ES2988943T3 - L2a5 antibody or functional fragment thereof against tumor antigens - Google Patents

Abstract

This invention provides a functional antibody or antibody fragment, or probe thereof, directed against a unique group of antigens identified in cancer. The present invention comprises nucleotide sequences derived from the monoclonal antibody L2A5. The functional antibody or antibody fragment, or probe thereof, includes a variable heavy chain domain and a variable light chain domain having an amino acid sequence provided herein. This DNA sequence/amino acid conjugation is unique and has never been described before. The present invention further provides a functional antibody or antibody fragment or conjugate or recombinant protein useful in the detection, treatment and prevention of human diseases, including cancer. (Automatic translation with Google Translate, no legal value)

Description

DESCRIPTION

L2a5 antibody or functional fragment thereof against tumor antigens

Technical field

The present invention provides a functional antibody or antibody fragments, or a probe thereof directed against a group of antigens identified in cancer.

State of the art

Sialyl Tn (STn) is a short O-glycan antigen, a disaccharide consisting of sialic acid linked to N-acetyl galactosamine, i.e. Neu5Aca-2,6GalNAc, which in turn is O-linked to serine or threonine residues in a polypeptide chain, in an alpha configuration through the GalNAc residue. Its relevance relates to the fact that it is absent in normal healthy tissues but detected at different frequencies in almost all types of carcinomas (Julien, Videira, & Delannoy, 2012). Furthermore, STn is a target for metastatic, drug-resistant and highly malignant cancer cells. Its motivating features include:

1) Association of STn expression with oncogenesis and metastatic capacity of human cancer cells and cancer-initiating cells (Okasakiet al.,2012).

2) correlation of STn with a poor prognosis in patients, with reduced overall survival and lack of response to chemotherapy (Choiet al.,2000) and 3) evasion of immune protection (Carrascalet al.,2014).

Alpha-2,6 sialic acids are frequently cancer-defining biomarkers. Short sialylated alpha-2,6 O-glycans are overexpressed in several types of cancer. In general, they are involved in cancer metastasis and progression. In addition, they may contribute to immune evasion through their recognition by several immune receptors such as sialic acid binding proteins (Siglecs) (Crocker, Paulson & Varki, 2007; Nicollet al.,2003).

The present invention therefore provides an antibody, functional antibody fragments or probes that specifically bind to cancer biomarkers. In addition to the specific identification of tumor cells, these antibodies have the potential to block the recognition of these ligands by host cell receptors, involved in the mechanism underlying tumor progression, including immune tolerance.

There are a significant number of antibodies that have been approved to treat cancer patients (https://www.cancer.org/). They include:

1. Monoclonal antibodies (mAbs) targeting specific (associated) cancer antigens. The recombinant humanized anti-HER2 mAb trastuzumab (HerceptinTM) is described in different scientific publications, e.g. Cancer Res., 1998, 58: 2825-2831. It targets the HER2 receptors expressed in almost 30% of breast cancer patients. WO0105425 describes an antitumor composition comprising an alkylating anthracycline coupled to the anti-HER2 antibody trastuzumab. However, these treatments are only available for a limited number of patients (30% of breast cancer patients). Also, the HER2 proteins targeted by the antibody trastuzumab are also expressed in relevant normal cells, such as cardiac cells, explaining the associated cardiotoxicity. The present invention enables the development of recombinant antibodies targeting a broader list of receptors beyond HER2, expressed by different cancer types, that are decorated with STn or a group of glycans terminated by alpha-2,6 sialic acids. This will potentially enable the development of therapies for a broader group of cancer patients. Since both STn and glycans terminated by alpha-2,6 sialic acids are highly overexpressed by cancer cells, it also has the potential for increased specificity and reduced toxicity.

2. MAbs that block molecules that stop the immune system from working. These are called immune checkpoint inhibitors and include drugs that block CTLA-4, PD-1, and PD-L1. Examples of PD-1 inhibitors are Pembrolizumab (Keytruda) and Nivolumab (Opdivo) which are approved to treat skin melanoma, non-small cell lung cancer, kidney cancer, bladder cancer, head and neck cancers, and Hodgkin lymphoma. However, immune checkpoint inhibitors do not provide an immediate anti-tumor response and are typically used as adjuvant therapy. Also, immune checkpoint inhibitors can allow the immune system to attack some normal organs in the body, which can cause serious side effects in some people.

3. MAbs that block cell signaling necessary for cancer cells to divide. These include Bevacizumab (Avastin®) which targets VEGF that affects blood vessel growth and Cetuximab (Erbitux®), an antibody that targets a cellular protein called EGFR, which is important for cell growth. However, these antibodies, which block cell signaling, also influence physiological mechanisms and are associated with toxicity such as high blood pressure, bleeding, blood clots and kidney damage.

There are some patent documents (EP 2014302 A1; WO 2015053871 A4; US 7423126 B2; EP 2993184 A1) claiming nucleic acids encoding human antibodies to tumor-associated carbohydrate antigens; however, none of the antibodies within the above-mentioned patent documents recognize the STn antigen, which is the target of the antibody of the present invention.

Patent application EP2680004 (A2) claims antibodies against sialylated glycans, including the STn antigen containing N-acetylneuraminic acid (NeuAc) or N-glycolylneuraminic acid (NeuGc), while the present invention relates to an antibody that is specific for the STn antigen mainly with NeuAc and for a group of glycans terminated by alpha-2,6 sialic acids.

Patent applications WO 1998046246 A1 and WO 1995029927 A3 claim methods for producing glycoconjugates or α-O-linked glycosides of tumor-associated carbohydrate antigens, including the 2,6-sialyl T antigen. While the possibility of using it to develop antibodies to treat cancer is mentioned, neither of these patent applications provides an antibody sequence for detecting the 2,6-sialyl T antigen, nor the group of related 2,6-sialic acid-containing antigens that are the subject of the present patent.

Several patent applications have claimed antibodies that bind sialic acid residues, such as CA 2743032 A1, which characterizes an IgM antibody that recognizes sialic acid residues including NeuGc, or US 20160184450 A1 and US 8148335 B2 which claim antibodies that specifically bind to a de-N-acetylated sialic acid, or patent applications EP 2302390 A1 and US 20120142903 A1 which include antibodies that recognize the NeuGc structure; whereas, the present invention claims an antibody that recognizes not only NeuAc bound to GalNAc (STn antigen) but also the NeuAc structure within a group of glycans terminated by alpha-2,6-linked sialic acids.

Patent application EP 2261255 A1 described the production of antibodies in chickens that recognise the monosaccharide NeuAc and patent application US 20110034676 A1 claims the production and purification of polyclonal antibodies against the Neu5Ac and Neu5Gc structures. However, the present invention claims nucleic acids encoding mAbs produced in mice that target the oligosaccharide antigen STn and a group of glycans terminated by alpha-2,6-linked sialic acids. This is an advantage, since the present invention will allow to distinguish glycans associated with tumors, and not only the monosaccharide NeuAc, which is also expressed in normal cells.

Furthermore, US patent application 2010/0034825 A1 claims the production of antibodies against the MUC1 glycoprotein, using immunization with long Tn- or STn-MUC1 tandem repeat glycopeptides. This work differs from the present invention, since the target structure is different, i.e. it targets the MUC1 glycoprotein and not glycan structures, STn or glycans terminated by alpha-2,6-linked sialic acids.

Liliana Loureiro et al. "Challenges in Antibody Development against Tn and Sialyl-Tn Antigens", BIOMOLECULES, vol. 5, no. 3, August 11, 2015, pages 1783-1809 refers to the development of antibodies against the carbohydrate antigens Tn and STn.

WO2017/040529A1 provides compositions for adoptive cellular therapies for cancers expressing the STn glycoepitope on TAG-72.

The antibodies of patent WO2016057916A1 recognize the STn antigen and not the glycans terminated by alpha-2,6-linked sialic acids.

As mentioned above, tumor antigens are often not tumor-specific due to their concurrent expression in normal cells. Therefore, targeted therapies often show associated toxicity due to off-target effects. Tumor antigens are not pan-tumor antigens and are limited to specific types or grades of cancers. Therefore, targeted therapies are usually limited to specific subsets of cancers.

Consequently, there is a need to develop functional antibodies or antibody fragments, or probes thereof, directed against a unique set of antigens only identified in cancer cells.

A unique nucleic acid sequence that allows producing a functional antibody or antibody fragments or a probe thereof to target sialylated glycans such as STn, 2,6-sialyl T, disialyl T and 2,6-sialo-N-acetyllactosamine, which are overexpressed in different types and subtypes of cancer cells, is very important.

The present invention relates to nucleotide sequences encoding mAbs against STn and a group of glycans terminated by alpha-2,6-linked sialic acids. These antigens are short-chain glycans that are overexpressed in cancer but not expressed by normal cells. These antigens are also ligands for immune receptors and dampen the activity of the immune system against tumor cells, so they can also be considered ligands for immune checkpoints.

Description

Monoclonal antibody (mAb) refers to an antibody that is produced by an individual B cell clone. mAbs can also be produced by a hybridoma, which is a hybrid between a B cell and a myeloma cell, or cell lines that express recombinant DNA encoding the immunoglobulin heavy and light chain and will therefore produce an individual, specific antibody.

Antibodies are expressed in the extracellular medium and then purified from there.

The specificity of an antibody is its ability to react with an antigen or a group of antigens that share a certain epitope. An epitope, also known as an antigenic determinant, is the part of an antigen that is recognized by the antibody.

An antibody belongs to the immunoglobulin class of proteins and is an assembly of two identical heavy chains (~50-70 kDa) and two identical light chains (~25 kDa). At the amino terminus of each heavy or light chain is a sequence of 100-130 amino acids encoding the variable region. At the carboxyl terminus of each heavy or light chain is a sequence encoding the constant region.

Each antibody binds twice to the same antigen, that is, it is bivalent.

Antigen-binding fragment (Fab) is the antibody fragment that binds to antigens. Each Fab is composed of one constant and one variable domain from each heavy and light chain of the antibody. The fragment crystallizable (Fc) region is composed of 2 or 3 domains from the carboxy terminus of the two heavy chains. While the Fab ensures antigen binding, the Fc region ensures that each antibody generates an effector immune response. The Fc region binds to different cellular receptors, such as Fc receptors and other molecules, such as complement proteins, mediating different physiological effects including opsonization to facilitate phagocytosis by phagocytes, cell lysis by natural killer cells, and degranulation of mast cells, basophils, and eosinophils.

The term "variable domain" or "variable region" is the amino-terminal portion of the light or heavy chains of an antibody that interacts with antigen. It is about 120 to 130 amino acids in length in the heavy chain and about 100 to 110 amino acids in length in the light chain. The sequences of each of the variable regions are substantially varied, particularly in the complementarity determining regions (CDRs), responsible for interaction with the specific antigen. The CDRs are flanked by less varied framework regions (FRs). The CDRs of each of the light and heavy chains are three in number. CDRs L1, L2, and L3 are within the light chain. CDRs H1, H2, and H3 are within the heavy chain.

The term "functional antibody fragment or probe" is a portion of the antibody that includes the variable region of the heavy chain and the light chain of the antibody, or includes either the variable region of the heavy chain or the variable region of the light chain of the antibody. The functional antibody fragment or probe retains most or all of the binding activity of the initial antibody from which the fragment or probe was derived. These functional antibody fragments or probes may include single chain Fv (scFv), diabody, triabody, tetrabody, and minibody.

The term "nucleotide sequences" refers to a sequence of nucleotides of any length, whether deoxyribonucleotides or ribonucleotides or analogs thereof.

The nucleotide sequence can be transcribed to produce mRNA, which is then translated into a polypeptide and/or a fragment thereof.

The potential treatment derived from the present invention relates to the use in the treatment, management or amelioration of a disease associated with the expression of STn or a group of sialylated alpha-2,6 glycans. In the present embodiments, a potential treatment relates to an antibody, functional antibody fragment or probe derived from the invention, in its native or modified form.

A possible treatment may also be included in the present invention in combination with an agent that is known to be useful for, or has been used or is currently being used for the treatment, management or amelioration of a disease.

Potential use as a diagnostic agent refers to a substance that when administered to a subject will aid in the diagnosis of a disease. These substances can be used to detect and/or define the location of a disease or a process that results from disease progression. It also includes a substance that can help predict the response to a certain treatment or the prognosis of a disease. In certain embodiments, the use of a diagnostic agent involves conjugation of the antibody or functional antibody fragment or probe of the present invention to a reporter molecule, such as fluorescence, enzyme, or secondary antibody.

Sialyl Tn, also known as STn, sialosyl Tn, sialylated Tn, Neu5Ac-a2,6GalNAca-O-Ser/Thr or also referred to as CD175s by the "cluster of differentiation" nomenclature is the simplest sialylated mucin-type O-glycan. STn is a truncated O-glycan containing an α-2,6 sialic acid (Neu5Ac) linked (via carbon 6) to N-acetyl-galactosamine (GalNAc) alpha-O-linked to a serine/threonine (Ser/Thr) (Neu5Ac-a2,6GalNAca-O-Ser/Thr). Sialylation prevents the formation of different core structures otherwise found in mucin-type O-glycans.

STn is associated with adverse outcome and poor prognosis in cancer patients. STn antigen biosynthesis has been linked to the expression of the sialyltransferase ST6GalNAc1 and to mutations or loss of heterozygosity of the COSMC gene.

STn is expressed in more than 80% of human carcinomas and is associated with a poor prognosis in cancer patients.

The invention provides nucleotide sequences encoding an antibody heavy or light chain or a functional antibody fragment or a probe thereof, wherein the antibody heavy or light chain or functional antibody fragment or probe thereof encoded by the nucleotide sequence of the invention has one or more of the CDRs listed in Tables 1 and 2. An antibody or functional antibody fragment or probe thereof that includes one or more of the CDRs is capable of specifically binding to STn or a sialylated alpha-2,6 glycan cluster. Specific binding includes specificity, affinity and/or avidity as provided in Example I.

In another aspect, an antibody or functional fragment thereof encoded by the polynucleotides of the invention may include complement-dependent cytotoxicity activity and/or antibody-dependent cell-mediated cytotoxicity (ADCC) activity.

The method for producing the antibody of the invention may include fusion between two cells that produce a hybridoma, introduction of a nucleotide sequence of the invention into a host cell, culturing the host cell under conditions and for a time sufficient to produce the encoded heavy and/or light chain of an antibody or functional fragment of the invention, and purifying the heavy and/or light chain of an antibody or functional fragment.

Recombinant expression of an antibody or functional antibody fragment or probe thereof of the invention that binds to STn or a group of sialylated alpha-2,6 antigens may include the construction of an expression vector containing a nucleotide sequence encoding the heavy and/or light chain of an antibody or functional antibody fragment or probe thereof of the invention.

The vector may be produced by recombinant DNA technology. These vectors may also include other coding nucleotide sequences, which give rise to a chimeric sequence. For example, it may include the nucleotide sequence encoding the constant region of the antibody molecule (WO 86/05807 and WO 89/01036) that allows the expression of a chimeric protein containing the amino acid sequence of the antibody, functional antibody fragment or probe thereof of the present invention followed by the entire heavy chain, or both the light and heavy chains, or both the entire heavy and light chains.

The expression vector may be transferred into a host cell by transfection/transduction techniques and the resulting cells produce the antibody or functional fragment thereof of the invention. Thus, the invention includes host cells containing nucleotide sequences encoding the antibody or functional antibody fragment or probe thereof of the invention.

The host cell can be chosen to modify the characteristics of the product derived from the inserted nucleotide sequences.

In one embodiment, these host cells may add glycosylation or phosphorylation sites or other modifications to the encoded proteins. In another embodiment, the host cells may provide for the correct processing and cellular trafficking/secretion of the proteins.

A single chain fragment variable (scFv) antibody refers to a functional antibody fragment containing only the VL and VH regions, which are joined by a linker, forming a monovalent antigen-binding site. Diabodies, tribobodies and tetrabodies are antibodies that include dimers, trimers or tetramers of scFv, i.e. containing two, three and four polypeptide chains, respectively, and which form two, three and four antigen-binding sites, respectively, which may be the same or different.

The resulting antibody, functional antibody fragments or probes thereof of this invention may have one or more binding sites. If it contains more than one binding site, these sites may be identical to each other or may be different. In case of two different binding sites, the antibody, functional antibody fragment or probe is called a "bispecific" antibody.

PCR-mediated site-directed mutagenesis resulting in amino acid substitutions can be used to introduce mutations into the CDR regions that improve affinity while retaining the specificity of the antibody of the invention.

In some embodiments, the antibody, functional antibody fragment or probe of the invention is conjugated or fused to one or more diagnostic agents, or therapeutic agent or any other desired molecule. The resulting conjugated antibody, functional antibody fragment or probe may be useful for monitoring or diagnosing the onset, development, progression and/or severity of a disease associated with the expression of STn or sialylated alpha-2,6 glycans.

As an example, but not limited to, the antibody may be conjugated to a therapeutic agent to induce cell death or other effect. A therapeutic agent may be a chemotherapeutic drug; a taxane; an antimetabolite, an alkylating agent, an antibiotic, an antimitotic agent, a hormone, a nucleoside analogue, a kinase inhibitor, a radioactive metal ion, a toxin, a cytokine, or an antiangiogenic agent.

The antibody or functional fragment of the invention can also be used to detect the expression of STn or sialylated alpha-2,6 glycans in any biological sample using classical immunohistological methods or immunoassays, such as enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay (RIA), flow cytometry and immunoblotting.

The antibody, functional antibody fragment or probe thereof of the invention may be included alone or conjugated or in combination in a pharmaceutical composition, provided in an effective concentration, to exert a therapeutically useful effect, with minimal side effects.

Summary of the invention

The present invention is defined in claims 1-15.

The present invention provides an antibody, or functional antibody fragments thereof, as claimed, that specifically binds to sialyl Tn (STn) and a group of glycans terminated by alpha 2,6-linked sialic acids.

The antibody, or functional antibody fragment thereof, binds to the antigen-binding site. An example of nucleotide sequences encoding the variable heavy and light chains are SEQ ID NO. 1 and 2, respectively.

The antibody was obtained after immunization of mice with mucins containing sialyl Tn (STn). The nucleotide sequences were derived from the monoclonal antibody L2A5. One nucleotide sequence provides the variable heavy chain (VH) (SEQ ID NO. 1) and comprises H-CDR1 (GGCTACTCCATCACCAGTGGTTATTAC, amino acid SEQ ID NO. 12), H-CDR2 (ATAAACTACGACGGTAGCAAT, amino acid SEQ ID NO. 14), and H-CDR3 (GCAAGAGGGGGGGACTAC, amino acid SEQ ID NO. 16). A nucleotide sequence provides a variable light chain (VL) (SEQ ID NO. 2) comprising the complementarity determining regions (CDRs): L-CDR1 (TCAAGTGTAAGTTAC, amino acid SEQ<i>D NO. 6), L-CDR2 (GACACATCC, amino acid SEQ ID NO. 8), and L-CDR3 (CAGCAGTGGAGTAGTGACCCACCCATGCTCACG, amino acid SEQ ID NO. 10).

The combination of the L-CDR1, L-CDR2, and L-CDR3 of the light chain and the H-CDR1, H-CDR2, and H-CDR3 of the heavy chain generates unique sequences encoding peptide sequences that recognize not only the STn disaccharide antigen, but unexpectedly, also recognize a set of glycans terminated by alpha-2,6 sialic acids also overexpressed in cancer. These peptide sequences are then used to generate additional antibodies, functional antibody fragments, or probes. This total set of glycans recognized by the antibodies, functional antibody fragments, or probes comprises the following:

Sialil Tn:

NeuAca-6GalNAca/pi-2,6-sialyl T:

Gal (31-3GalNAcal-

NeuAca2-6

disialyl T:

NeuAca2-3Gal¡31-3GalNAcal

NeuAca2-6

2,6-sialo-N-acetyl-lactosamine:

NeuAca2-6Gal|31-4Glc|31-Antibodies with this specificity are particularly interesting due to their high tumor specificity and low or no reactivity to normal cells, unlike current antibodies and antibody-based anti-cancer therapies.

The isolated polynucleotide as disclosed herein may also include a nucleic acid sequence provided herein, wherein the nucleic acid sequence encodes the variable heavy and light chain domain of the antibody, functional antibody fragments, or probes. Examples of the nucleic acid sequence are SEQ ID NO. 1 and 2, respectively.

In another aspect, the present invention provides an antibody, or functional antibody fragments thereof, as claimed, that specifically binds to STn and a group of glycans terminated by alpha 2,6-linked sialic acids for use in medicine, or for use in the treatment of cancer, including a method for detecting a tumor in a subject.

The invention also provides pharmaceutical compositions comprising the antibody, or functional antibody fragments thereof, as claimed, and a pharmaceutically acceptable carrier.

The invention may provide pharmaceutical compositions comprising the antibody, functional antibody fragments or probes that block cell-cell or receptor-ligand interactions.

The invention provides a pharmaceutical composition of the invention as claimed for use in the treatment or prevention of a disease in a subject in need, which treatment may comprise administering a therapeutically effective amount of a pharmaceutical composition of the invention.

This disclosure presents accompanying drawings of illustrated embodiments for easier understanding.

Description of the drawings

Figure 1 shows the titer and binding of the L2A5 antibody to animal mucins using indirect ELISA. Antibody titration was performed using different concentrations of bovine submaxillary mucins (BSM) coating previously treated or not with sialidase. Phosphate buffer was used as a negative control and binding of the anti-STn B72.3 antibody to BSM as a positive control. Binding of mAb L2A5 was detected with goat anti-mouse IgG conjugated to horseradish peroxidase.

Figure 2 shows the binding of L2A5 to the cell surface of tumor cells. The binding of the L2A5 antibody was assessed by flow cytometry and using cancer cell lines transduced or not (wild type) with the ST6GalNAc1 gene and therefore overexpressing the STn antigen. The figure shows a representative histogram of the relative cell count of the L2A5 antibody in the STn-positive and WT breast cancer cell line MDA-MB-231. Antibodies 3F1 (anti-STn antibodies) or the secondary antibody anti-mouse Ig-FITC (gray profile) were used. To assess the specificity of binding to sialylated antigens, STn-expressing cell lines were desialylated by treatment with sialidase. The solid and dashed lines represent the histograms for untreated sialidase and the treated cell, respectively. The X-axis represents fluorescence, related to STn expression.

Figure 3 shows the reactivity of the L2A5 antibody to membrane-bound proteins and recombinant human mucin 1 (MUC1). Western blot analysis of anti-STn antibodies 3F1 and L2A5 binding to membrane extracts from the STn-expressing MDA-MB-231 cell line and to the MUC1 chimeric protein heavily decorated with STn plus the Fc region of human Ig. MDA-MB-231 STn+ membrane extracts (A) and the MUC1 STn-IgG chimeric protein (B) were stained with 3F1 and L2A5 antibodies. In addition to blotting of untreated membrane extracts and chimeric protein (NT), membrane extract samples were desialylated using sialidase (T) or nonglycosylated MUC1 STn-IgG protein (Ung) was used.

Figure 4 shows the reactivity of the L2A5 antibody, measured by immunohistochemistry, in paraffin-embedded bladder cancers. (a) L2A5 staining (left) shows a high extent and strong intensity, whereas the mAbs B72.3 (middle) and TKH2 (right), which bind only to STn, show less sensitivity, transduction at low intensity and reduced extent of specific staining. (b) Sensitivity of L2A5 compared to available antibodies. L2A5 (left) recognizes a reduced amount of antigen (arrows), unlike B72.3 (middle) and TKH2 (right) which show different reactivity in the same regions. (c) Effect of sialidase treatment of cancer tissues on antibody reactivity. After sialidase treatment and incubation with L2A5, L2A5 staining completely disappears. Left: without sialidase; Right: with sialidase.

Figure 5 shows the reactivity of the L2A5 antibody, measured by immunohistochemistry, in paraffin-embedded colorectal cancers. The figure shows the reactivity of L2A5 compared to antibodies against the STn antigen. L2A5 (left) shows increased reactivity in terms of extent and intensity compared to the reactivity of B72.3 (center) and TKH2 (right).

Figure 6 shows the reactivity of the L2A5 antibody, by immunohistochemistry in paraffin-embedded bladder cancers. The figures show immunohistochemical evidence of high specificity of the L2A5 antibody compared to anti-STn antibodies B72.3 and TKH2, a) - Tumor specificity of L2A5 in the metastatic bladder cancer sample. L2A5 is mainly present in tumor cells (arrows), there is no staining in the lymphocytic population, vessels and connective tissues. b) - Reactivity of the L2A5 (left) and B72.3 (right) antibodies in normal colorectal tissues. L2A5 shows weak reactivity with enterocytes (arrow, left) while B72.3 reacts with goblet cells (right).

Figure 7 shows the glycan antigen sequences bound by the L2A5 antibody. Glycan specificity was determined by glycan microarray analysis.

Figure 8 shows the in vivo antitumor capacity of a targeting module (TM) composed of a portion containing amino acids encoded by the nucleic acids of L2A5, object of the present invention. The TM derived from L2A5 is an antibody fragment and has the same reactivity as L2A5, i.e. anti-STn TM. Killing of STn-positive cells by UniCAR T cells was target-specific and strictly dependent on the presence of TM.

Figure 9 shows the in vivo antitumor capacity of a targeting module (TM) composed of a portion containing amino acids encoded by the L2A5 nucleic acids, object of the present invention. The L2A5-derived TM is an antibody fragment and has the same reactivity as L2A5, i.e. anti-STn TM. Adoptive transfer of anti-STn TM and UniCAR T cells (Koristka et al. 2014) to an animal model expressing STn+ tumors shows effective and TM-dependent eradication of STn-positive tumors. Figure 8 shows that the use of UniCAR T cells armed with TM specific for the STn antigen kills tumor cells. Statistical analysis was performed using one-way ANOVA with Bonferroni's multiple comparison test (**p<0.01).

Detailed description of the invention

The present invention provides an antibody, or functional antibody fragments thereof, as claimed, that specifically binds to STn and a group of glycans terminated by alpha 2,6-linked sialic acids.

The antibody, or functional antibody fragment thereof, binds to the antigen-binding site. An example of nucleotide sequences encoding the variable heavy and light chains are SEQ ID NO. 1 and 2, respectively.

The isolated polynucleotide as disclosed herein may also include a nucleic acid sequence provided herein, wherein the nucleic acid sequence encodes the variable heavy and light chain domain of the antibody, functional antibody fragments, or probes.

The present invention further provides compositions as claimed. These can be used to produce an antibody, functional antibody fragments or probes thereof that specifically bind to STn and a group of glycans terminated by alpha 2,6-linked sialic acids.

The compositions include nucleotide sequences encoding the antigen binding site of an antibody, a functional antibody fragment, or probes. The compositions include nucleotide sequences encoding the variable heavy and light chains.

The isolated polynucleotide as disclosed herein may also include a nucleic acid sequence provided herein, wherein the nucleic acid sequence encodes the variable heavy and light chain domain of the antibody, functional antibody fragments, or probes.

The invention may provide pharmaceutical compositions comprising the nucleotide sequences encoding the antibody, or functional antibody fragments thereof, as claimed and a pharmaceutically acceptable carrier.

The method comprises the following steps:

a) Staining a biological sample obtained from a subject possibly having a tumor with an antibody that specifically binds to STn and a group of glycans terminated by alpha 2,6-linked sialic acids, in which the aforementioned staining is carried out under conditions suitable for the specific binding of the antibody, or functional antibody fragments or probes thereof to STn, 2,6-sialyl T, disialyl T or 2,6-sialolactosamine;

b) And where the presence or absence of antibody binding is indicative of the presence or absence of tumor cells expressing cell surface STn, 2,6-sialyl T, disialyl T, or 2,6-sialolactosamine.

Biological sample, as used herein, comprises isolated cells, or tissue, or tumor-derived proteins.

Sialylated glycans are overexpressed in several types of cancer cells compared to matched healthy cells, where their expression was negligible. The highest frequencies of STn are found in pancreatic, colorectal and ovarian cancers, where almost 100% of cancer cells express STn (Julien et al 2012). The frequency in bladder cancers is 75% (Ferreira et al 205). Lung adenocarcinoma expresses almost 80% and cervical cancer, cholangiocarcinoma, esophageal, colon and breast cancer have frequencies between 50 and 70%. Furthermore, STn overexpression occurs earlier in carcinogenesis and loss of cell differentiation, which is frequently involved in high histological grade grading, positively modulates STn expression (Julien et al).

Sialylated glycans can be targeted by antibodies, functional antibody fragments or probes with high affinity and specificity. The present invention further provides compositions for producing antibodies, functional antibody fragments or probes that bind to STn and a group of glycans terminated by alpha-2,6 sialic acids.

These compositions were obtained by immunization of 6-week-old female Balb/c mice with ovine serum mucins, using methods such as those described in Example I.

Serum from mice showing reactivity for STn-positive cell lines, STn-positive mucins or cell lysates was selected by methods such as those described in Examples II or III or VI. Splenocytes from those immunized mice showing serum reactivity for STn were harvested and fused with a myeloma cell (Sp2/0) to obtain an antibody-expressing immortalized hybridoma cell.

Methods for the hybridoma technique, such as those described in Example IV, are well described in the art.

Hybridoma supernatants were examined for the presence of antibodies against STn by methods such as those described in Examples II or III or VI. Selected hybridomas were expanded for antibody production and characterization. Of the several anti-STn mAbs obtained, mAb L2A5 was selected as a lead candidate and used for further analysis. Reactivity against mucin proteins with a high STn content and the antibody titer of mAb L2A5 were determined using methods as described in Example II and depicted in Figure 1. In addition, desialylation by sialidase treatment was performed to assess the recognition of sialylated structures by antibody L2A5. As shown in Figure 1, the reactivity of mAb L2A5 increases with mAb concentrations in a logarithmic manner. High immunoreactivity to BSM was observed, reaching an endpoint titer of 6,000. Furthermore, sialidase treatment clearly demonstrated a reduction in the reactivity of this antibody to BSM, showing specific and dependent binding to sialylated structures. It is noteworthy that, in methods similar to those described in Example II, but where the STn-bearing mucin proteins were replaced by desialylated mucins (asialo), the L2A5 antibody showed no reactivity.

Sialylated glycans are overexpressed in cancer cells and can be targeted by antibodies, functional antibody fragments or probes with high affinity and specificity.

The present invention provides compositions for producing an antibody, functional antibody fragments or probes thereof that bind to STn and a group of glycans terminated by alpha-2,6 sialic acids.

Glycosylation is critical for the quality and development of therapeutic mAbs. Glycosylation patterns change with the selected expression system or culture conditions with a significant impact on their pharmacokinetics and pharmacodynamics. Therefore, control of glycosylation is essential to ensure the safety and efficacy of molecules. For therapeutic targeting of cancer cells, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated phagocytosis (ADCP) and direct cell apoptosis are critical for efficacy.

mAbs produced in the SP2/0 murine myeloma cell line can add sugars not naturally found in normal human IgG with an impact in terms of immunogenicity.

Changes in the glycan composition of the IgG molecule can be made at the glycosylation sites Asn88 and Asn297 through manipulation of mannose, sialic acids, fucose and galactose residues in order to increase the efficacy of the therapeutic antibody (reviewed in Liming Liu 2015).

Non-human antibodies may be humanized, which refers to the construction of chimeric immunoglobulins, which contain the amino acid sequence of interest derived from the original non-human antibody (e.g., mouse antibody) embedded in a human immunoglobulin (recipient antibody). In humanized antibodies, the CDR amino acid residues of the human antibody are replaced by CDR residues from a non-human species (e.g., mouse antibody) having the desired specificity, affinity, and capacity. In general, humanized antibodies will comprise at least one, and usually two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human antibody and all or part of the FR regions are those of a human immunoglobulin.

Immune cells such as T cells can be modified to express antibody (all or part) or receptors that bind to an antibody, i.e., a chimeric antigen receptor (CAR). CARs are molecules that combine antibody specificity for a desired antigen (e.g., tumor antigen) with an intracellular T cell receptor activation domain to generate a chimeric receptor that exhibits specific antitumor cellular immune activity. The antigen recognition domain of these modified T cells can bind to a tumor-associated antigen. It may be related to adoptive cell transfer of T cells modified to express a CAR.

The CAR can be produced using a variety of techniques known in the art, including but not limited to the use of RNA-guided endonucleases, in particular the Cas9/CRISPR system, for T cells specifically modified to express the CARs (WO 2014191128 A1). A targeting module composed of a binding moiety specific for a certain human cell surface protein and a tag can be used, where the tag is derived from any human nuclear protein, preferably from the human nuclear protein La (WO2016030414 A1).

Also, the antibody or functional fragment of the invention can be linked to nanoparticles, inserted into the liposome membrane in order to be used as a specific vector to administer toxic compounds in situ that induce apoptosis, as well as metal ions useful for hyperthermia therapy.

Compositions for producing an antibody, functional antibody fragments or probes thereof of the present invention were obtained by immunizing 6-week-old female Balb/c mice with ovine serum mucins using methods such as those described in Example I. Serum from mice showing reactivity for STn-positive cell lines, STn-positive mucins or cell lysates was selected by methods such as those described in Examples II or III or VI. Splenocytes from those immunized mice showing serum reactivity for STn were harvested and fused with a myeloma cell to obtain an immortalized hybridoma cell expressing anti-STn antibodies. Methods for the hybridoma technique such as those described in Example IV are well described in the art. Hybridoma supernatants were examined for the presence of antibodies against STn by methods such as those described in Examples II or III or VI. The selected hybridomas were expanded for antibody production and characterization. Of the several anti-STn mAbs obtained, mAb L2A5 was selected as a lead candidate and used for further analysis. Reactivity against mucin proteins with a high STn content and the antibody titer of mAb L2A5 were determined by using methods as described in Example II and depicted in Figure 1. In addition, desialylation was performed by sialidase treatment to assess the recognition of sialylated structures by the L2A5 antibody. As shown in Figure 1, the reactivity of mAb L2A5 increases with mAb concentrations in a logarithmic manner.

High immunoreactivity to BSM was observed, reaching an endpoint titer of 6000. Furthermore, sialidase treatment clearly demonstrated a reduction in the reactivity of this antibody to BSM, showing specific and dependent binding to sialylated structures. It is noteworthy that, in methods similar to those described in Example II, but where the STn-bearing mucin proteins were replaced by desialylated mucins (asialo), the L2A5 antibody showed no reactivity.

Binding of mAb L2A5 to viable MDA-MB-231 cancer cells was confirmed by using methods as described in Example III. MDA-MB-231 cancer cells expressing the STn antigen, due to overexpression of the ST6GalNAc1 gene, were used as a model. As shown in Figure 2, mAb L2A5 shows high reactivity to 80 to 88% of STn+ cells. The reactivity decreased significantly after treatment of the cancer cell lines with sialidase. As also shown in Figure 2, L2A5 showed no binding to wild-type MDA-MB-231 cancer cells, which do not express the STn antigen. Taken together, these results confirmed the specificity and selectivity of mAb L2A5 against the STn antigen presented on a cancer cell surface. As also depicted in Figure 2, other anti-STn antibodies, which also react with STn, show slightly different binding profiles, relative to the MDA-MB-231 cancer cell line.

To confirm the binding specificity of mAb L2A5 to membrane proteins bearing STn, assays as described in Example VI were performed. As shown in Figure 3, mAb L2A5 reacted with proteins derived from the MDA-MB-231 cell line overexpressing ST6GalNAc1 and therefore STn. The profile showed reactivity to proteins with molecular weights greater than 245 kDa and approximately 160, 85, 50 and 40 kDa. Upon desialylation of the membrane proteins, a decrease or abolition of the reactivity is observed, confirming the binding of mAb L2A5 to sialylated proteins. Membrane proteins derived from wild-type cancer cells, which do not express the STn antigen, did not provide positive reactivity with mAb L2A5. As also shown in Figure 3, L2A5 demonstrated robust binding to MUC1 STn-IgG (approximately 180 kDa) but not to non-glycosylated MUC1 STn-IgG. Taken together, the results indicate that mAb L2A5 recognizes STn antigen in membrane extracts of STn-expressing cancer cells as well as STn-carrying proteins such as MUC1.

A series of 30 cases with 15 bladder tumors (eight cystectomies and seven metastases) and 15 colorectal tumors (adenocarcinomas and adenomas) were stained with L2A5 and two anti-STn mAbs. As shown in Figure 4, all bladder tumors were positive for all mAbs tested, compared to the metastatic cases. Three of them were positive and three were negative for the mAbs L2A5, B72.3 and TKH2. One case showed reduced staining with L2A5 and not with the other mAbs. A slightly higher sensitivity of antigen detection for L2A5 is noted. As shown in Figure 4, while TKH2 and B72.3 showed similar reactivity, L2A5 revealed higher reactivity in bladder cancer cases. This specificity and sensitivity was abolished after enzymatic treatment of the cancer tissue with sialidase.

As shown in Figure 5, all colorectal cancer cases were positive for anti-STn mAb and for L2A5, but exhibited different patterns in terms of extent and intensity. L2A5 binding (left) shows similar intensity and extent for most pathological tissues (approximately 70%), compared to the staining pattern of B72.3 (center) or TKH2 (right).

As shown in Figure 6 a), in the metastatic bladder cancer sample, L2A5 is mainly reactive with tumor cells and not with the lymphocyte population, vessels or connective tissues. In Figure 6 b), normal colorectal cancer tissues, L2A5 (left) shows nonspecific staining in enterocytes, whereas B72.3 (right) reacts with goblet cells.

In the bladder tumor model, L2A5 staining is exclusively tumoral. L2A5 exhibits specific staining in urothelial tumor cells, including additional staining at invasive and metastatic sites with low STn density.

In colorectal samples, L2A5 reacts with cancerous tissues, but also with non-pathological tissue. Staining is essentially localized in enterocytes, whereas nonspecific staining obtained with B72.3 or TKH2 is present in goblet cells. In colorectal samples, there is no specific location of staining, although L2A5 was able to detect the presence of weak staining in dysplastic tissue with L2A5.

To examine carbohydrate binding specificity in more detail, the antibodies were analyzed using glycan microarrays composed of structurally diverse glycan probes printed on a suitable solid surface. The results confirmed specificity with selective recognition of STn, but binding was also observed to:

Sialil Tn:

NeuAca-6GalNAca1/pi

2,6-sialyl T:

Gal(3l-3GalNAcal-

di-sialyl T:

NeuAca2-3Gaipi-3GalNAcal-AO

2,6-sialolactosamine:

NeuAca2-6Gal|31-4Glc|31

and virtual absence of binding to other antigen sequences that were present on the microarray. The bound sequences are summarized in Figure 7.

To determine the amino acid sequence of the variable regions CDR (SEQ ID NO. 6, 8, 10, 12, 14, 16) and FR (SEQ ID NO. 5, 7, 9, 11, 13, 15) of the light and heavy chains of mAb L2A5, a method described in Example IX was used.

Examples

The following examples are provided merely as illustrative of different aspects of the present disclosure and should not be construed to limit the disclosure in any way. They relate to the characterization, selection and production of antibodies.

EXAMPLE I

Antibody Production - Immunizations

Example methods for producing antibodies are provided, but any other standard method may be used.

Monoclonal antibody (mAb) production was performed according to hybridoma technology. Six-week-old female Balb/c mice (Harlan, UK) were immunized intraperitoneally with 10 µg of ovine submaxillary mucin (OSM) emulsified 1:1 (V/V) with complete Freund's adjuvant (Sigma-Aldrich) followed by 2 additional injections of OSM emulsified with incomplete Freund's adjuvant (Sigma-Aldrich) at 21-day intervals. Cheek blood samples were collected from mice and the collected serum was examined for STn binding specificity by ELISA. If the serum showed the desired and specific immune response, a final booster injection would be given to the corresponding mice three days before killing and harvesting the spleen.

EXAMPLE II ELISA

Mouse serum titers and examination of hybridoma supernatants were determined by ELISA against bovine submaxillary mucin (BSM), a protein that expresses STn. Wells of a 96-well plate were coated with 50 µl of BSM (3 pg/ml) dissolved in phosphate-buffered saline (PBS) and incubated overnight at 4 °C. To assess the specific binding of the examined hybridoma supernatants to sialylated structures, 50 µl of sialidase, from Clostridium perfringens (Roche) at 25 mU/ml diluted in sialidase buffer (10 mM Na2HPO4, pH=6.0), was added to a subset of wells and incubated for 90 min at 37 °C. After sialidase treatment, plates were washed three times with PBS containing 0.05% Tween 20 (PBS-T), followed by blocking with 5% nonfat dry milk for 60 min. After PBS-T washes, diluted mouse sera or hybridoma supernatants were added to the wells and incubated for 90 min. Plates were washed four times with PBS-T, followed by incubation with horseradish peroxidase (HRP)-conjugated goat anti-mouse Ig (1:1000) (BD Pharmingen) for 60 min. After three additional washing steps, 50 µl of tetramethylbenzidine substrate (Thermofisher Scientific) was added to each well, plates were incubated in the dark, and the reaction was stopped by the addition of 50 µl of 1 M HCl. Optical density was measured at 450 nm in a microplate reader. The mouse producing the highest titer of antibodies of interest was selected for fusion. To examine antibody production from hybridoma cells, the same procedure was implemented.

EXAMPLE III

Flow cytometry analysis and preparation

Binding of antibodies or hybridoma supernatants was determined by flow cytometry using human bladder and breast cell lines stably expressing STn and parental cells not expressing STn. Approximately 3*105 cells per condition were harvested and resuspended in PBS buffer. To assess the specific binding of the examined hybridoma supernatants and antibodies to sialylated structures, samples were treated with sialidase at 100 mU/ml, for 90 min at 37 °C. After sialidase treatment, cells were washed and incubated for 30 min at 4 °C with anti-STn mAbs B72.3, 3F1, TKH2 and hybridoma supernatants. Subsequent washing steps were performed and primary antibodies were detected with FITC-conjugated anti-mouse Ig (Dako; 1:10 dilution) for 15 min in the dark. After washing, data for each sample were acquired using a flow cytometer for each sample.

Example IV

Antibody production - Hybridoma technology

Splenocytes from the immunized mouse were mixed with Sp2/0 myeloma cells (ATCC, USA) at a 3:1 ratio and fused in the presence of polyethylene glycol/dimethyl sulfoxide using a standard protocol. Cells were then plated into 96-well flat-bottom microplates (Orange Scientific) and maintained in RPMI medium supplemented with HAT (1*10.4 M hypoxanthine, 4*10.7 M aminopterin, 1.6*10.5 M thymidine, Sigma-Aldrich), 10% FBS, 2 mM L-glutamine, 0.2 mg/ml gentamicin (Sigma-Aldrich), 1 mM sodium pyruvate (Gibco), 1% (v/v) MEM non-essential amino acids (Gibco) and incubated at 37 °C for 7-12 days. Hybridoma cells producing antibodies reactive against BSM were expanded and screened by indirect ELISA and cloned by the limiting dilution method at least three-fold to obtain stable single-clone cell lines. The selected hybridomas were cultured in the selection medium without HAT supplementation at 37 °C. An L2A5 hybridoma specific for sialylated structures, particularly STn, was selected and cloned by four-fold limiting dilution.

EXAMPLE V

Immunohistochemical analysis of STn expression

A series of 30 cases with 15 colorectal tumors (adenocarcinomas and adenomas) and 15 bladder tumors (eight cystectomies and seven metastases) were obtained in accordance with the local ethics committee. Additionally, five cases of normal colorectal tissue adjacent to the tumor were included. Formalin-fixed, paraffin-embedded (FFPE) tissues were examined for STn by immunohistochemistry (IHC) using the biotin/streptavidin system. Briefly, FFPE tissue sections were deparaffinized with xylene, rehydrated with a graded series of alcohol washes, and subjected to heat-induced antigen retrieval using citrate buffer pH 6.0 (Vector, Burlingame, USA) for 15 min in the microwave, after preheating the solution at maximum rated power for 5 min. Sections were incubated with 0.3% hydrogen peroxide (Merck KGaA, Darmstadt, Germany) for 25 min, blocked with UV Block® (Thermo Scientific, Fremont, USA) and incubated overnight at 4 °C in a humid chamber with anti-STn mAbs B72.3, TKH2 (Kjeldsen et al., 1988) and L2A5. After washing with PBS-Tween, secondary antibody was added to the tissue sections, prior to incubation with streptavidin. STn was visualized by incubation with 3,3'-diaminobenzidine (ImmPACT™ DAB) (Vector, Burlingame, USA) for 4 min. Finally, the nuclei were counterstained with hematoxylin for 1 min. STn expression was assessed using anti-STn mAbs B72.3, TKH2, and L2A5 hybridoma culture supernatant, diluted 1:5, 1:5, and 1:3 in 5% BSA in PBS, respectively. Positive and negative control sections were tested in parallel. Negative control sections were performed without primary antibodies. STn+ tumor tissues were used as positive controls. Tumors were scored as positive when immunoreactivity of the anti-STn antibody TKH2 was observed by microscopic presence of brown chromogenic product in tumor cells. STn expression and L2A5 staining were assessed in a double-blind manner by two independent observers and validated by an experienced pathologist. Whenever there was disagreement, slides were reviewed and a consensus was reached. In order to assess antibody specificity, sialidase treatment was performed after incubation with hydrogen peroxide, in which sialic acids are removed from the STn antigen, thus impairing antibody recognition. Therefore, positive staining after this enzymatic treatment (4 h at 37 °C; 0.2 U/mL) was considered nonspecific.

EXAMPLE VI

Western Blot (WB)

Membrane proteins were isolated from cell lines using membrane protein extraction kits, according to the manufacturer's instructions. The amount of protein obtained was estimated using protein assay kits, following the manufacturer's recommendations. Membrane protein extracts (50 pg) or purified proteins containing STn (1 pg) - BSM and MUC1 STn-IgG- - were denatured and loaded onto 8% gradient acrylamide gel, subjected to SDS-PAGE electrophoresis under reducing conditions and electrophoretically transferred to polyvinylidene difluoride (PVDF) membranes (Amersham Hybond P 0.2 |jm PVDF, GE Healthcare Life Sciences) according to standard procedures. Membranes were blocked with 10% nonfat dry milk in TBS 0.1% Tween (TBS-T) for 1 h, followed by incubation with supernatant of anti-STn B72.3, 3F1, or L2A5 primary antibodies diluted in TBS-T overnight at 4 °C. After washing with TBS-T, labeled proteins were revealed using HRP-conjugated goat anti-mouse Ig diluted 1:2500 in TBS-T for 1 h. After washing, labeled proteins were revealed by Lumi-Light Western Blotting Substrate (Roche) and then exposed to X-ray film.

EXAMPLE VII

mRNA isolation and cDNA synthesis

Between 1*10® and 5*10® hybridoma cells were used for RNA isolation. Cells were centrifuged for 5 min at 300xg and the supernatant was discarded. The cell pellet was washed with PBS and total RNA was isolated using the GenElute™ Mammalian Total RNA Miniprep Kit (Sigma-Aldrich), according to the manufacturer’s instructions. Extracted total RNA was quantified using Nanodrop and up to 2 µg was used for reverse transcription as described in the High Capacity cDNA Transcription Kit (Applied Biosystems). cDNA synthesis was performed using the following thermal cycling conditions: 25 °C for 10 min, followed by 37 °C for 120 min and 85 °C for 5 sec. Finally, reactions were held and cooled to 4 °C.

EXAMPLE VIII

Antibody sequencing - scFv fragments

The heavy (V<h>) and light (V<l>) chain variable domains of MAb L2A5 were amplified from cDNA using the primer pair V<h>forward (TTTTTGGATCCSARGTNMAGCTGSAGSAGTCWGG)/V<h>reverse (ATTGGGACTAGTTTCTGCGACAGCTGGATT) and V<l>forward (TTTTTGAATTCTGAYATTGTGMTSACMCARWCTMCA)/V<l>reverse (TTTTTGGGCCCGGATACAGTTGGTGCAGCATC). All PCRs were performed using the Advantage HF 2 PCR kit (Clontech). The following thermal cycling conditions were used: initial melting at 94 °C for 3 min, followed by 95 °C for 45 s, 70 °C for 1 min, and 68 °C for 2 min. Reactions were then held at 68 °C for 5 min and cooled to 4 °C. Purified PCR products were further cloned into the pGEM-Teasy cloning vector (Promega) according to the manufacturer's protocols. Plasmids were isolated using the QIAGEN plasmid plus midi kit (QIAGEN) according to the manufacturer's protocol. Sequencing was performed by Seqlab (Gottingen, Germany) using the T7 promoter primer for the pGEM-Teasy vector.

These compositions can be produced with consistent quality for clinical and diagnostic applications.

EXAMPLE IX

Antibody domains

The heavy chain (VH) and light chain (VL) variable nucleotide sequences encoding the amino acid sequences of the FR and CDR domains of mAb L2A5 were determined by searching the IMGT V domain delineation system (ImMunoGeneTics international database; http://imgt.cines.fr) using the IgBLAST sequence analysis tool.

Example X

Cloning of nucleic acids into a target module

The anti-STn targeting module (TM) was made as described (Cartellieri et al 2016) but replacing the CDR regions with LA25 nucleic acid sequences. T cell-mediated tumor killing was measured using standard chromium release assays. MDA-MB-231 and MCR STn+ cell lines were incubated with T cells engrafted with either the vector control (vector backbone encoding only the EGFP marker protein), the UniCAR stop construct (lacking intracellular signaling domains), or the α-E5B9 signaling construct (UniCAR 28/Z) (Mitwasi 2017). Both cell lines were cultured with the respective genetically modified T cells in the presence or absence of 80 nM anti-STn TM (α-STn TM) for 24 h at an effector-to-target (E:T) ratio of 5:1.

Example XI

Antitumor activity in vivo

MDA-MB-231 STn cells were transduced to express firefly luciferase (Luc) resulting in MDA-MB-231 STn-Luc cells. The anti-STn targeting module (TM) was made as described (Cartellieri et al 2016) but replacing the CDR regions with LA25 nucleic acid sequences. Per mouse, 1.5*106 tumor cells were mixed with 1*106 UniCAR 28/Z T cells and 10 |jg of anti-STn TM. MDA-MB-231 STn-Luc cells (1.5*106) were used alone or mixed with 1*106 UniCAR 28/Z T cells without TM as untreated controls. The respective mixture was injected subcutaneously into female NMRI-Foxn1nu/Foxn1nu mice resulting in three groups of animals each consisting of five mice. Luminescence imaging of anesthetized mice was performed 10 min after i.p. injection of 200 µl of D-luciferin potassium salt (15 mg/ml) starting on day 0 and followed on days 1, 3, 6 and 8.

Table 1 shows an example of the nucleotide sequence of the variable heavy (VH) and variable light (VL) chains of clone L2A5 identified as SEQ ID NO. 1 and 2, respectively, and the encoded amino acid sequence of the variable heavy (VH) and variable light (VL) chains identified as SEQ ID NO. 3 and 4, respectively.

Table 1: Example of nucleotide sequence and encoded amino acid sequence of the variable light (VL) and variable heavy (VH) chains of clone L2A5.

Table 2 shows the encoded amino acid sequence of the six framework regions (FR) and six complementarity determining regions (CDR) (H-CDR1, H-CDR2 and H-CDR3, L-CDR1, L-cDr2, L-cDr3) for clone L2A5 and are identified as the nucleotide sequence of SEQ ID NO: 5 to 16.

Table 2: Encoded amino acid sequence of the three framework regions (FR) and three complementarity determining regions (CDR) (H-CDR1, H-CDR2 and H-CDR3, L-CDR1, L-CDR2, LCDR3) for clone L2A5.

Claims (15)

1. An antibody comprising a combination of a light chain variable region (VL) and a heavy chain variable region (VH), where: the VL comprises complementarity determining regions (CDRs) L-CDR1, L-CDR2 and L-CDR3 as set forth in SEQ ID NO. 6, 8 and 10, respectively; and, the VH comprising the Cd R H-CDR1, H-CDR2 and H-CDR3 as set forth in SEQ ID NO. 12, 14 and 16, respectively. 2. An antibody according to claim 1, wherein the VL comprises SEQ ID NO. 5, 7 and 9; and the VH comprises SEQ ID NO. 11, 13 and 15. 3. An antibody according to claim 1 or 2, wherein the VL comprises SEQ ID NO. 4 and the VH comprises SEQ ID NO. 3. 4. An antibody according to any preceding claim that binds to STn and a group of glycans terminated by alpha 2,6-linked sialic acids. 5. An antibody according to any preceding claim, wherein the glycans terminated by alpha 2,6-linked sialic acids comprise STn, 2,6-sialyl T, di-sialyl T or 2,6-sialolactosamine. 6. An antibody according to any preceding claim that is subject to glycan changes at glycosylation sites. 7. An antibody according to any preceding claim which is a monoclonal antibody, chimeric antibody or a humanized antibody. 8. An antibody according to any preceding claim which is a functional antibody fragment thereof, which binds to STn and a group of glycans terminated by alpha 2,6-linked sialic acids. 9. A pharmaceutical composition comprising an antibody according to any preceding claim and a pharmaceutically acceptable carrier. 10. A method for detecting a tumor in a subject using an antibody according to any preceding claim, the method comprising: staining a biological sample in vitro, obtained from a subject, with the antibody according to any preceding claim under conditions suitable for specific binding of the antibody, wherein the presence or absence of binding of the antibody is indicative of the presence or absence of tumor cells expressing cell surface STn, 2,6-sialyl T, di-sialyl T or 2,6-sialolactosamine, optionally wherein the biological sample comprises isolated cells, or tissue, or proteins derived from tumors. 11. An antibody according to any one of claims 1 to 8, or a pharmaceutical composition comprising the antibody, for use in medicine. 12. An antibody according to any one of claims 1 to 8, or a pharmaceutical composition comprising the antibody, for use in the treatment of cancer. 13. An expression vector comprising a polynucleotide encoding an antibody according to any one of claims 1 to 8, optionally wherein the polynucleotide comprises SEQ ID NO. 1 and/or SEQ ID NO. 2; nucleotide sequences T<c>AAGTGTAAGTTAC, GACACAT<c>C and CAGCAGTGGAGTAGTGACCCACCCATGCTCACG; or nucleotide sequences GGCTACTCCATCACCAGTGGTTATTAC, ATAAACTACGACGGTAGCAAT and GCAAGAGGGGGGGACTAC. 14. A host cell comprising an expression vector according to claim 13. 15. A method of producing an antibody according to any one of claims 1 to 8, which method comprises using a host cell according to claim 14.