies of the anti-STn-MMAE ADC. Tumor volume and body weight are measured twice weekly, and individual mice are sacrificed when tumor size reaches the target end size (>1500 mm) for the vehicle control group or when the mice are moribund. Mice from each group are assessed for tumor STn expression by IHC and flow cytometry after completion of the study.

Based on the results of these studies, the toxin/linkers are further optimized to enhance the in vivo and in vitro efficacy of the anti-STn ADC.

Example 16. Selection of CRC PDX models.

Micropanels (TMAs) of CRC tumors from patient-derived tumor (PDX) cells are assessed for STn expression using humanized anti-STn antibodies to identify susceptible models for PDX studies. Well characterized commercial dos

- 104 045483 blunt formalin-fixed paraffin-embedded (FFPE) PDX TMA (CrownBio, Santa Clara, CA), comprising a total of over 200 samples, were analyzed for STn expression by IHC methods. These TMAs consist of a variety of untreated and treatment-resistant PDX models, including those resistant to AP24534 [ponatinib, (RET inhibitor)], cetuximab, bevacizumab (AVASTIN®), trastuzumab (HERCEPTIN®), sorafenib, 5- fluorouracil, cisplatin, docetaxel, gemcitabine, irinotecan or paclitaxel. TMA is stained with an unconjugated anti-STn antibody, an isotype control antibody, or a secondary antibody alone. All TMA IHC assays are scored by microscopic examination in a blinded manner for antibody staining intensity, frequency, and location of staining. Correlations between STn expression and CRC progression are studied and used to select models.

Based on the IHC data, 10 PDX are selected for in vitro testing of the anti-STn ADC candidate. These 10 PDX models are evaluated for cytotoxicity and colony formation in the viability and colony formation assays described previously. The analysis includes a control ADC, three standard treatment drugs (irinotecan, cetuximab, and bevacizumab, as separate agents), and a vehicle control. Three PDX models [including 1 standard-of-care (SOC) resistant PDX model] are selected for in vivo studies based on STn expression and in vitro response to anti-STn ADC.

Example 17 Single dose PK studies.

Prior to in vivo studies, the anti-STn ADC candidate is evaluated for PK properties in an in vivo single-dose study in mice. This single-dose study uses two dose levels (2.5 and 5 mg/kg) and 10 time points (0, 1, 4, 8, 24, 48, 72, 168, 336, and 672 hours post-dosing). A total of nine mice are included in each treatment group, with three subgroups of mice for a staggered blood sample collection schedule. At scheduled time points, blood samples are collected from mice and serum is collected in serum separation tubes. The blood is allowed to clot for a minimum of 30 minutes, followed by centrifugation (3500 rpm for 10 minutes at 5°C) within 1 hour of sample collection. After centrifugation, the serum is separated and used in immunoassays to determine the anti-STn ADC present in the serum. The terminal elimination half-life (HL), area under the curve (AUC), maximum observed plasma concentration ( _Cmax ), steady-state volume of distribution ( _Vss ) and terminal phase volume of distribution ( _Vz ) are determined. PK parameters are used to determine the optimal dosing regimen for in vivo PDX studies.

Example 18: In Vivo Efficacy Assessment in PDX Mouse Models.

PDX studies in mice are conducted using three PDX models identified by PDX TMA immunohistochemistry to evaluate the in vivo efficacy of anti-STn ADC single-agent therapy candidates. PDX mice randomized into 6 groups (10 mice per group) were treated with anti-STn ADC at 1, 2.5 or 5 mg/kg, isotype control ADC (5 mg/kg), standard treatment (irinotecan). 40 mg/kg), cetuximab (10 mg/kg) or bevacizumab (10 mg/kg)), or vehicle only. Doses are administered to animals once a week for four weeks. STn expression is determined at baseline and after completion of the study using IHC and flow cytometry.

Example 19: Combination Treatment in PDX Mouse Models.

Anti-STn ADC candidates are being evaluated for combination therapy in combination with standard of care (SOC) in a selected PDX mouse model using either co-administration or sequential drug administration.

For co-administration, a total of 9 treatment groups are tested, with 10 mice per group, including: vehicle only, anti-STn ADC (1 mg/kg), anti-STn ADC (5 mg/kg), SOC only, SOC+ vehicle, SOC+anti-STn ADC (1 mg/kg), SOC+anti-Sτn ADC (5 mg/kg), SOC+non-conjugated anti-STn (1 mg/kg) and SOC+non-conjugated anti-STn ( 5 mg/kg). Animals are dosed weekly for four weeks. STn expression in tumors is determined at the beginning of the study and after completion of the study using IHC and flow cytometry.

In the case of sequential administration, mice first receive SOC. After treatment for four weeks, or after the average tumor size has decreased to <25% of the initial tumor volume, mice are randomized into 6 groups as follows: anti-STn ADC (1 mg/kg), anti-STn ADC (5 mg/kg ), non-conjugated antibody (1 mg/kg), non-conjugated antibody (5 mg/kg), one isotype control ADC and vehicle control. Animals are dosed weekly for four weeks. STn expression in tumors is determined at the beginning of the study and after completion of the study using IHC and flow cytometry.

Example 20: Repeated dose pilot toxicology study.

A multiple-dose study was conducted to evaluate the pharmacokinetic characteristics and toxicity of hSIA101-MMAE administered to cynomolgus monkeys. Studies included lifetime assessments such as mortality/morbidity, clinical observations, body weight,

- 105 045483 food intake, body temperature, local irritation, ophthalmological signs, as well as assessment of clinical pathology. These studies used 9 males (age 2-4 years), 3 monkeys per treatment group, see Table. 14.

Table 14 Treatment groups

Group Dose (mg/kg) Dose volume (ml/kg) Dose concentration, (mg/ml) Animals in group 1 1 1.2 0.833 3 2 3 1.2 2.5 3 3 6 1.2 5 3

Animals in all groups were dosed a total of 2 times by intravenous bolus injection, once on day 1 and again on day 22. Blood samples were collected for pharmacokinetic studies on days 1 and 22 using the following schedule: pre-dose (0) and then thereafter. 1, 3, 6, 24, 48, 72, 96 and 168 hours after dosing. Additional blood samples were collected at 240, 336, and 504 hours post-dosing on Day 1 of dosing only. Blood samples were collected for clinical pathology assessment (clinical chemistry, hematology, and coagulation) using the following schedule: predose (pretest), day 8, day 22 (before second dose), and day 29 before euthanasia. Clinical biochemical parameter assessments included sodium, creatinine, total protein, potassium, alkaline phosphatase, triglycerides, chloride, alanine aminotransferase, total bilirubin, calcium, aspartate aminotransferase, albumin, inorganic phosphorus, glucose, globulin, urea nitrogen, cholesterol, and albumin/globulin ratio. . Hematologic assessments included hematocrit, mean red cell hemoglobin concentration, hemoglobin, reticulocyte count (absolute and relative), platelet count, red blood cell count, mean platelet volume, total white blood cell count, mean red blood cell hemoglobin, differential white blood cell count (absolute and relative). relative), average erythrocyte volume and relative width of distribution of erythrocytes by volume. K3-EDTA was used as an anticoagulant. Coagulation assessments included assessment of prothrombin time and activated partial thromboplastin time. All groups of animals were euthanized on day 29, and organs were collected for study.

Sera from subjects' blood samples were used for pharmacokinetic (PK) assessment of human IgG levels. PK parameters were assessed using Phoenix pharmacokinetic analysis software (Certara, USA) using a non-compartmental approach consistent with the intravenous bolus route of administration. All parameters were derived from individual human IgG serum concentrations obtained on days 1 and 22. Parameters were estimated using nominal sample collection times relative to the end of each dose. The concentration at time zero on day 1 was back extrapolated from the first two observed serum concentrations for parameter estimation purposes. Samples with unquantifiable concentration values were considered missing samples.

Serum antibody concentrations obtained relative to the first dose are presented in FIG. 2 and the values obtained relative to the second dose are presented in FIG. 3. The area under the concentration-time curve (AUC) was calculated using the linear trapezoidal method with linear/linear interpolation. AUC was not calculated for PK profiles with less than 3 quantifiable test substance concentrations at individual time points. When appropriate, a curve of the final elimination phase of each concentration versus time was generated using at least the last three concentration values observed. The slope of the curve for the final elimination phase was determined using log linear regression on unweighted concentration data. Average values with standard deviation (SD; in parentheses) are given in table. 15 and table. 16. C _max is the maximum observed concentration measured after dosing.

AUCi _ast is the area under the concentration-time curve from the start of the dose to the last observed quantifiable concentration using the linear or linear/log trapezoidal method. AUC _{day 0-7} is the same but does not include the area after day 7. AUC _INF is the area under the concentration-time curve from dosing initiation to time extrapolated to infinity. CL represents the apparent clearance rate of the antibody from the assay matrix. HL represents the apparent terminal half-life. Vss is the apparent volume of distribution at steady state.

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Table 15

PK parameters obtained after the first dose

Dose (mg/kg) Rate (µg/ml) Mean (SD) AUC _last (day*µg/m l) Mean (SD) AICo-7 _day (day*µg/ml) Average (SD) auc _№F (day* µg/m l) Mean (SD) CL (ml/kg/day) Mean (SD) HL (days) Average (SD) v _ss (ml/kg) Average (SD) 1 19.13 (1.78) 57.31 (7.5) 42.6 (0.96) 62.62 (7.81) 16.13 (1.96) 4.66 (1.43) 96.83 (17.29) 3 81.24 (11.17) 228.39 (25.02) 157.34 (4.94) 267.39 (56.39) 11.57 (2.52) 7.47 (3.91) 96.16 (30.64) 6 151.37 (20.97) 419.05 (50.18) 342.22 (32.58) 440.63 (28.26) 13.65 (0.86) 3.53 (051) 61.62 (10.98)

Table 16

PK parameters obtained after the second dose

Dose (mg/kg) Stach (mcg/ml); Mean (SD) AUCiast (day*µg/ml) Mean (SD) 1 22.16 (0.01) 41.59 (4.68) 3 75.27 (5.18) 145.48 (51.93) 6 117.23 (45-7) 172.36 (141.18)

On day 1, the increase in systemic exposure of human IgG was linear and dose proportional at doses of 1-6 mg/kg/dose, with a slight trend towards being more than dose proportional. At day 22, the increase in systemic exposure was nonlinear at doses of 1-6 mg/kg/dose. A decrease in systemic exposure of human IgG, in terms of AUC( _0-t ), was observed when comparing day 1 and day 22, with an increase with increasing dose level.

Animals surviving to day 29 were euthanized and necropsied. Tissues required for microscopic analysis were minced, processed in the usual manner, embedded in paraffin and stained with hematoxylin and oesin, followed by examination under a light microscope.

No drug-related serious pathologies were found. The observed severe pathologies were considered to be coincidental, often observed in cynomolgus monkeys of this lineage and at this age; and/or occurred with approximately equal frequency in control and drug-treated animals and were therefore considered unrelated to antibody administration. There was no weight loss or death observed in the animals in the study, and there were no serious pathologies in all organs examined. Histopathological changes were limited to the bone marrow, an effect associated with MMAE. A minimal or weak decrease in cellular abundance and a moderate decrease in the ratio of myeloid to erythroid cells were observed. Changes in hematologic parameters (ie, mild neutropenia) were consistent with changes observed with other MMAE antibody-drug conjugates and were not considered to be target related. Finally, all clinical biochemistry results remained normal throughout the study.

Example 21. Preparation of a cell line.

To produce antibodies for clinical use, a stable Chinese hamster ovary (CHO) cell line was generated using the GIBCO® FREEDOM® CHO-S® kit (Thermo Fisher Scientific, Waltham, MA). CHO-S cells are transfected with a construct containing genes encoding a humanized anti-STn antibody. Stable clones are selected using a two-phase selection scheme. Clones with high levels of production are selected for further characterization. The stable cell line is transfected under Good Manufacturing Practice (GMP) conditions to produce antibodies for GLP toxicology testing and Phase I clinical testing.

Example 22 Tissue cross-reactivity studies.

Tissue cross-reactivity studies were performed to evaluate the binding profile (both target and potentially non-target binding) to human tissues and relevant species used in non-clinical safety studies. Humanized anti-STn antibodies were tested for binding to a panel of normal human, rat, mouse and cynomolgus macaque tissues. The study was carried out on sections of 10 normal frozen tissues: heart, brain, kidney, stomach, lung, small intestine, colon, liver, pancreas

- 107 045483 milk gland, spleen and bone marrow, using doses of 1-3 mcg/ml. A human pancreatic neoplasm containing STn-positive cancer cells served as a positive control, and normal stromal cells contained in the same sample served as a negative control. Staining of all normal tissues was limited to the cytoplasm. Additional analyzes were performed without the primary antibody or using IgG isotype controls as negative controls.

Of all the antibodies tested, hSIA101 had the lowest cross-reactivity with normal human tissue. Moderate binding activity was observed in cardiac tissue vessels, gastric epithelial cells, small intestinal epithelial cells and splenic vessels. The antibodies were also found to cross-react with similar tissues from cynomolgus monkey (see Table 17) and rat (not shown). In the table: 1+ = weak staining; 2+ = moderate staining; 3+ = strong staining; 4+ = intense staining; neg. = negative; frequent = frequent (staining in >75-100% of cells); epi. = episodic (staining in >25-50% of cells) and rare. means staining in 1-5% of cells.

Table 17

Tissue cross-reactivity study results

Textile Human Cynomolgus macaque Heart vessels 2+ rare (cytoplasm) neg. Gastric epithelial cells 2-3+ parts, (cytoplasm) 2-3+ epi. (cytoplasm) Stomach vessels neg. 2+ epi. (cytoplasm) Lung epithelium neg. 2-3+ parts, (cytoplasm) Epithelial cells of the small intestine 2-4+ parts, (cytoplasm) 3-4+ parts, (cytoplasm) Colon epithelial cells 1+ parts, (cytoplasm) neg. Vessels of the colon neg. 4+ parts, (cytoplasm) Pancreatic vessels neg. 2-3+ parts, (cytoplasm) Vessels of the spleen 2-3+ parts, (cytoplasm) neg.

Staining was not observed on cell membranes in any of the tissues. The cerebral hemispheres, cerebellum, kidneys and liver were not stained. There was no staining when using the isotype control and the secondary antibody alone, while strong membrane staining with hSIA101 was observed only in the positive control tissue (pancreatic neoplasm).

Example 23 Comparison of anti-STn antibody treatment and cisplatin treatment in a PDX ovarian tumor model.

Anti-STn ADC candidates were compared to standard of care (SOC) in a tumor xenograft model using human patient-derived ovarian cancer tumors from two different patients. Cells in patient samples have previously been confirmed to express STn by immunohistochemical staining; and tumors from one of the chemotherapy-naïve patients (no chemo) and tumors from a chemotherapy-naïve patient were implanted into immunodeficient athymic nude mice. Mice with successfully growing tumors were selected and treated with antibodies (5 mg/kg dose) by weekly i.v. injection; cisplatin (dose 3 mg/kg) by weekly intraperitoneal injection (limit 3 doses); or solvent control. Antibodies administered included hSIA101, hSIA101-ADC (MMAE conjugate), or isotype control IgG MMAE conjugate (isotype-ADC). Mice with chemo-free tumors received antibody treatment for 4 weeks, while mice with chemo-resistant tumors received antibody treatment for 6 weeks. Tumor volume in mice treated with hSIA101-ADC or isotype-ADC was monitored for 4-5 weeks after completion of treatment to assess tumor regression.

Volumes of cisplatin-sensitive (Fig. 4, top panel) and cisplatin-resistant (Fig. 4, bottom panel) tumors were measured over time. The introduction of hSIA101-ADC resulted in the most effective reduction in tumor volume compared to other types of treatment. Tumors derived from cisplatin-resistant cells were significantly reduced in size upon administration of hSIA101-ADC compared with administration of cisplatin (Fig. 4, bottom panel). Accordingly, administration of hSIA101-ADC can be used to effectively treat cisplatin-sensitive and cisplatin-resistant tumors. Interestingly, 75% of chemo-free mice treated with hSIA101-ADC had complete tumor resolution four weeks after completion of treatment, while tumor regression of greater than 50% was observed in 75% of mice with chemo-resistant tumors 5 weeks after completion of treatment. hSIA101-ADC.

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Claims (3)

CLAIM 1. A method of treating cancer resistant to treatment with platinum compounds in a subject, comprising administering to the subject an antibody-drug conjugate (ADC), wherein the cancer resistant to treatment with platinum compounds (i) is selected from breast cancer, melanoma, colon cancer , liver cancer, lung cancer, bladder cancer, cervical cancer, stomach cancer and prostate cancer, (ii) is resistant to cisplatin or oxaliplatin, or (iii) is ovarian cancer and resistant to cisplatin, and said ADC contains: a) an anti-STn antibody, wherein said anti-STn antibody contains: 1) a heavy chain variable domain (VH) with the amino acid sequence SEQ ID NO: 7 and a light chain variable domain (VL) with the amino acid sequence SEQ ID NO: 8; 2) VH with the amino acid sequence SEQ ID NO: 9 and VL with the amino acid sequence SEQ ID NO: 10; or 3) VH with the amino acid sequence SEQ ID NO: 11 and VL with the amino acid sequence SEQ ID NO: 12; And b) a cytotoxic agent conjugated to an anti-STn antibody. 2. The method of claim 1, wherein said cytotoxic agent is selected from auristatin, maytansine, tubulisin, vinca alkaloid, pyrrolobenzodiazepine dimer, camptothecin, duocarmycin, amanitine, phosphoinositide 3-kinase inhibitor (PI3K) and mitogen-activated protein kinase inhibitor (MEK) . 3. The method according to claim 1 or 2, wherein said cytotoxic agent is conjugated to an anti-STn antibody using a linker. 4. The method of claim 3, wherein the linker is a cleavable linker. 5. The method according to claim 3 or 4, wherein said linker contains one or more polymers selected from poly(ethylene glycol) (PEG), poly(N-(2-hydroxypropyl)methacrylamide) (polyHPMA), poly(amino acid), carbohydrate polymer, glycopolysaccharide, glycolipid, glycoconjugate, polyglycerol, polyvinyl alcohol, poly(acrylic acid), polyketal, polyacetal and poly(1hydroxymethylethylene hydroxymethylformal) (PHF). 6. Method according to any one of claims 3-5, where the linker contains valine-citrulline. 7. The method according to any one of claims 1 to 6, wherein the anti-STn antibody contains a VH with the amino acid sequence SEQ ID NO: 7 and a VL with the amino acid sequence SEQ ID NO: 8. 8. The method according to any one of claims 1 to 7, wherein the cancer resistant to treatment with platinum compounds is ovarian cancer and resistant to cisplatin. 9. Method according to any one of claims 1 to 7, wherein the cancer resistant to treatment with platinum compounds is colon cancer. 10. The method of claim 9, wherein said colorectal cancer is resistant to treatment with at least one of cetuximab, panitumumab, bevacizumab, ramucirumab, trastuzumab, ponatinib, sorafenib, 5-fluorouracil, docetaxel, gemcitabine, irinotecan or paclitaxel. 11. Method according to any one of claims 1 to 10, where the cytotoxic agent contains MMAE. 12. The method according to any one of claims 1 to 11, wherein the anti-STn antibody comprises a VH having the amino acid sequence SEQ ID NO: 7 and a VL having the amino acid sequence SEQ ID NO: 8, and the cytotoxic agent contains MMAE. 13. Method according to any one of claims 1 to 12, where ADC is administered at a dose of 1 to 6 mg/kg. - 109 -