WO2019197974A1

WO2019197974A1 - Anti-telomerase reverse transcriptase antibodies and use thereof

Info

Publication number: WO2019197974A1
Application number: PCT/IB2019/052886
Authority: WO
Inventors: Meir Lahav; Orit Uziel; Dan Peer; Olga KARPOV
Original assignee: Mor Research Applications Ltd.; Ramot At Tel Aviv University Ltd.
Priority date: 2018-04-09
Filing date: 2019-04-08
Publication date: 2019-10-17
Also published as: EP3774924A1

Abstract

Antibodies that recognize and attach to human telomerase reverse transcriptase (hTERT) peptides presented by tumor cells are provided. These antibodies are useful in detection and isolation of circulating tumor cells (CTCs), in diagnosing cancer and in monitoring response to cancer therapy in a subject, in cancer-type independent manner.

Description

ANTI-TELOMERASE REVERSE TRANSCRIPTASE ANTIBODIES AND USE THEREOF FIELD AND BACKGROUND

The present disclosure, in some embodiments thereof, relates to anti- telomerase antibodies, more particularly, but not exclusively, to anti-human telomerase reverse transcriptase antibodies and use thereof for diagnosing cancer.

In spite of major advances in understanding the biological pathways underlying malignant transformation and subsequent development of rationalized drugs to combat the disease, the diagnosis area still lags behind and presents an unmet need. Liquid biopsies and solid biopsies are examples of some of the current diagnosis technique. Solid biopsies obtained from the tumor mass itself involve body penetration, cause pain and clinical complications, and contain a heterogeneous population of tumor as well as other non-cancer cells, which may be lead to miss- interpretation (e.g., false negative or false positive diagnosis). Circulating tumor cells (CTCs) provide liquid biopsies which may be applicable is non-invasive diagnosis, genetic analysis and therapeutic decisions in cancer, and as a possible tool for detection and quantification of tumor mass. Available methods for isolation of CTCs are specific for certain types of cancer. No universal, cancer type-independent marker is currently known that enables a successful identification and/or isolation of CTCs of any type of ancer.

Telomeres are highly conserved genetic elements located at the ends of all eukaryotic chromosomes, which preserve genome stability and cell viability by preventing aberrant recombination and degradation of DNA. In humans, the telomeric sequence is composed of 15-20 kilobases of TTAGGG repeats. There is increasing evidence that gradual loss of telomeric repeat sequences may be a timing ("clock") mechanism limiting the number of cellular divisions in normal somatic cells. In contrast, immortal cells are capable of maintaining a stable telomere length by upregulating or reactivating telomerase, an enzyme able to add TTAGGG repeats to the ends of chromosomes.

Telomerase is a multi-subunit ribonucleoprotein enzyme, consisting of one RNA component and one protein subunit, both of which are necessary for its activity. The catalytic subunit, termed telomerase reverse transcriptase (TERT), exhibits reverse transcriptase activity and utilizes the RNA template to catalyze the addition of telomeric DNA to chromosomal ends.

Most cells in adult humans do not exhibit telomerase activity, except for germ line tissues (sperm cells and oocytes) and certain blood cells. However, telomerase activity has been detected in at least 90% of primary human tumors tested from a variety of tissue types (Kim et al., (1994) Science, 266: 2011-2015; Shay and Bacchetti, (1997) European Journal of Cancer, 33: 787-791). The detection of high telomerase activity in human cells or tissues almost always correlates with indefinite proliferation capability (immortalization). U.S. Paten. No. 5,648,215 describes the presence of telomerase activity in somatic cells as indicative of the presence of immortal cells, such as certain types of cancer cells, which are used for determining the presence of cancer even when the cells would be classified as non-cancerous by pathology.

Peptides of TERT are expressed on the outer membranes of cancer cells but not on membranes of normal somatic cells, in which the gene coding for telomerase is suppressed. TERT is thus the hallmark of a cancer cell. Few of the TERT peptides are well characterized, including PEP 544, LMSVYVVELLRSFFYVTE (hTERT (548-566)), PEP 508; KRAS LLDILDTAGHEEYSAMRDQ ((52-70) Q61H); 1540 ILAKFLHWL; HR2822 ILAKFLHWL (hTERT 540-548), RLVDDFLLV (hTERT 865-873); and GV1001 CEARPALLTSRLRFIPK (hTERT (610-626). Some of these hTERT antigens (for example, GV1001) were used for producing vaccines against human hTERT.

SUMMARY

As liquid biopsies, particularly circulating tumor cells (CTCs) biopsies, are emerging promising approaches in cancer diagnosis, there are yet at least two challenges in CTCs-based diagnosis of cancer: isolation of CTCs and a method for detection of cancer, which will not be cancer type specific, and as such will enable detection of cancer in vivo and ex vivo both in liquid and solid tissues. Active telomerase, a common and unique property of almost all cancer cells is utilized, in accordance with the present disclosure, for providing an "antipersonalized" universal tool for detecting almost all types of cancer. Aspects of the present disclosure relate to the provision of antibodies designed to recognize and attach to human telomerase reverse transcriptase (hTERT) peptides presented by tumor cells originating from about 90% of the various cancers. Based on recognition of telomerase active cells in the human body, sine qua non of cancer cells, use of these anti-hTERT antibodies for the in vivo and ex-vivo isolation of CTCs, as well as in vivo identification and diagnosis of almost all types of cancers is provided herein.

As a non-limiting example, the production and utilization of a novel antibody (herein designated LU-001) which recognizes and attaches to the synthetic peptide GV1001 (EARPALLTSRLRFIPK), corresponding to the hTERT (611-626) fragment is described. GV1001 is a promiscuous HLA class II epitope, a property that has made this peptide a candidate for a cancer vaccine that may be administered without prior HLA typing of the patients. The antibodies provided herein attach to CTCs as well as to various other types of cancers.

In an aspect of the present disclosure, there is provided an antibody that specifically binds one or more telomerase reverse transcriptase (TERT) peptides, wherein the antibody comprises a heavy chain comprising the nucleic acid sequence of SEQ ID NO:4, and a light chain comprising the nucleic acid sequence of SEQ ID NOG, a fragment thereof or a homolog thereof. In exemplary embodiments, the light chain comprises a variable light region (V_L) comprising the amino acid sequence of SEQ ID NO:6.

In some embodiments, the anti-TERT antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NOG, and a light chain comprising the amino acid sequence of SEQ ID NO: 1, a fragment thereof or a homolog thereof.

The anti-TERT fragment is an antigen-binding fragment that specifically binds one or more TERT peptides, for example, Fab, F(ab')2, Fd, Fv, dAb, scFv, and a complementarity determining region (CDR). In exemplary embodiments, the antigen binding fragment is F(ab')2.

In some embodiments, the TERT peptide is a human telomerase reverse transcriptase (hTERT) peptide. In exemplary embodiments, the hTERT peptide is the peptide GV1001 comprising the amino acid sequence identified herein as SEQ ID NOG.

In some embodiments, the anti-TERT antibody is a monoclonal antibody. A contemplated antibody may be a chimeric antibody comprising an antigen binding fragment that specifically binds one or more TERT peptides, fused to a human IgG or IgM constant region.

In some embodiments, a homolog of a contemplated anti-TERT antibody or a of a fragment thereof may have 80-99.9% identity.

A contemplated anti-TERT antibody or an antigen-binding fragment thereof may be, in some embodiments, a dual specificity antibody, namely, it may specifically bind two or more different but structurally related TERT peptides, for example, peptide is a peptide of SEQ ID NO: 5.

A disclosed anti-TERT antibody or an antigen-binding fragment thereof may conjugated to one or more of: (a) a tag useful for purification and detection; (b) a chemical moiety that alters the physical properties of the antibody; (c) an immunoadhesion peptide or protein; and (d) a drug, e.g., and anti-cancer drug.

In an aspect of the present disclosure there is provided a composition comprising at least one of a contemplated anti-TERT antibody, a fragment thereof or a homolog thereof, and a physiologically acceptable excipient. The composition may be useful for diagnosis and/or for therapy.

In yet another aspect of the present disclosure, methods for diagnosing cancer, treating cancer and isolating CTCs are provided. Some embodiments pertain to a method for diagnosis of cancer in a subject, the method comprising: (a) obtaining a biological sample suspected of containing cancer cells from the subject; (b) contacting the sample with an anti-TERT antibody, a fragment thereof or a homolog as described herein; and (c) measuring binding of the antibody, fragment or homolog thereof to TERT -presenting cells in the sample, wherein detection of binding indicates the presence and amount of cancer cells in the biological sample.

In some embodiments, a method for isolating and detecting cancer cells, for example, CTCs, in a sample is provided. A biologic sample is obtained and contacted with an anti-TERT antibody, a fragment thereof or a homolog as described above, the cells bound to the anti-TERT antibody are subjected to isolation means, isolated and binding of the antibody is detected and measured. Detection of binding indicates the presence and amount of cancer cells in the biological sample. The isolation means may be, for example, magnetic beads, and isolation of cells attached thereto may be achieved by exposing the sample to an external magnetic field.

In some embodiment, the present disclosure provides a method for diagnosing cancer and monitoring response to cancer therapy in a subject, wherein a diagnostically effective amount of a contemplated anti-TERT antibody, a fragment thereof or a homolog thereof is administering to a subject suspected of having cancer, or a subject subjected to anti-cancer treatment, and the binding of the anti-TERT antibody, a fragment thereof or a homolog thereof is measured. Detection of binding indicates the presence or level of cancer in the subject. Optionally, the anti-TERT antibody, fragment thereof or homolog thereof is labeled with one or more detectable substances such as an enzyme, a prostatic group, a fluorescent material, a luminescent material, a radioactive material, a contrast agent, an isotope and/or inorganic micro- or nanoparticles.

In some embodiments, a method for treating cancer in a subject is provided, the method comprising administering to a subject inflicted with cancer a therapeutically effective amount of a contemplated an anti-TERT antibody, a fragment thereof or a homolog thereof that specifically recognizes one or more telomerase antigens presented by cancer cells according, wherein the anti-TERT antibody, fragment thereof or homolog thereof is conjugated to an anti-cancer drug, thereby treating cancer in the subject.

In an aspect of the present disclosure, a nucleic acid molecule, is provided, selected from (i) a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:3, a fragment thereof or a homolog thereof; (ii) a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:4, a fragment thereof or a homolog thereof; (iii) a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:3 and a nucleotide sequence of SEQ ID NO:4, a fragment thereof or a homolog thereof. Further provided are recombinant or engineered cells or cell lines, vectors, plasmids and artificial chromosomes which comprise the disclosed nucleic acid. In exemplary embodiments, the recombinant or an engineered cell or cell line is a cancer cell or a cancer cell line, or an immortalized cell line.

In a further aspect, the present disclosure provides a hybridoma that can produce an antibody or a fragment thereof that specifically binds one or more TERT peptides. In exemplary embodiments, the antibody is the anti-hTERT antibody herein identified as LU-001.

In yet a further aspect of the present disclosure, there is provided a kit comprising at least one of: an anti-TERT antibody, an antigen- binding fragment thereof and/or as contemplated herein; reagents required for the detection and quantification of the antibody; and instructions for use. A contemplated kit may be useful in in vitro and in vivo diagnosis of cancer.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the described herein, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the present disclosure are herein described, by way of example only, with reference to the accompanying drawings. It is stressed that the particulars shown in the drawings are by way of example and for purposes of illustrative discussion of embodiments described herein. The description taken with the drawings makes apparent to those skilled in the art how embodiments of the present disclosure may be practiced.

In the drawings:

Fig. 1 is a bar graph showing level of binding of the anti-hTERT antibody LU- 001 and of the mouse IgGl isotype (control) to the hTERT peptide GV1001 (SEQ ID NO:5). The optical density (OD; Y axis) indicates the level of binding;

Fig. 2 is a bar graph showing percentage of binding of the anti-hTERT antibody LU-001 to various cells in vitro. Cell-binding was measured by FACS;

Figs. 3A-3B show FACS analysis of ex-vivo binding of anti-hTERT mAh LU- 001 to cancer cells isolated from chronic lymphatic leukemia (CLL) patients. Fig. 3 A is a histogram showing the count of cells bound to fluorescently labeled anti-LU-OOl antibody (red, right line), and cells bound to an isotype control (green, left line), versus fluorescence intensity of FITC. Fig. 3B is dot plot showing fluorescence intensity obtained from CLL cells bound to different anti-LU-OOl fluorescent antibodies or to an iso type control;

Fig. 4 is a FACS dot plot showing fluorescence intensities obtained from ovary cancer cells bound (ex vivo ) to anti-hTERT antibody LU-001. Red dots (upper right quarter): cells bound to FlTC-conjugated anti-LU-OOl antibody; blue dots (lower right quarter): cells bound to FITC-conjugated anti-LU-OOl antibody stained with a control dye; orange dots (left upper quarter): cells bound to isotype controls conjugated to FITC; green dots (lower quarter): cells bound to isotype controls conjugated to FITC and stained with a control dye;

Fig. 5 is a FACS histogram showing the count of healthy lymphocyte cells bound to FITC-conjugated anti-LU-OOl antibody from 2 different batches (red and purple lines), and cells bound to a fluorescently labeled isotype control (green line), versus fluorescence intensity of FITC;

Figs. 6A-6B show purification and binding affinity of F(ab')₂ fragment of anti- hTERT mAh LU-001. Fig. 6A is a photograph of two SDS gel panels showing the separation and purification of the anti-hTERT mAh LU-001 (left panel), and of its F(ab')₂ fragment (right panel) (“M” denotes a marker). Fig. 6B is a bar graph showing binding level to the immobilized hTERT peptide GV1001 (SEQ ID NO:5) of F(ab')₂ fragments, mouse IgGl isotype (control), and a binding buffer (negative control, no Abs), as measured in two ELISA assays. The optical density (OD; Y axis) specifies the level of binding;

Figs. 7A-7D show ex vivo binding of anti-hTERT mAh F(ab')₂ fragment to chronic lymphocytic leukemia (CLL) as assessed by flow cytometry. FACS dot plots showing fluorescence intensities of Alexa Fluor® 647 (conjugated anti-mouse secondary antibody) obtained from CLL lymphocytes bound to: a mouse IgGl isotype (negative control) (7 A), anti-hTERT antibody LU-001; positive control (7B), and F(ab')₂ fragment of two different preparations (7C, 7D);

Fig. 8 is a schematic representation of the step-wise process for isolating circulating tumor cells (CTCs) in a biological sample using magnetic beads that contact anti-telomerase antibodies attached to the CTCs via bonding to telomerase antigens presented by the CTCs; and

Fig. 9 is FACS dot plot showing fluorescence intensities obtained from chronic lymphocytic leukemia (CLL) lymphocytes isolated from a blood sample of a CLL patient by (ex vivo ) attachment to magnetic beads conjugated to anti-hTERT antibody LU-001.

DETAILED DESCRIPTION

The present description, in some embodiments thereof, relates to anti- telomerase antibodies, more particularly, but not exclusively, to anti-human telomerase reverse transcriptase antibodies and use thereof for diagnosing cancer and isolating circulating tumor cells.

The term“anti-hTERT antibody”, as used in the context of various aspects and embodiments of the present disclosure, encompasses monoclonal antibodies and fragments thereof produced against peptides derived from the catalytic subunit telomerase reverse transcriptase (TERT) protein and are presented on the membrane of most cancer cells. These peptides, currently, have no known catalytic activity of their own. Non-limiting examples of antigenic hTERT peptides useful for the purpose of embodiments described herein include PEP 544, LMSVYVVELLRSFFYVTE (hTERT (548-566)), PEP 508; KRAS LLDILDTAGHEEYSAMRDQ ((52-70) Q61H); 1540 ILAKFLHWL; HR2822 ILAKFLHWL (hTERT 540-548),

RLVDDFLLV (hTERT 865-873); and GV1001 CEARPALLTSRLRFIPK (hTERT (610-626). The anti-hTERT antibodies described herein are to be distinguished from the anti-hTERT antibodies described in U.S. Patent Nos. 7,078,491 and 6,639,057, which are produced against the endogenous catalytic protein itself (as a whole) or any polypeptide derived therefrom and have catalytic properties of the catalytic subunit of human telomerase.

It has been envisaged by the present inventors that telomerase reverse transcriptase peptides presented on the membrane of most cancer cells could be utilized for the development of an anti-hTERT antibody-based tool for isolating circulating tumor cells (CTCs), and for the diagnosis and identification of cancer cells and cancer tissues both in situ, namely in the patient’s body, and ex-vivo in a biological sample obtained from the patient. The present inventors have synthesized and characterized new anti-hTERT specific monoclonal antibodies (mAbs) which presented specificity only to TERT positive cells, both in vitro and ex vivo, and did not bind to cells which did not present the corresponding antigenic peptides for example, non-malignant cells.

In the process of realization of the embodiments described herein, the present inventors have validated the specificity of the anti-hTERT antibodies to CTCs in blood samples of different patients with cancers of various origins, both solid cancers such as ovarian, lung, hepatobiliary, breast, prostate, pancreas, gastric and colon cancers, as well as hematological cancers such as various types of lymphoma, acute leukemia, and chronic myeloid leukemia. As shown in the Examples section herein, an exemplary mouse anti-hTERT mAb, newly synthesized by the present inventors, termed herein“LU-001”, and fragments thereof, bound and identified cancer cells of hematological malignancies such as chronic myeloid leukemia and acute leukemia as well as circulating tumor cells of various solid cancers. For example, the antibody was able to bind, in vitro, cells of cancerous cell lines such as MCF-7, Jurkat, K562, A549 and U266 cells. This exemplary antibody did not bind cells from a non-cancer origin.

Anti-telomerase antibodies

Described herein are novel antibodies specific for peptides or proteins derived from the telomerase catalytic subunit, and presented by cancer calls, e.g., as antigenic determinants in the membrane of intact cells (also termed herein“membrane antigens” and“cell surface antigens”). The contemplated antibodies are termed herein“anti- TERT antibodies”. In some exemplary embodiments, the anti-TERT antibody is anti human TERT antibody (“anti-hTERT antibody”).

An antibody (Ab), also referred to herein and in the art as immunoglobulin is a glycoprotein molecule produced mainly by plasma cells (differentiated B cells) of the humoral immune system in response to the presence of antigens which may be foreign substances, for example, pathogens such as bacteria and viruses, or autologous substances such as peptides and proteins produced by the body. The region of an antigen that interacts with an antibody is called the epitope. Many of the key structural features of antibodies can be highlighted using immunoglobulin G (IgG) as a model since IgG is the most abundant antibody in serum and the major Ig in extravascular spaces. The classical representation of an antibody is as a Y-shaped molecule composed of four polypeptide subunits with two identical heavy and light chains. N- terminus of each heavy chain associates with one of the light chains to create two antigen-binding domains. These are the arms of the“Y” shape and are each of which is termed a“fragment antigen binding” (Fab) domain. The C-termini of the two heavy chains combine to form the fragment crystallization (Fc) domain, which represents the tail or leg of the“Y”. The Fc domain is important for the antibody’s interaction with effector cells such as macrophages and for activation of the complement cascade. The four polypeptide chains are held together by covalent disulfide bridges and non-covalent bonds.

IgG light chains are approximately 220 amino acids in length (approximately 25 kDa) and can be divided into two equal 'variable' and 'constant' regions. The highly variable N-terminal region is termed“VL”, and a constant C-terminal region is termed “CL”. Fleavy chains are about 440 amino acids in length (approximately 50 kDa) and are subdivided into four 110 amino acid segments - one variable region and three constant regions. The variable and constant regions of the heavy chain are termed “VH” and“CH”, respectively.

The sequence heterogeneity within the variable regions is not random but occurs at three short (5-30 amino acids in length) segments within each chain called the hypervariable regions (Hvl, Hv2, Hv3). Since the residues in hypervariable regions form the actual binding site for the antigen, they are also known as the “complementarity determining regions” or“CDRs”. Four framework regions (FR) which have more stable amino acids sequences separate the HV regions. The variable regions of both light and heavy chains localize to the Fab domains, providing the structural basis for the antigen and epitope-selectivity of antibodies. The three constant regions of the heavy chain identified as CHI , CH2 and CH3 form the Fc domain.

The terms“antigen binding segment” or“antigen binding fragment” as used herein, as interchangeable, and encompass, for example, (i) fragment antigen-binding (Fab) as defined herein; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab’ fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains; (v) a dAb fragment, which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules known as single chain Fv (scFv). The VH and VL fragment may be linked by a spacer peptide such as a glycine-serine linker, having, for example, 5 to 20 amino acid residues (for example, the sequence GGGGS or GGGSAAA).

Other forms of single chain antibodies, such as diabodies are also encompassed. Diabodies are bivalent, bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but using a linker that is too short to allow for pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains of another chain and creating two antigen binding sites (see e.g., Flolliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak et al. (1994) Structure 2: 1121-1123).

The light chains of an antibody can be classified as either kappa (K) or lambda (l) type based on small differences in polypeptide sequence, with kappa light chains being the more common of the two. In mammals, antibodies are divided into five isotypes: IgG, IgM, IgA, IgD and IgE, based on the number of Y units and the type of Fc fragment. The isotypes differ in their biological properties, functional regions and ability to interact with different antigens. There are five types of mammalian Ig heavy chain denoted by Greek letters: a, d, e, g and m which are found in IgA, IgD, IgE, IgG and IgM isotypes, respectively. Isotype IgA may comprise al or a2 heavy chain and is identified as isotypes IgAi or IgA₂, respectively. IgG consists of four human subclasses (IgGi, IgG₂, IgGi and IgG₄) which are highly homologous but they differ in their Fc region, in the hinge region (IgGi and IgG₄ contain two inter-chain disulphide bonds in the hinge region, IgG₂ has 4 and IgG_¾ has 11 inter-chain disulphide bonds), and the extent to which they activate the host immune system. In mice, the IgG class is divided into five sub-classes (IgGi, IgG_2A, IgG_2B, IgG₂c and IgG₃) and in rat there are four (IgGi, IgG_2A, IgG_2B, IgG₂c). Sub-class nomenclature has arisen independently for each species and so there is no general relationship between the sub-classes from each species. Fc regions of IgG and IgM can bind to receptors on the surface of immunomodulatory cells such as macrophages and stimulate the release of cytokines that regulate the immune response.

A contemplated anti-TERT antibody described herein is an IgG isotype, for example, anti-hTERT isotype designated herein“a anti-hTERT” isotype, which may be cleaved with suitable proteases to thereby obtain fragments of anti-TERT antibody. For example, cleavage of a contemplated antibody with the protease papain, produces two identical Fabs and an Fc fragment, each conferring the biological activity of the antibody such as binding to a TERT peptide or binding to Fc-receptor bearing cells. Other enzymes such as pepsin and Immunoglobulin degrading enzyme from Streptococcus pyogenes (IdeS) cleave below the hinge region to form a F(ab')2 fragment and a pFc' fragment (as referred to in Example 4 herein). The F(ab')2 fragment can be split into two Fab' fragments by mild reduction.

Each of the F(ab’)2, Fab and Fc can be further cleaved using known means to obtain, e.g., separate variable and constant regions. Additionally or alternatively, specific regions, such as CDRs, hypervariable, framework, and/or constant regions of the sequenced antigen may be synthesized and some fragments of interest may be fused together to thereby obtain modified and chimeric anti-TERT antibodies presenting higher affinity, higher selectivity, multiple affinity/selectivity to one or more antigens, and/or reduced immunogenicity. For example, the variable regions of the heavy and light chains can be fused together to form a single-chain variable fragment (scFv), which is only half the size of the Fab fragment, yet retaining the original specificity of the parent immunoglobulin.

Alternatively, a single antibody molecule may comprise such fragments derived from two separate antibodies to create a dual-specific or heterodi meric molecule. The anti-TERT Ab is also referred to herein in terms of its functional fragments Fab, F(ab')2, scFv, pFc' and Fc as defined herein.

The anti-TERT antibodies described herein are monoclonal antibodies (mAh). The term“monoclonal antibodies”, as used herein, refers to antibodies of one type, namely copies of one type of antibody, presenting the same specificity.

A contemplated Ab or a fragment thereof specifically binds one or more human or non-human TERT peptides. In some embodiments, the antibody is an anti human TERT (hTERT) antibody. The term“human antibody” refers herein to antibody having variable and constant regions corresponding to, or derived from, human germline immunoglobulin sequences. The human anti-human TERT antibodies described herein may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, particularly in CDR3. In some embodiments, the anti-hTERT Ab is a fully human monoclonal antibody, namely it comprises a human IgG constant region fused to a human variable region that specifically binds to one or more TERT antigens.

Some embodiments for productions of anti-TERT mAbs, for example anti- hTERT mAbs, employ hybridoma methods well known in the art. Some alternative embodiments for production, expression and isolation of anti-TERT mAbs and any fragments thereof employ recombinant means such as, but not limited to, transgenic animals, recombinant expression vectors transfected into a host cell and any other means that involve splicing and ligation of particular immunoglobulin gene sequences to other DNA sequences. For example, human anti-hTERT mAbs may be produced by transgenic mice, namely mice which have undergone inactivation (silencing) of endogenous mouse genes and insertion of exogenous human immunoglobulin. Use of transgenic mice expressing human immunoglobulins avoids human anti-mouse antibody responses and maintains the technical advantages of mouse hybridomas. Alternatively, human anti-TERT mAbs may be produced by recombinant expression of human antigen-binding fragments in a bacteriophage or a lentivirus and subsequent selection based on desirable antigen-binding properties.

Human anti-TERT antibodies are useful for in vivo detection and identification of cancerous tissues and circulating cancer calls, while causing a minimal immunological response when administered to humans. Reducing further the immunogenicity of human anti-TERT mAbs may be achieved, for example, by production of allotypical variants of the mAbs to match the specific immunoglobulin gene segment alleles found in the genomes of distinct patient populations, since phenotypes and immunoglobulin G allotypes differ within and between populations. Additionally or alternatively, protein engineering on the CDR of the mAh may be employed in order to reduce immunogenicity of the mAbs. In some embodiments, the monoclonal anti-TERT antibodies may comprise glycoproteins of the IgG type solely of animal origin, for example originating from mouse, rabbit or goat. These m Ahs are referred to herein as“non-human monoclonal antibodies”. Non-human anti-TERT mAbs, are highly useful in in vitro and ex-vivo detection, identification and isolation of CTCs, and in in vitro detection of cancerous tissues. However, non-human antibodies may be immunogenic, causing an undesired human anti-antibody response, which may result in their removal from the circulation. Non-human antibodies with reduced antigenicity may be obtained by certain modification of the non-human Ab such as replacing immunogenic regions with corresponding non-immunogenic regions originating, for example, from human immunoglobulin, to thereby obtain a reduction, i.e. abrogation or decrease in a statistically or biologically significant manner, in antigenicity of the antibodies when presented to the human immune system.

In some embodiments, the anti-TERT m Ahs described herein are chimeric monoclonal antibodies (cmAbs). “Chimeric antibody” (cAb), as used herein, refers to an antibody made by fusing the antigen binding region, for example, Fab, (Fab’)₂ or the variable domains of the heavy and light chains, VH and VL, respectively, of one species like a non-human mammal (e.g., murine, rat, rabbit), with the constant domain (Fc) of human (for example, a human IgG or IgM constant region). Non-limiting examples of such chimeric antibodies include rabbit/human cmAb, and mouse/human cmAb.

In some embodiments, the chimeric anti-telomerase mAh is a“CDR-grafted antibody”, namely an antibody in which the sequences of one or more CDR regions of the VH and/or VL of a non-human species are replaced with CDR sequences of human. For example, antibodies having murine constant regions and murine heavy and light chain variable regions in which one or more of the murine CDRs (e.g., CDR3) has been replaced with human CDR sequences. The more human components are fused with non-human antibody, the more “human-like” or “humanized” the antibody becomes. In a humanized Ab, a larger part of the immunoglobulin is derived from human sequences then in non-humanized Ab, for example, an antibody which comprises heavy and light chain variable region sequences from a non-human species (e.g., a mouse) in which at least a portion of the VH and/or VL sequence has been altered to be more similar to human germline variable sequences. Chimeric antibodies retain the original antibody’s antigen specificity and affinity and may be valuable for in vivo and in vitro TERT-based detection and isolation of cancer cells and cancer tissues.

In some embodiments, the anti-TERT antibody is a humanized antibody. For example, an anti-TERT antibody comprising a human IgG or IgM constant region fused to humanized mouse variable region in which human CDR sequences are introduced into mouse VH and VL sequences to replace the corresponding e.g., mouse CDR sequences. A further humanized anti-TERT Ab is exemplified by an antibody comprising the CDRs of a non-human, for example, murine, monoclonal antibody grafted into a human conserved framework region. In some embodiments, the humanized antibodies comprise 5-10% sequences derived from non-human antibodies and 90-95% sequences derived from human antibodies.

The human IgG heavy and light constant domains may be derived from any one of IgGl, IgG2, IgG3, and IgG4 subclasses of human IgG antibodies, and may comprise one, two or three intact or truncated constant domains (CH1 -3), which may optionally be mutated to alter effector function or provide for heteromultimer formation, or be modihed post-translationally (e.g. glycosylation) to improve the half- life of the antibody. In some embodiments, the IgG constant region is a human IgGl constant region.

A contemplated anti-TERT Ab, e.g., anti-hTERT mAh, may be specific to a single epitope of a TERT peptide. Alternatively, the anti-TERT Ab may have predictable dual or multiple specificities, namely, the antibody may have true specificities for two, three, four, five or more different epitopes. The two or more different epitopes may be parts of two or more structurally-related, yet different, TERT peptides, or the two or more different epitopes may be parts of the same TERT peptide.

In some embodiments, a dual specificity anti-TERT antibody or an antigen binding fragment thereof are provided, that specially bind to epitopes present on at least two TERT antigens. The term“dual specihcity antibody”, as used herein, is intended to include antibodies that specihcally recognize even more than two different but structurally related antigens, such as antibodies that recognize three, four, hve or more structurally related but distinct antigens. The term“different but structurally related antigens”, as used herein, is intended to include TERT antigens whose overall structures are related or similar as well as TERT antigens which share one or more structurally-related regions but are otherwise unrelated or dissimilar.

A dual specificity antibody described herein may display similar or equal binding activities toward two or more different but structurally related TERT antigens to which it binds or, alternatively, the dual specificity antibody may bind with high spcificity to one antigen, yet still have specificities towards the other two or more related antigens as compared to unrelated antigens.

Many designs have been put forward for creating dual-specific antibodies. One example is based on the so called“knobs into holes” concept (see, for example, U.S. Patent No. 7,642,228 and No. 8,653,242). For example, variable regions having specificity to a first TERT antigen may fused, for example, to one IgG heavy chain molecule and other variable regions, having specificity to a second TERT antigen, may be fused to another IgG heavy chain, and the two IgG heavy chain molecules may be associated directly or indirectly, e.g., via an interface molecule. A heteromultimer is thus formed having binding-specificity for both the first and the second TERT antigens. An exemplary dual specificity antibody is a heteromultimer comprising (a) a single chain variable fragment (scFv) that specifically binds to a first TERT antigen, fused, via a peptide linker, to the C-terminus of the Fc-region of a first (human) IgG heavy chain molecule; and (b) a scFv that specifically binds to a second TERT antigen, fused, via the same or a different peptide linker, to the C-terminus of the Fc-region of a second (human) IgG heavy chain molecule. The length of such a linker peptide is in the range of from 5 to 15 amino acid residues. For example, a linker peptide may have the amino acid sequence PGSAGGSGG, or it may consist of two or three consecutive peptides of the sequence GGGGS.

A dual specificity antibody is further exemplified by an antibody which may be chimeric or non-chimeric, having specificity for a first TERT antigen, fused via a linker peptide to a scFv that specifically binds to a second TERT antibody. When the Ab having specificity for a first TERT is a chimeric antibody, it may be a humanized Ab. The scFv may be humanized to minimize its immunogenicity or it may be derived from a human monoclonal antibody. A yet further dual specificity antibody contemplated herein, also referred to herein as a“bispecific” antibody, comprises two scFvs having different binding specificities that are interconnected via a linker peptide. In principle, a bispecific antibody maybe combined with a wild type, chimeric or humanized antibody to form a dual or triple specificity Ab.

Further non-limiting examples of dual specificity antibodies include: (i) an antibody composed of a human IgG constant region fused to non-human (e.g., mouse) variable regions of an anti-TERT monoclonal antibody that specifically binds a first TERT antigen, and the antibody is connected at the C-terminus of its Fc region, via linker peptides, to two scFv fragments that specifically bind to a second TERT antigen; a linker peptide may be of 5 to 15 amino acid residues, for example, one or more repeats of GGGGS (ii) an antibody comprising two scFvs that specifically bind to a first TERT antigen fused via a linker peptide to two other scFvs that specifically bind to a second TERT antigen; and (iii) a quadroma, an antibody wherein each light/heavy chain pair has a different binding specificity, or an antigen binding fragment such as F(ab’)₂ fragment or a Fab fragment of a quadroma.

In some embodiments, a contemplated dual specificity antibody binds to at least two different but structurally related hTERT peptides. In exemplary embodiments, at least one of the structurally related hTERT peptides is the peptide GV1001 herein identified as peptide of SEQ ID NO:5.

As shown below in Example 8, the nucleotide sequences of the light and heavy chains of an exemplary mouse anti-hTERT antibody herein designated“LU- 001” have been determined and the constant and variable regions have been identified. It should be understood that any combination of CDRs and framework regions that confer to the antibody specificity towards at least one hTERT antigen as defined herein is encompassed by the present disclosure.

In some embodiments, the anti-hTERT antibody is a homolog of anti-hTERT Ab that comprises framework regions having at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or at least 99.9% identity to the framework region of a monoclonal anti-TERT antibody contemplated herein (for example, LU-001), and each one of the CDR regions has at least 80%, at least 85%, at least 90%, at least 95% or at least 98% or at least 99.9% identity to the corresponding CDR regions of a monoclonal anti-TERT antibody contemplated herein (for example, LU-001), and any intermediate value therebetween. In some exemplary embodiments, the variable regions comprising homolog CDRs and framework regions confer specific binding to at least the antigen of SEQ ID NO: 5.

Sequence homology between protein or DNA sequences is defined herein in terms of shared ancestry between structures or genes. Homology among proteins or DNA may be inferred from their sequence similarity. A homolog antibody or a homolog fragment of an antibody, as referred to herein, is a molecule that differs from the corresponding parent compound by an increment in the chemical structure, nucleotide sequence and/or amino acid sequence.

In a non-human antibody or a homolog thereof, the framework regions or even CDR’s may be modified to remove putative T-cell epitopes, a procedure called“de immunization”, while maintaining binding specificity.

Non-human, human, chimeric, humanized and dual specificity anti-TERT antibodies contemplated herein may be produced using any of the suitable in vivo and/or in vitro technologies known in the art, and commonly provided by specialized commercial laboratories. Some of these techniques are further discussed in the following section.

The binding activity of the anti-telomerase antibodies and any antigen-binding fragments thereof described herein toward their related antigens, as well as toward unrelated antigens, can be assessed using standard in vitro immunoassays, such as an enzyme linked immunosorbent assay (ELISA) or Biacore™ bioassay analysis (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.).

The anti-hTERT antibody or antigen-binding portion thereof is selected to have desirable binding kinetics (e.g., high affinity, low dissociation, slow off-rate) for one or more TERT antigens to which it specifically binds. Binding may be quantified by means of the dissociation constants I and k„_//, wherein the term“IQ” as used herein is intended to refer to the dissociation constant of a particular antibody-antigen interaction, and“k _OJy” as used herein is intended to refer to the off-rate constant for dissociation of an antibody from the antibody/antigen complex.

One quantitative assessment of non-specific binding of the anti-hTERT mAh and any antigen-binding fragment thereof is the ratio of IQ for dissociation from a structurally unrelated antigen to the K_d for dissociation from a structurally related antigen. In some embodiments, the ratio K_d (structurally unrelated antigcn)/K,_/ (structurally related antigen) is at least 3, at least 5, at least 10, at least 50, at least 100, at least 200, at least 300, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900 or at least 1000 or higher, and any intermediate value therebetween.

In alternative quantitative terms, the difference between background binding and specific binding of the anti-TERT mAh and any antigen-binding fragment thereof is one of level or degree. For example, background binding is at a low level, e.g., less than 5%, less than 3%, less than 2%, less than 1.5%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1% and any intermediate value therebetween.

In some embodiments, an anti-hTERT antibody, or portion thereof may bind one or more (structurally related) antigens with a k„_// rate constant of 0.1 s ¹ or less, for example, a k„_jj rate constant of lxlO V¹ or less, k„_jj rate constant of lxlO V¹ or less, k„_// rate constant of lxlO V¹ or less, or even karate constant of lxlO V¹ or less.

Since it is shown hereinafter in the examples that the non-human anti-hTERT antibody LU-001 binds specifically and with high affinity to human cancer cells presenting TERT antigens, it can be expected that the human, chimeric, humanized and dual-specific antibodies as described herein comprising the variable regions of LU-001 or homologs thereof, would be at least as effective in binding to TERT antigens presented on human cancer cells.

Methods of generating monoclonal antibodies

Methods for making monoclonal antibodies using in vivo approaches, in vitro approaches, or a combination of both, are described in the following subsections.

In vivo Approaches

Antibodies can be prepared by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal, including transgenic and knockout versions of such mammals) with an immunogenic preparation of an antigen to thereby expose the in vivo antibody repertoire to the antigen, followed by recovery of an antibody or antibodies of interest from the animal. An appropriate immunogenic preparation can contain, for example, a chemically synthesized or recombinantly expressed antigen. The preparation can further include an adjuvant, such as Freund’s complete or incomplete adjuvant, or similar immunostimulatory compounds. Moreover, when used to raise antibodies, in particular by in vivo immunization, a telomerase reverse transcriptase peptide antigen can be used alone, or used as a conjugate with a carrier protein. Such an approach for enhancing antibody responses is well known in the art. Examples of suitable carrier proteins to which a TERT antigen can be conjugated include keyhole limpet haemocyanin (KLH) and albumin.

Antibody-producing cells can be obtained from the subject and used for the preparation of monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (. Nature 256:495-497, 1975) and later by Brown et al. (/. Immunol 127: 539-546, 1981) and Yeh et al. ( Int . J. Cancer 29:269-275, 1982), or by the human B-cell hybridoma technique (Kosbor et al., Immunol. Today 4:72, 1983), and EBV-hybridoma technique (Cole et al. Monoclonal Antibodies and Cancer Therapy, Alan R Liss Inc. pp 77-96, 1985), or other means known in the art. Briefly, an immortal cell line (typically a myeloma) is literally fused to lymphocytes (typically splenocytes or lymph node cells or peripheral blood lymphocytes) from a mammal immunized with an immunogen to thereby obtain fused cells termed“hybridomas”, and the culture supernatants of the resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody with specificity for the immunogen of interest. Any of the many well-known protocols used for fusing lymphocytes and immortali ed cell lines can be applied for the purpose of generating the monoclonal antibodies described herein. Moreover, the ordinary skilled artisan will appreciate that there are many variations of such methods, which also would be useful. Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. For example, murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation, with an immortalized mouse cell line. Often used immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine (‘ΉAT medium”). Any of a number of myeloma cell lines may be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/0-Agl4 myeloma lines. These myeloma lines are available, e.g., from the American Type Culture Collection (ATCC®, Rockville, Md). Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol (“PEG”)· Hybridoma cells resulting from the fusion are then selected using HAT medium, in which unfused and unproductively fused myeloma cells die (unfused splenocytes die after several days because they are not transformed). Hybridoma cells producing monoclonal antibodies that specifically recognize the molecules of interest are identified by screening the hybridoma culture supernatants for such antibodies, e.g., using a standard ELISA assay.

Depending on the type of antibody desired, various animal hosts may be used for in vivo immunization. A host that itself expresses an endogenous version of the antigen(s) of interest can be used or, alternatively, a host can be used that has been rendered deficient in an endogenous version of the antigen(s) of interest. For example, it has been shown that mice rendered deficient for a particular endogenous protein via homologous recombination at the corresponding endogenous gene (i.e.,“knockout” mice) elicit a humoral response to the protein when immunized with it and thus can be used for the production of high affinity monoclonal antibodies to the protein.

For production of non-human antibodies against a TERT antigen, various non human mammals are suitable as hosts, including but not limited to mice, rats, rabbits and goats (and knockout versions thereof), with mice more often used for hybridoma production. Furthermore, for production of fully human antibodies against a human TERT antigen, a host non-human animal can be used that expresses a human antibody repertoire. Such non-human animals include transgenic animals (e.g., mice) carrying human immunoglobulin transgenes, hu-PBMC-SCID chimeric mice, and human/mouse radiation chimeras.

In some embodiments, mouse is the animal that is immunized with a telomerase reverse transcriptase antigen such as, but not limited to, the peptide GV1001 (SEQ ID NO:5). In some embodiments, the animal, e.g., mouse, is transgenic for human immunoglobulin genes such that it makes human antibodies upon antigenic stimulation. In such animals, typically, human germline configuration heavy and light chain immunoglobulin transgenes are introduced that have been engineered so that their endogenous heavy and light chain loci are inactive. Upon antigenic stimulation of such animals with a human TERT antigen, antibodies derived from the human immunoglobulin sequences are produced, and human monoclonal antibodies can be made from lymphocytes of such animals by standard hybridoma technology. Further description of human immunoglobulin transgenic mice and their use in the production of human antibodies is disclose, for example, in U.S. Patent Nos. 5,939,598, 5,625,126, and 5,814,318; Green (/. Immunol. Methods 231:11-23, 1999); Yang et al. (/. Leukoc. Biol. 66:401-410, 1999); and Gallo et al. (Eur. J. Immunol. 30:534-540, 2000).

In some embodiments, the animal that is immunized with a TERT antigen of interest may be a mouse with severe combined immunodeficiency (SCID) that has been reconstituted with human peripheral blood mononuclear cells or lymphoid cells or precursors thereof. Such mice, referred to herein as hu-PBMC-SCID chimeric mice, produce human immunoglobulin responses upon antigenic stimulation (Hutchins et al., Hybridoma 18:121-129, 1999; Murphy et al., Clin. Immunol. 90:2- 27, 1999; and Heard et al., Molec. Med. 5:35-45, 1999).

In some embodiments, the monoclonal antibodies described herein are non human monoclonal antibodies prepared using hybridoma methods as descried in the Examples section herein. A monoclonal antibody secreted by a hybridoma prepared by hybridoma technology, is termed herein“hybridoma-derived monoclonal antibody”.

B. In vitro Approaches

In some embodiments the anti-telomerase antibodies described herein are identified and obtained by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library or recombinant antibody libraries expressed on the surface of yeast cells or bacterial cells) with a TERT antigen such as, but not limited to, GV1001 peptide of SEQ ID NO:5 to thereby isolate immunoglobulin library members that bind specifically to these molecules of interest. Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAP™ Phage Display Kit, Catalog No. 240612). The phage display library may be a scFv library or a Fab library. The phage display technique for screening recombinant antibody libraries has been described extensively in the art. Once an anti-TERT antibody of interest has been identified from a combinatorial library, DNAs encoding the light and heavy chains of the antibody may be produced by standard molecular biology techniques and then amplified, such as by polymerase chain reaction (PCR), using PCR primers either specifically designed or commercially available.

In some embodiments, an anti-telomerase antibody or a fragment thereof having been identihed, e.g., in a combinatorial library, is produced by recombinant expression of the immunoglobulin light and heavy chain genes in a host cell. The term “recombinant expression” as referred to herein relates to the procedure wherein DNA from a donor genome is extract and cut up into fragments containing from one to several genes that are allowed to insert themselves individually into opened-up small autonomously replicating DNA molecules such as bacterial plasmids, which thereby become carriers or vectors or the inserted DNA fragments. The vector molecules with their inserts are interchangeably referred to herein as “recombinant DNA” or “recombinant expression vectors”, “expression cassettes” or “artificial chromosomes”.

To express an antibody recombinantly, a host cell is transfected with one or more recombinant expression vectors carrying genes encoding the immunoglobulin light and heavy chains of the antibody such that the light and heavy chains are expressed in the host cell and secreted into the medium in which the host cells are cultured. From this medium, the antibodies can be recovered. Standard recombinant DNA methodologies are well known to a skilled person.

Once DNA fragments encoding the VH and VL segments of the anti- telomerase antibody of interest are obtained, these DNA fragments can be further manipulated by standard genome editing techniques, such as, but not limited to, clustered regularly interspaced short palindromic repeats (CRISPR) and/or by standard chemical crosslinking methods. Additionally, or alternatively, various parts of the Ab can be fused or linked to other antibody parts. For example, a VL- or VH- encoding DNA fragment may be operatively linked to another DNA fragment encoding, e.g., a constant region or a flexible linker. For example, isolated DNA encoding the VH region can be converted to a full-length heavy chain gene by operatively linking the V_H-encoding DNA to another DNA molecule encoding heavy chain constant regions (Cn 1 , CH2 and CH3). Isolated DNA encoding the VL region can be converted to a full-length light chain gene (as well as a Fab light chain gene) by operatively linking the V_L-encoding DNA to another DNA molecule encoding e.g., the human light chain constant region, CL- The term“operatively linked”, as used in this context, is intended to mean that the two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame. The sequences of human heavy and light chains constant region genes are known in the art.

To create a scFv gene of an anti-TERT Ab, the VH- and V_L-encoding DNA fragments may be operatively linked to another fragment encoding a flexible linker, e.g., encoding the amino acid sequence (Gly₄-Ser)₃, such that the VH and VL sequences can be expressed as a contiguous single-chain protein, with the VL and VH regions joined by the flexible linker (see for example, Bird et al. Science 242:423-426, 1988; and McCafferty et al. Nature 348:552-554, 1990).

To express the recombinant anti-telomerase antibodies or fragments thereof described herein, DNAs encoding partial or full-length light and heavy chains, obtained as described herein, can be inserted into expression vectors such that the genes are operatively linked to transcriptional and translational control sequences. In this context, the term“operatively linked” is intended to mean that an anti-TERT Ab gene is ligated into a vector such that transcriptional and translational control sequences within the vector serve their intended function of regulating the transcription and translation of the anti-TERT antibody gene. The expression vector and expression control sequences are chosen to be compatible with the expression host cell used. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector. The antibody genes are inserted into the expression vector by standard methods (e.g., ligation of complementary restriction sites on the antibody gene fragment and the vector, or blunt end ligation if no restriction sites are present).

Prior to insertion of the light or heavy chain sequences, the expression vector may already comprise nucleotides sequences of antibody constant region (CH segment(s)). For example, one approach to converting the VH and VL sequences to full-length anti-telomerase antibody genes is to insert them into expression vectors already encoding heavy chain constant light chain constant regions, such that the VH segment is operatively linked to the constant region of the heavy chain within the vector and the VL segment is operatively linked to the constant segment of the light chain (CL) within the vector.

In addition to the anti-TERT antibody genes, a contemplated recombinant expression vectors can carry regulatory sequences that control the expression of the antibody genes in a host cell. The term“regulatory sequence” is intended to include promoters, enhancers and other expression control elements (e.g., polyadenylation signals) that control the transcription or translation of the antibody chain genes. A regulatory sequence may further be a sequence encoding a signal peptide that facilitates secretion of the anti-TERT antibody chain from a host cell. Regulatory sequences contemplated with embodiments of the present disclosure are described, for example, in Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, Calif., 1990. It will be appreciated by those skilled in the art that the design of the expression vector, including the selection of regulatory sequences may depend on such factors as the choice of the host cell to be transformed, the level of expression of protein desired, etc. Non-limiting examples of regulatory sequences for mammalian host cell expression include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40), adenovirus, (e.g., the adenovirus major late promoter (AdMLP)) and polyoma.

In addition to the anti-TERT antibody genes and/or regulatory sequences, a contemplated recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes. The selectable marker gene facilitates selection of host cells into which the vector has been introduced. For example, typically, the selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, to a host cell into which the vector has been introduced. Selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR-host cells with methotrexate selection/amplification), and the neo gene for neomycin selection. For expression of the light and heavy chains, the expression vector(s) encoding the heavy and/or light chains is transfected into a host cell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniques commonly used for the introduction of exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, calcium-phosphate precipitation, DEAE- dextran transfection and the like. Although it is theoretically possible to express the anti-TERT antibodies described herein in either prokaryotic or eukaryotic host cells, expression of antibodies in eukaryotic cells, and particularly in mammalian host cells, is usually desired because such eukaryotic cells, and in particular mammalian cells, are more likely than prokaryotic cells to assemble and secrete a properly folded and immunologically active antibody. Non-limiting examples of mammalian host cells for expressing recombinant anti-TERT antibodies include Chinese Hamster Ovary (CHO cells) optionally comprising a DHFR selectable marker (DHFR-CHO cells), NSO myeloma cells, COS cells and SP2 cells. When recombinant expression vectors encoding anti-TERT antibody genes are introduced into host cells, the antibodies are produced by culturing the host cells for a period of time sufficient to allow for expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. It will be understood that variations on the above procedures are within the scope of the present disclosure.

In some embodiments, host cells are also employed for production of portions or fragments of anti-TERT antibodies such as Fab fragments or scFv molecules. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an anti-TERT antibody described herein. Recombinant DNA technology may also be used for construction and production of dual specificity and/or bifunctional antibodies as described herein.

As a non-limiting example of a system for recombinant expression of a contemplated anti-TERT antibody or antigen-binding portion thereof, a recombinant expression vector encoding both the antibody heavy chain and the antibody light chain is introduced into DHFR-CHO cells by calcium phosphate-mediated transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are each operatively linked to CMV enhancer/ AdMLP promoter regulatory elements to drive high levels of transcription of the genes. The DHFR gene in the vector allows for selection of CHO cells that have been transfected with the vector using methotrexate selection/amplification. The selected transformant host cells are cultured to allow for expression of the antibody heavy and light chains, and anti-TERT antibody or antigen-binding portion thereof are recovered from the culture medium.

Combination Approaches

In some embodiments, anti-hTERT antibodies are prepared using a combination of in vivo and in vitro approaches, such as methods in which the TERT antigen is originally exposed to an antibody repertoire in vivo in a host animal to stimulate production of antibodies that bind the antigen but wherein further antibody selection and/or maturation (i.e., improvement) is accomplished using one or more in vitro techniques. For example, recombinant antibodies may be generated from single, isolated lymphocytes using a procedure referred to in the art as the selected lymphocyte antibody method (SLAM), as described in U.S. Patent No. 5,627,052, and Babcocket al. ( Proc . Natl. Acad. Sci. USA 93:7843-7848, 1996). In this method, as applied to the anti-TERT antibodies described herein, first, a non-human animal (e.g., a mouse, rat, rabbit, goat, or transgenic version thereof, or a chimeric mouse) is immunized in vivo with a TERT antigen to stimulate an antibody response against the antigen. Then single cells secreting antibodies of interest, e.g., specific for the TERT antigen, are selected using, for example, an antigen-specific hemolytic plaque assay. In this assay, the TERT antigen itself, or the structurally-related molecules of interest, are coupled to sheep red blood cells using a linker, such as biotin, thereby allowing for identification of single cells that secrete antibodies with the appropriate specificity using. Following identification of antibody-secreting cells of interest, heavy- and light- chain variable region complementary DNAs (cDNAs) are rescued from the cells by reverse transcriptase-PCR and these variable regions can then be expressed, in the context of appropriate immunoglobulin constant regions (e.g., human constant regions), in mammalian host cells, such as COS or CFlO cells. Conjugates of anti-TERT antibodies

The monoclonal antibodies and antigen-binding parts thereof described herein may be“naked monoclonal antibodies”, namely, antibodies that do not have any drug or marking agent (e.g., radioactive material, fluorophore) attached thereto. Alternatively or additionally, an antibody described herein may be a“conjugated monoclonal antibody”, namely the antibody or antigen-binding portion thereof may be part of a conjugate formed by covalent or noncovalent association of the antibody or antibody portion with one or more organic and/or inorganic moieties such as a drug (e.g., chemotherapeutic drug), a marking agent, a radioactive particle, or a magnetic particle. In some embodiments, conjugated m Ahs are useful as homing or targeting devices to take the substances conjugated thereto directly to the cancer cells.

Some embodiments of the present disclosure pertain to an anti-TERT antibody or an antigen binding fragment thereof attached to one or more of the following moieties: (a) a tag useful for purification and detection; (b) a chemical moiety that alters the physical properties of the antibody such as stability; (c) an immunoadhesion peptide or protein; and (d) a drug, for example, an anti-cancer drug.

A contemplated anti-TERT Ab or antigen-binding part thereof may be conjugated to a tag that enables efficient purification such as, but not limited to, a histidine-tag (a string of six to nine histidine residues-tagged proteins can be purified and detected easily because the string of histidine residues binds to several types of immobilized metal ions, including nickel, cobalt and copper, under specific buffer conditions) or an antigenic peptide tag, and/or the antibody or antigen-binding part thereof may be covalently linked to a nonproteinaceous polymer, such as polyethylene glycol (PEG) to increase the stability of the antibody and change the rate at which the antibody is eliminated from a subject after administration thereto. The PEG may be a substituted or unsubstituted polymer having a molecular weight of from about 1000 to about 5000 Da or more. Other non-limiting examples of such polymers are poly(propylene glycol), or poly (oxyalkylene).

Some embodiments feature immunoadhesion molecules comprising an anti- TERT Ab or a fragment thereof conjugated to a protein such as streptavidin. Nucleic acid molecules, hybridomas and recombinant DNAs

In an aspect of the present disclosure, a nucleic acid molecule is provided comprising nucleotide sequences which encode an anti-TERT antibody or an antigen binding fragment thereof as described herein. In exemplary embodiments, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO:4, a part thereof or a homolog thereof encoding the heavy chain of the antibody or a part thereof, and/or the nucleic acid sequence of SEQ ID NO: 3 a part thereof or a homolog thereof encoding the light chain of the mouse antibody or a part thereof.

In some embodiments, the nucleic acid molecule comprises nucleotide sequences which encode one or more scFvs, for example, one scFv that specifically binds to a first TERT antigen and a second scFv that specifically binds to a second TERT antigen.

In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes the heavy chain of a chimeric antibody that specifically binds to a first TERT antigen and a nucleotide sequence that encodes a scFv that specifically binds to a second TERT antigen, and/or a nucleotide sequence that encodes the light chain of a chimeric antibody that specifically binds to first or a second TERT antigen. In some embodiments, the nucleic acid molecule comprises a nucleotide sequence that encodes a human-mouse chimeric heavy chain and/or human-mouse chimeric light chain.

In another aspect of the present disclosure relates to a recombinant expression vectors, a plasmid, an any form of an artificial chromosome are provided comprising or having contained therein the nucleic acid molecule defined hereinbefore, particularly, but not exclusively, the heavy and light chains of SEQ ID NOs:3 and 4 of the anti-hTERT Ab described herein, and optionally a nucleotide sequence encoding a heterologous polypeptide such as an antigenic peptide tag or enzyme, operably linked to at least one expression control sequence such as a promoter capable of driving the expression of said nucleic acid molecule.

Further contemplated by the present disclosure is a host cell, which is a recombinant or an engineered cell or cell line such as mouse myeloma NSO and Chinese Hamster Ovary cells (CHO), which comprises at least one recombinant expression vectors, a plasmid, an any form of an artificial chromosome as defined herein and which produces an anti-TERT antibody or antigen-binding fragment thereof according to the embodiments described herein. The cell is usually, but not exclusively, a cancer cell or a cancer cell line, or an immortalized cell line. The recombinant expression vectors, plasmid, and/or the artificial chromosome are stably integrated into the cell’s chromosome, or are stably episomally expressed (i.e., can exist autonomously in the cytoplasm).

Also contemplated by the present disclosure are non-human transgenic organisms that comprise the recombinant or the engineered cell described above.

In a yet further aspect, the present disclosure provides a hybridoma such as a mouse or goat, which produces an anti-TERT antibody or a fragment thereof as described herein in exemplary embodiments, a contemplated hybridoma produces anti-hTERT Ab specific to antigen of SEQ ID NO:5.

Pharmaceutic compositions

In an aspect of some embodiments of the present disclosure, there is provided a pharmaceutic composition comprising one or more anti-TERT antibodies or antigen-binding fragments thereof, and a pharmaceutically acceptable carrier.

In some embodiments, the pharmaceutical composition is for diagnosis of cancer. In accordance with these embodiments, a contemplated pharmaceutical composition may further comprise at least one additional diagnostic agent for diagnosis of cancerous cells or tissues and/or a therapeutic agent.

In some embodiments, the pharmaceutical composition is for treatment of cancer. In accordance with these embodiments, a contemplated pharmaceutical composition may further comprise at least one therapeutic agent.

The pharmaceutical compositions described herein may be formulated for administration to a subject and as such may comprise a pharmaceutically acceptable carrier.

The term “pharmaceutical composition”, as used herein, refers to a composition essentially comprising one or more anti-TERT antibodies or antigen binding fragments thereof as defined herein, for example the LU-001 Ab, which may be adjusted for medicinal utilization such as, but not limited to, therapeutic or diagnostic utilization.“Formulation”, as used herein, refers to any mixture of different components or ingredients, at least one of which is an anti-TERT antibodies or an antigen-binding fragment thereof, e.g., LU-001, prepared in a certain way, i.e., according to a particular formula so as to be applicable for administration to a subject. Such formulation is termed herein“anti-TERT Ab formulation”. For example, an anti-TERT Ab formulation may be formulated for treatment or diagnosis of cancer and may include an anti-TERT Ab and/or an antigen-binding fragment thereof combined or formulated together with, for example, one or more carriers, excipients, stabilizers, contrast agents, therapeutically active agents and the like. Usually, an anti- TERT Ab a formulation comprises one or more pharmaceutically or physiologically acceptable carriers, which can be administered to a subject (e.g., human or non-human subject) in a specific form, such as, but not limited to, tablets, linctus, ointment, infusion, injection. The anti-TERT Ab formulation may further comprise other active agents such as chemotherapy agents, one or more additional antibodies that bind other antigens, anti-inflammatory agents, antibiotics and the like. A diagnostic anti-TERT Ab formulation may be used in combination with two or more other known diagnosing agents. Such combination diagnosis may utilize lower dosages of the administered diagnostic agents, thus avoiding possible toxicities or complications associated with various diagnosis methods.

As used herein, the terms“pharmaceutically acceptable”,“pharmacologically acceptable” and“physiologically acceptable” are interchangeable and mean approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. These terms include formulations, molecular entities, excipients, carriers and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, or a human, as appropriate. For human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by, e.g., the U.S. Food and Drug Administration (FDA) agency, and the European Medicines Agency (EMA).

Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition or formulation to further facilitate process and administration of the active ingredients.“Pharmaceutically acceptable excipients”, as used herein, encompass approved preservatives, antioxidants (e.g., ascorbic acid (vitamin C) or a salt thereof; cysteine or a cysteine derivative; lipoic acid; uric acid; carotenes; a-tocopherol (vitamin E); and ubiquinol (coenzyme Q)), surfactants (e.g., Tween®-20, Tween®-40, Tween®-60 and Tween®-80), a buffer (e.g., histidine buffer, acetate buffer, sodium acetate buffer, sodium succinate buffer, sodium citrate buffer, sodium phosphate or potassium phosphate buffer, Tris buffer, sodium hydroxide buffer, or a mixture thereof), coatings, isotonic agents, absorption delaying agents, carriers and the like, that are compatible with pharmaceutical administration, do not cause significant irritation to an organism and do not abrogate the biological activity and properties of a possible active agent. Physiologically suitable carriers in liquid formulations may be, for example, solvents or dispersion media. The use of such media and agents in combination with antibodies is well known in the art.

As used herein, “pharmaceutically acceptable carrier” and“diagnostically acceptable carrier” include any and all solvents, dispersion media, coatings, isotonic agents (e.g., sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride) absorption delaying agents, and the like that are physiologically compatible and approved for use in animals, and more particularly in humans. Examples of pharmaceutically acceptable carriers include water, saline, phosphate buffered saline, dextrose, glycerol, ethanol and the like, as well as combinations thereof.

The antibodies and antibody-portions described herein can be incorporated into a diagnostic composition suitable for parenteral administration. Often, the antibody or antibody-portions will be prepared as an injectable solution containing 0.1-250 mg/ml antibody. The injectable solution can be composed of either a liquid or lyophilized dosage form in a vial, ampule or pre-filled syringe. The buffer can be L- histidine, 1-50 mM (optimally 5-10 mM), at pH 5.0 to 7.0 (optimally pH 6.0). Other suitable buffers include but are not limited to, sodium succinate, sodium citrate, sodium phosphate or potassium phosphate. Sodium chloride can be used to modify the toxicity of the solution at a concentration of 0-300 mM (optimally 150 mM for a liquid dosage form). Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Bulking agents can be included for a lyophilized dosage form, principally 1-10% mannitol (optimally 2-4%). Stabilizers can be used in both liquid and lyophilized dosage forms, principally, 1-50 mM E-methionine (optimally 5-10 mM). Other suitable bulking agents include glycine, arginine (0- 0.05%), polysorbate-80 (optimally 0.005-0.01%).

The compositions provided herein may be in a variety of forms. These include, for example, liquid, semi-solid and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, hard or soft- shell gelatin capsule, buccal tablets, troches, capsules, elixirs, suspensions, syrups, powders, liposomes and suppositories. The selected form depends on the intended mode of administration and diagnostic or therapeutic application. In some embodiments, the compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies.

Diagnostic and therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. Sterile injectable solutions can be prepared by incorporating the active compound (i.e., antibody or antibody portion) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile, lyophilized powders for the preparation of sterile injectable solutions, the common methods of preparation are vacuum drying and spray-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

Methods of diagnosis, methods for isolating cells and methods of treatment

Given their ability to bind one or more TERT antigens presented by more than 90% of various cancer cells, the anti-TERT antibodies or antigen-binding portions thereof, described herein can be used for diagnostic purposes, to detect cancer in vitro, e.g., in a biological sample, such as serum, plasma or a tissue biopsy, as well as in vivo by administration to subjects suspected of having cancer a diagnostic composition comprising an anti-TERT antibody and/or an antigen-binding portion thereof. In vitro detection may be conducted using conventional immunoassays, such as, but not limited to, an enzyme linked immunosorbent assay (ELISA), a radioimmunoassay (RIA), Western blots or tissue immunohistochemistry, and/or sorting assays such as fluorescence activated flow cytometry (FACS).

In an aspect of some of any of the embodiments of the present disclosure, a method for detecting cancer cells in a biological sample of a subject is provided. In some embodiments, the in vitro method for diagnosing cancer comprises at least the following steps:

(a) obtaining a biological sample suspected of containing cancer cells from the subject;

(b) contacting the sample with an anti-TERT antibody, a fragment thereof or a homolog thereof, as described herein, that specifically recognizes one or more TERT antigens presented by cancer cells; and

(c) measuring binding of the antibody, fragment or homolog thereof to TERT- presenting cells in the sample, wherein detection of binding indicates the presence and, optionally, the amount or level of cancer cells in the biological sample.

In some embodiments, the cancer cells are circulating cancer cells (CTCs).

The term“presence”, as used herein in the context of the method for diagnosis or detection of cancer, indicates that the method is a qualitative method, providing critical information regarding the presence or absence of cancer cells. The terms “amount” and“level” indicates that the method can further be, if so desired by a skilled person, a quantitative method producing information regarding the amount or number of cancer cells present in the patient sample. Thus, monitoring e.g., the progression of cancer or the regression of a cancerous disease following anti-cancer treatment is achieved by assessing the level of cancer cells over time, subsequent to the initiation of anti-cancer treatment.

In some embodiments, the in vitro method for detecting cancer additionally comprises isolation, separation or segregation of cancer cells, e.g., CTCs in a sample. Embodiments of isolating cancer cells, e.g., CTCs, comprise contacting conjugated anti-TERT Abs with the cancer cells in a sample, followed by contacting the Ab binding-cells with isolation means and then operating a certain manipulation on the isolation means to thereby isolate or segregate the isolation means from the sample together with the cancer cells attached thereto.“Isolation means” as used herein refers to substances, such as particles, e.g., non-proteinaceous particles, that can attach to cancer cells, for example, via bonding to antibodies or to cell receptors. A non limiting example of such isolation means are magnetic particles wildly used for the purpose of sedimentation, purification and isolation of cells, such as magnetic bead further discussed herein below.“Manipulation”, as referred to herein, is the subjection of the sample to an element that affects or acts upon the isolation means. For example, when the isolation means are magnetic beads, the manipulation is a magnetic field or a magnet.

In some embodiments, a method for isolating and detecting cancer cells, e.g., CTCs, in a sample is provided, the method comprising at least the following steps:

(a) obtaining a biological sample suspected of containing cancer cells from a subject;

(b) contacting the sample with an anti-TERT antibody, a fragment thereof or a homolog thereof as described herein, that specifically recognizes one or more telomerase antigens presented by cancer cells;

(c) subjecting the cells bound to the anti-TERT antibody, a fragment thereof or a homolog thereof to isolation means; and

(d) isolating the cells and measuring binding of the antibody, a fragment thereof or a homolog thereof to isolated, TERT-presenting cells, wherein detection of binding indicates the presence and, optionally, level or amount of cancer cells in the biological sample.

Steps (c) and (d) above, directed subjecting the cells bound to the anti-TERT antibody, a fragment thereof or a homolog thereof to isolation means, and isolating the cells, may comprise, in some exemplary embodiments, at least the following steps: (i) adding isolation means to the sample and allowing for the binding or attachment of the isolation means to the cells; (ii) subjecting the sample to a manipulation operable on the isolation means to thereby isolate or separate the isolation means from the sample together with the cancer cells attached thereto; and (iii) optionally, releasing the isolated (or separated) cells from the isolation means. It is shown in Example 7 herein that Dynabeads® magnetic beads can reversibly bind to anti-hTERT Abs attached to CTCs, thereby, when the sample is exposed to a magnetic field, cells with magnetic beads attached thereon are attracted to the magnet, and separate, mechanically, from the sample by residing as a sediment, in the sample tube (see Fig. 8 herein). Dynabeads® are uniform polystyrene spherical beads of exactly the same size, typically 1 to 5 micrometers in diameter. Dynabeads®, produced by Invitrogen™ are frequently used for cell isolation.

Magnetic particles such as Dynabeads® have been made magnetisable and superparamagnetic, meaning they are only magnetic in a magnetic field. Due to this property, the beads can easily be resuspended when the magnetic field is removed. Magnetic beads may be covalently linked to an antibody that recognizes a specific protein on the surface of the target cell, and be used for positive cell isolation. For example, the contemplated anti-TERT Abs, homologs and/or antigen-binding parts thereof may be attached to the beads directly or indirectly. Alternatively, the beads mat be coated with other antibodies that recognize other cell surface antigens. The attachment of target-specific antibodies to the surface of the beads allows capture and isolation of intact cells directly from a complex suspension such as blood, and other unprocessed samples, avoiding the need for column separation techniques or centrifugation.

The antibodies may be attached to the surface of the magnetic beads via a DNA linker, which provides a cleavable site to release and remove the beads from the cells after isolation. Alternatively, the magnetic particles may attach to the cell indirectly, either via, e.g., streptavidin on the magnetic particle linking to a biotinylated primary antibody attached to the cells, or a secondary antibody on the magnetic beads linking to the primary antibody on the cell surface. Non-limiting examples of secondary antibodies include Sheep anti-Mouse IgG, Goat Anti-Mouse IgG, Rat Anti-Mouse IgG, Sheep Anti-Rabbit IgG, and the like.

Further non-limiting examples for some known magnetic beads that can be useful for isolating cancer cells in a sample include mono-disperse, silica-based superparamagnetic beads (Silica-coated iron oxide magnetic beads) covalently conjugated with recombinant protein A, recombinant Protein G or fusion protein A/G on the surface (e.g, BcMag™ Protein A, EcoMag™ Protein G, and EcoMag™ Protein A/G Magnetic Beads, and the like). Such magnetic beads are useful when a selected primary antibody is used, such that the antibody on the cell surface attaches via its Fc portion to protein A (or protein G or fused protein A/G) anchored onto the beads. Isolated cells are released from the beads by the use of an appropriate Elution Buffer (e.g., a solution containing 0.2 M Glycine/HCl, pH 2.5).

The biological sample can be, for example, an in vitro sample, such as a sample of cells, tissue biopsy or bodily fluids (e.g., blood, plasma, urine, saliva etc.).

The anti-TERT antibodies described herein or compositions comprising them may be useful for in vivo diagnosis of cancer, for example, by imaging of tissues labeled with a contemplated antibody or a fragment thereof, e.g., conjugated to a labeling moiety as described herein. Such diagnosis approach may be useful in first diagnosis of cancer in a subject suspected of being inflicted with the disease, as well as in monitoring anti-cancer treatment progression in a subject already diagnosed as having a cancerous disease.

In an aspect of some of any of the embodiments of the present disclosure, a method for diagnosing cancer and monitoring response to cancer therapy in a subject is provided. In some embodiments, a contemplated method comprises at least the following steps:

(a) administering to a subject suspected of having cancer, or a subject subjected to anti-cancer treatment, a diagnostically effective amount of an anti-TERT antibody, a fragment thereof or a homolog thereof as described herein, that specifically recognizes one or more telomerase antigens presented by cancer cells; and

(b) measuring binding of the anti-TERT antibody, a fragment thereof or a homolog thereof, wherein detection of binding indicates the presence and, optionally, the level of cancer in the subject, thereby diagnosing or monitoring response to cancer therapy in the subject.

The term “diagnosis” as used herein denotes identifying, detecting or uncovering the existence of a cancerous disease or cancerous process in a subject. The use of this term applies to the first discovery of a cancer disease in a subject as well as to uncovering recurrence of the disease. Diagnosis as used herein also encompasses monitoring and following-up on the course of anti-cancer treatment and regression or recurrence of the disease. Imaging tests suitable for diagnosing cancer using the anti-TERT antibodies (or fragments or homolog thereof) are, for example, magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), and PET- CT.

Contemplated anti-TERT antibodies used for in vitro or in vivo detection of cancer may be directly or indirectly labeled with a detectable substance to facilitate detection of the bound cancer cells or cancer tissues. Suitable detectable substances include various enzymes, fluorescent materials, luminescent materials, radioactive materials, isotopes, contrast agents, prosthetic groups and inorganic micro- or nanoparticles. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, b-galactosidase, or acetylcholinesterase. “Prosthetic groups” as used herein refers to tightly bound, specific non-polypeptide units (i.e., certain cofactors) required for the biological function of some proteins. Prostatic groups can be organic such as, but not limited to, vitamins (for example the B-vitamin biotin), sugar, streptavidin, avidin, biotin, or lipid, or inorganic such as metal ions, but are not composed of amino acids.

“Fluorescent material”,“fluorophore” and“fluorescent dye” are terms used herein interchangeably, referring to a fluorescent chemical compound that can re-emit light upon light excitation. Examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate (FITC), rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin (PE), peridinin chlorophyll a protein (PerCP)-Cy5.5, PE-Texas, Red Alexa Fluors, Cys dyes, Allophycocyanin (APC), green fluorescent protein (GFP). Fluorophores, particularly useful for FACS include, but not limited to, DyLight 405, DyLight 405, Pacific Blue, FITC, DyLight 550, PE, APC, Alexa Fluor 647, DyLight 650, PerCP and Alexa Fluor 700.

“Luminescent materials”, as used herein, are compounds such as phosphors- containing compounds that spontaneously emit light (infrared to ultraviolet) under external energy excitation by incident energy in the form of high energy electrons (e.g., in a chemical reaction), photons, or electric field, but not as a result of heat. A non-limiting example of a luminescent material includes luminol. “Radioactive material”,“radioactive tracer” and radioactive label” are ter s used herein interchangeably, referring to a chemical compound in which one or more atoms have been replaced by a radioisotope (an atom, e.g., hydrogen, carbon, phosphorus, sulphur, and iodine, having an imbalance of neutrons and protons resulting in excess nuclear energy that can be emitted from the nucleus either as gamma radiation, alpha particles or beta particles). By virtue of its radioactive decay, a radioisotope can be used, inter alia, to track the distribution of a substance within a natural system such as a cell or tissue. Radionuclides occur naturally or can be artificially produced in radionuclide generators. Non-limiting examples of suitable radioactive material include compounds containing radioisotopes such as ¹²⁵I, ¹³¹I, ³⁵S, ³H, P³² and S³³.

For diagnosing by an imaging method, for example MRI, the anti-TERT Ab or antigen-binding fragment thereof may be conjugated to a contrast agent (contrast enhancer), selected from the multitude of contrast agents suitable for each technique as well known in the art.

A contemplated anti-TERT antibody or an antigen-binding fragment thereof may be conjugated to an anticancer drug and be used as means to target or home the anti-cancer drug to cancerous tissues or cells.

In an aspect of some of any of the embodiments of the present disclosure, a method for treating cancer in a subject is provided. In some embodiments, a contemplated method comprises administering to a subject inflicted with cancer a therapeutically effective amount of an anti-TERT antibody, a fragment thereof or a homolog thereof that specifically recognizes one or more telomerase antigens presented by cancer cells as described herein, wherein the anti-TERT antibody, fragment thereof or homolog thereof is conjugated to an anti-cancer drug, thereby treating cancer in the subject.

Treating a disease, as referred to herein, means ameliorating, inhibiting the progression of, delaying worsening of, and even completely preventing the development of a disease, for example, inhibiting the progression or metastasis of a tumor in a subject with cancer. Treatment refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or a pathological condition after it has begun to develop. “Administration”, as referred to herein, is introduction of a contemplated Ab or a formulation comprising it, as defined herein, into a subject by a chosen route. Administration can be local or systemic. Examples of local administration include, but are not limited to, topical administration, subcutaneous administration, intramuscular administration, or administration to the nasal mucosa or lungs by inhalational administration, intravitreal administration. In addition, local administration includes routes of administration typically used for systemic administration, for example by directing intravascular administration to the arterial supply for a particular organ. Thus, in particular embodiments, local administration includes intra-arterial administration and intravenous administration when such administration is targeted to the vasculature supplying a particular organ. Systemic administration includes any route of administration designed to distribute an active compound or composition widely throughout the body via the circulatory system. Thus, systemic administration includes, but is not limited to intra-arterial and intravenous administration, oral administration, topical administration, subcutaneous administration, intramuscular administration, or administration by inhalation, when such administration is directed at absorption and distribution throughout the body by the circulatory system.

An effective amount, for example, a therapeutically effective amount or diagnostically effective amount, of an anti-TERT antibody, an antigen-binding fragment thereof or a homolog thereof, or a composition comprising same, as referred to herein, is a quantity, e.g., number or concentration, of the Ab or a fragment thereof, which is sufficient to achieve a desired effect in a subject being treated or being diagnosed. An effective amount of the Ab or a fragment thereof, can be administered in a single dose, or in several doses, for example once or several times, during a course of treatment. The effective amount of will depend on the subject being treated or diagnosed, the severity and type of the affliction, and the manner of administration of the transplant.

The term“subject” as used herein encompasses human and non-human mammals (e.g., dog, cats, monkeys, horses, cows, rats, rabbits etc.). Kits

In yet another aspect, the present disclosure provides a kit for detecting cancer cells, comprising at least one of: an anti-TERT antibody, an antigen-binding fragment thereof and/or a homolog thereof according to any one of claim 1 to 15; reagents required for the detection and quantification of the antibody; and instructions for use.

A contemplated kit is useful in in vitro and in vivo diagnosis of cancer.

In some embodiments, a contemplated kit is useful for diagnosis of cancer in a subject, in accordance with a method described herein.

In some embodiments, a contemplated kit is useful for isolating and detecting cancer cells in a sample obtained from a subject, in accordance with a method described herein.

In some embodiments, a contemplated kit is useful for monitoring response to cancer therapy in a subject, in accordance with a method described herein.

In some embodiments, a contemplated kit is useful for treating cancer in a subject, in accordance with a method described herein.

In some of any of the embodiments described herein, the cancer cells are circulating tumor cells (CTCs).

Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

The terms "comprises", "comprising", "includes", "including",“having” and their conjugates mean "including but not limited to".

Throughout this application, various embodiments may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

It is appreciated that certain features of the present disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the present disclosure. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present disclosure as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples. EXAMPLES

Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the present disclosure in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in embodiments described herein include molecular, chemical, biochemical, microbiological and/or recombinant DNA techniques. Such techniques are thoroughly explained in the literature. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference.

Materials and Methods

Cell culture growth. Cells were grown under standard growth conditions. The cells tested were cancer cell lines: THP1 (acute monocytic leukemia), Jurkat cells (T cell leukemia), U266 (multiple myeloma), JeKo 1 (mantle cell lymphoma), A549 (adenocarcinoma), SUP-T1 (T cell lymphoblastic lymphoma), K-562 (Leukemia), MCF-7 (breast carcinoma) and HCT116 (colorectal carcinoma). Human mononuclear cells (MNCs) from healthy volunteers and human fibroblasts served as a negative control (non-cancer cells). All cell lines were obtained from the American Type Culture Collection (ATCC™). All cells (except A549 cells) were cultured in RPMI- 1640 supplemented with 10% fetal bovine serum (FBS) and containing 100 units/ml L-glutamine and 1% penicillin/streptomycin (Biological Industries Beit Haemek, Israel). A549 cells were cultured in DMEM with 10% FBS supplemented with L- glutamine and penicillin/streptomycin as indicated above.

Antibody production. Telomerase reverse transcriptase antigens for antibodies production are all commercially available. In the Examples described herein, the 17- amino acid hTERT antigen GV1001 having the sequence CEARPALLTSRLRFIPK, herein identified as SEQ ID NO:5 (MW 1971.41, purity: 95.43% (F1PLC), peptide ID: 3286265) was used as antigen for production of anti-hTERT Ab.

Monoclonal antibodies were produced using the known somatic cell hybridization technique. This technique involves fusing certain myeloma cells (cancerous B cells), which can multiply indefinitely but cannot produce antibodies, with plasma cells (noncancerous B cells), which are short-lived but produce a desired antibody. The resulting hybrid cells, called hybridomas, grow at the rate of myeloma cells but also produce large amounts of the desired antibody. Briefly:

(1) Animal immunization. Five 6-week old BALB/C mice (females) were immunized with the antigenic peptide of SEQ ID NO:5 conjugated to a carrier (Keyhole Limpet Flemocyanin (KLH), a large, multi-subunit, oxygen carrying, metalloprotein which is a potent immune-stimulating molecule used extensively as a carrier protein in the production of antibodies). Mice with the best immonuresponse were chosen for the following steps (spleen of these mice produced plasma cells that secrete antibodies against peptide of SEQ ID NO:5)

(2) Hybridoma production. Freshly harvested spleen cells from mice were mixed with myeloma cells in the presence of polyethylene glycol that facilitated fusion of adjacent plasma membranes. In order to select for the (rare) successful fusions, fused cells were transferred to a special selection medium containing hydroxanthine, aminopterin, and thymidine (HAT). Myeloma cells used for hybridoma production were lacking the ability to synthesize the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) that allows them to grow independently in HAT medium. This enzyme enables cells to synthesize purines using an extracellular source of hypoxanthine as a precursor. Ordinarily, in absence of HGPRT, cells use the alternative de novo purine synthesis pathway. However, exposure to aminopterin (a folic acid analog) in the growth medium HAT silences the de novo pathway and myeloma cells become fully dependent on HGPRT for survival. Thus, whereas unfused myeloma cells lacking HGPRT would not grow in HAT, and unfused normal spleen cells would not grow indefinitely because of their limited life span, hybridoma cells (produced by successful fusions) were able to grow indefinitely because the spleen cell partner supplied HGPRT and the myeloma partner was immortal.

(3) The incubation medium was then diluted into multi-well plates to such an extent that each well contains only one cell. Since the antibodies in a well are produced by the same B cell, they will be directed towards the same epitope, and are thus monoclonal antibodies. The next stage was a rapid primary screening process, which identified and selected only those hybridomas that produced the desired antibodies. Cell culture supernatants of the hybrid cell colonies were screened by ELISA, and positive cell lines were sub-cloned in 96-well plates. After 2-3 rounds of subcloning, supernatant from each positive clone was tested again (FACS and ELISA) for the desired antibodies. Established clones were continuously grown. Clones of hybridoma living cells were kept in medium containing IMDM +10% FBS, at room temperature.

(4) For mass production, cultures of the successful clones (i.e., the most productive and stable clones) were scaled up to yield 10-60 pg/ml monoclonal antibodies per hybridoma cell clone.

Enzyme-linked immunosorbent assay (ELISA). ELISA is a plate-based assay technique in which an antigen is immobilized to a solid surface and then complexed, directly or indirectly with an antibody that is linked to an enzyme. Detection is accomplished by assessing the conjugated enzyme activity via incubation with a substrate to produce a measurable product. The ELISA procedure results in a colored end product which correlates to the amount of analyte present in the original sample. The most crucial element of the detection strategy is a highly specific antibody- antigen interaction. Binding of mono clonal antibodies (mAbs) to the hTERT peptides was assessed using the ELISA, wherein tested mAbs were allowed to react with hTERT peptides adsorbed to the assay plate. Unbound antibodies were removed by washing. The nonspecific binding was blocked with bovine serum albumin (BSA). Subsequently, the bounded antibodies were detected with horse radish peroxidase (HRP)-conjugated anti-mouse IgG secondary antibody and a colored reaction was developed with a 3,3',5,5'-tetramethylbenzidine (TMB) substrate. The successful binding of anti-hTERT mAh to the antigen was detected by direct visualization of the TMB substrate. The optical density was measured at 450 nm by microplate reader.

An isotype control is an antibody that maintains similar properties to the primary antibody but lacks specific target binding. Used in place of the primary antibody, this negative control helps determine the contribution of non-specific background to staining. Background staining refers to signal detection in the absence of a specific antibody- antigen interaction. An isotype control determines the non specific binding of the test antibody to Fc receptors found on cells, ensures the observed staining is due to specific binding rather than an artifact, and reveals other non-specific binding of the antibody or fluorophores to cellular components. Isotype controls should closely match the properties of the primary antibody and be used under identical experimental conditions to best determine the presence and extent of non specific binding. Isotype controls are from the same host species, Ig class and subclass (isotype), and used at the same working concentration as the primary antibody. When using a labeled primary antibody, the isotype control should also have the same conjugate and ideally, with the equivalent label-to-antibody ratio. For example, if a mouse IgGl monoclonal antibody that is conjugated to FITC is used, a mouse IgGl isotype control conjugated to FITC would be selected. During analysis, the signal intensity of the isotype control sample is compared with the experimental sample, probed with the primary antibody.

The isotype control should have negligible staining when background signal is not affected by Fc receptor binding, endogenous enzymes, and reactive epitopes. Isotype controls are commonly used in flow cytometry experiments and immunoassays such as ELISA Fluorescence activated cell sorting (FACS). Flow cytometry is an analytical cell-biology technique that utilizes light to count and profile cells in a heterogenous fluid mixture. Flow cytometry analysis was used to evaluate binding of antibodies to various cancer cells. In this assay, a test antibody was added to a cell suspension and cells which bound the antibody were visualized by a fluorescently labeled secondary antibody that bound to the test antibody. Accordingly, samples were first“labeled” with the test (primary) anti-hTERT mAb, then incubated with fluorescein isothiocyanate (FITC) or Alexa Fluor® 647 conjugated to anti-mouse secondary Ab (Jackson) for hTERT positive cells visuali ation.

The immunophenotyping of chronic lymphocytic leukemia (CLL) subpopulation was based on surface membrane antigen expression patterns, as a means of distinction between CLL and other chronic lymphoid leukemias, using flow cytometry. The two CLL antigens monitored were CD5 and CD 19 using commercial mouse monoclonal anti-CD 19 or anti-CD5 antibodies conjugated to fluorescent dyes: Streptavidin-Phycoerythrin (SAPE) and Peridinin-chlorophyll a protein (PerCP) (BioLegend®, San Diego, CA, USA). Streptavidin-phycoerythrin conjugates excel in cellular fluorescence detection assays based on biotin labeling. Phycoerythrin is a pink-colored protein purified from seaweed. The emission maximum is at 578 nm with a high quantum yield. PerCP is a fluorescent peridinin-chlorophyll a protein complex isolated from dinoflagellates. The peridinin molecules absorb light in the blue-green wavelengths (470 to 550 nm) and transfer energy to the chlorophyll molecules with extremely high efficiency.

In order to confirm the specificity of binding, and further in order to correct for non-specific or background staining, isotypes control mouse IgGs were added to cell suspensions at the same concentration as the test Abs. To deduct the non-specific or background staining from the positive staining due to direct mAh binding to the antigen, the level of back ground staining was assessed by the use of the same fluorochrome as that used for staining the anti-hTERT mAh.

Ah cells were prepared for FACS analysis according to a routine procedure: for cell lines, floating cells were collected from the growth media by centrifugation; adherent cells were treated with trypsin to detach them from the surface of the growth dishes. For whole blood (CLL patients and healthy volunteers), mononuclear cells (MNCs) were separated by Lymphoprep sedimentation. Cells were washed with phosphate buffer.

The binding was analyzed on the flow cytometer (FACS Calibur, Becton Dickinson) using the same setting/compensation for all healthy and CLL patients MNCs samples.

Protein Sequencing

(i) Edman degradation. Sequencing of the protein which comprises the light chain (less than 50 amino acids) of the test antibody was performed using Edman degradation, as described, for example, in“ PTH Amino Acid Analysis”, Hunkapiller, ABI User Bulletin Issue No. 14, (1985). Briefly: identification of the amino acid sequence was performed step-wise, staring with the first N-terminal amino acid of the light chain protein, by reacting it with phenyl isothiocyanate (PTH), under mildly alkaline conditions, to form a cyclical phenylthiocarbamoyl derivative, followed by cleavage of this derivative, under acidic conditions, to obtain a thiazolinone amino acid. The thiazolinone amino acid was then selectively extracted into an organic solvent and treated with acid to form the more stable phenylthiohydantoin (PTH)- amino acid derivative that was identified using chromatography or electrophoresis. The next amino acids of the remaining protein were identified sequentially, repeating the PTH labeling, cleavage and PTH-amino acid derivative formation steps.

(ii) In-gel protein digestion and liquid chromatography-mass spectrophotometry (LC-MS) measurements. Basically, the heavy chain of the purified test antibody was broken up into peptides by enzymatic in-gel digestion (trypsinization), then mass spectroscopy analysis was performed on the individual peptides, and the information obtained was stitched together to reveal the protein identity. Key steps in this strategy included: (1) preparation of lysate samples from biological specimens; (2) subjecting the lysate to SDS polyacrylamide gel electrophoresis (SDS-PAGE) to denature and separate the proteins in a sample. After electrophoresis, visualizing protein bands or spots using Coomassie fluorescence; (3) collecting gel slices that contained the desired band(s) and de-staining them; (4) extracting the proteins in these gel plugs and then reducing them (namely reducing the disulfide groups using a reducing agent such as tris(2-carboxyethyl) phosphine (TCEP) or dithiothreitol (DTT); (5) alkylating the free sulfhydryl groups on the cysteine residues of the extracted proteins with reagents such as iodoacetamide or iodoacetic acid to irreversibly prevent the free sulfhydryls from reforming disulfide bonds; (6) digesting (with trypsin) the denatured, reduced and alkylated proteins in situ by endoproteinases, which hydrolytically break peptide bonds so as to fragment the heavy chain proteins of the mAbs into peptides; (7) extracting the peptides from the gel matrix and preparing for MS analysis; and (8) running LC-MS. Further fragmentation of the peptides inside the mass spectrometer was used to gain information about the peptides’ sequences.

Nucleic acid sequencing. The nucleotide sequence was obtained using degenerated primers based on predicted sequences. Degenerate primers are a mix of unique oligonucleotide sequences wherein one or more positions along the sequence of each oligonucleotide contain a number of possible or optional bases, giving a population of primers with similar (but not identical) sequences that cover all possible nucleotide combinations coding for a given protein sequence. The commercially available kit KAPA HiFi HotStart PCR was used for the polymerase chain reaction (PCR) analysis, and the procedure was performed as described in the manufacturer’s protocol.

EXAMPLE 1

Anti-hTERT monoclonal antibody binding to immobilized hTERT peptide

The specificity of the newly synthesized anti-hTERT mAh LU-001 was tested by assessing its ability to bind to the 17-amino acid telomerase peptide GV1001 (SEQ ID NO:5), the peptide against which it was designed.

LU-001 binding was assessed by ELISA as described above in Materials and Methods. As shown in Fig. 1, LU-001 specifically bound to the hTERT peptide, while the isotype control mouse IgGl antibody did not bind to the hTERT peptide.

EXAMPLE 2

Anti-hTERT monoclonal antibody in vitro binding to various cancer cell

Cancer cells present TERT peptides on their membrane. The ability of the newly synthesized anti-hTERT mAh LU-001 to bind various cancer and non-cancer (normal) cells in vitro was assessed using a flow cytometry procedure which included binding of primary and secondary antibodies, washing after binding and then binding to specific cancer markers. The following cells were assayed: THP1 (acute monocytic leukemia), Jurkat cells (T cell leukemia), U266 (multiple myeloma), JeKo 1 (mantle cell lymphoma), A549 (adenocarcinoma) and SUP-T1 (T cell lymphoblastic lymphoma). Fibroblast cells served as control. Binding percentage was assessed by FACS measurement as described above in Material and Methods.

As shown in Fig. 2, LU-001 specifically bound to various cancer cell lines, albeit at different percentages (varying affinity). Fibroblasts, which do not express TERT, did not bind the antibody.

EXAMPLE 3

Ex-vivo binding of anti-hTERT mAb to cancer cells isolated from patients

Binding of LU-001 antibody to cancer cells isolated from chronic lymphatic leukemia (CLL) patients was tested ex vivo as described in Materials and Methods.

Fig. 3 A shows an exemplary FACS histogram obtained for ex vivo LU-001 antibody binding to CLL cells. As seen, about 70% of the CLL cells were bound to the LU-001 antibody. Conversely, as shown in the dot plot depicted in Fig. 3B, about 73% of the telomerase antibody positive lymphocytes were identified as CLL.

As shown in Fig. 4, LU-001 could bind to ovary tumor cells obtained from ovary cancer patient as well. Cells were labelled with a control dye so as to exclude unspecific binding directly to the secondary antibody.

Flowever, this antibody did not bind to healthy lymphocytes, as shown in Fig. 5: lymphocytes bound to the control isotype (green line) and lymphocytes bound to LU-001 mAh purified from 2 different batches (red and purple lines), presented the same fluorescence intensity.

EXAMPLE 4

F(ab')₂ fragment of anti-hTERT mAb: purification and binding assay

In order to optimize the binding affinity of the anti-hTERT mAh LU-001 to the TERT antigen, the F(ab')2 fragment (a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region) was cleaved off from the complete antibody using known means (pepsin cleavage). The F(ab')2 fragment was purified by gel electrophoresis (SDS-PAGE), and its binding to the immobilized telomerase peptide antigen was assayed by ELISA, as described in Material and Methods.

Purification of LU-001 mAh and of the F(ab')2 fragment is demonstrated in Fig. 6A (right and left panels, respectively), and the results of binding assays of F(ab')2 fragment obtained from two different preparations are demonstrated in Fig. 6B. As clearly shown in Fig. 6B, F(ab')2 fragment presented a substantially higher binding affinity as compared to the isotype IgGl control.

EXAMPLE 5

Ex vivo binding of anti-hTERT mAb F(ab')2 fragment to chronic lymphocytic leukemia (CLL)

Having demonstrated enhanced in vitro binding, F(ab')2 fragments were assessed for their ex vivo binding to CLL lymphocytes. The FACS dot plots obtained for CLL lymphocytes bound to either mouse IgGl isotype (Fig. 7A), intact LU-001 Ah (Fig. 7B), or F(ab')2 fragment (obtained from two different cleavage and purification processes; Figs. 7C-7D), show that a substantially larger population of CLL lymphocytes bound the F(ab')2 fragment as compared to LU-001 and isotype control.

EXAMPLE 6

Isolation of circulating tumor cells (CTCs)

Anti-TERT antibodies conjugated to cells which are, in turn, bound to magnetic particles, constitute a means for ex vivo isolation of circulating tumor cells (CTCs) presenting TERT antigenic peptides on their surface. Thus, by way of contacting (e.g., reversibly conjugating) magnetic beads (for example, magnetic Dynabeads) to CTCs, and by the following use of a magnet to attract and thereby segregate the“magnetic” CTCs from the sample, these CTCs may be isolated.

The isolation process is schematically presented in a scheme shown in Fig. 8. Briefly: the anti-TERT antibody conjugated to streptavidin or avidin is added to a whole blood sample containing CTCs, or mononuclear suspended cells obtained as described in Material and Methods. After lO-minute incubation, secondary antibodies (anti anti-TERT antibody) labeled with DSB-X biotin are attached to the cells. DSB- X biotin is a derivative of desthiobiotin, a stable biotin precursor that has the ability to bind biotin-binding proteins, such as streptavidin and avidin. Magnetic Dynabeads are then added and allowed to conjugate to the DSB-X biotin antibodies adhered to CTCs. After 15-minute incubation, the sample tube is contacted with an external magnet. Unspecific binding and unbound cells are washed away, the magnet is removed, and a releasing antigen is added, in order to release the Dynabeads from the isolated CTCs onto which they were bound. Following a 10-minute incubation, the sample is contacted again with an external magnet to thereby attract the free Dynabeads from the sample and remove them. The beads-free CTCs are collected and transferred to a separate tube.

EXAMPLE 7

Isolation of chronic lymphocytic leukemia (CLL) cells from whole blood sample

The magnetic beads-antibody conjugation means for isolation of cells that bind anti-telomerase antibodies was applied to a blood sample containing CLL cells, using the step-wise procedure described in Example 6 above. LU-001 antibodies were added to a blood sample of a CLL patient, then contacted with secondary antibodies subsequently followed by contacting with Dynabeads. Cells bound to the magnetic beads were isolated from other sample constituents by the attracting force of an external magnetic field. Bound cells were washed, released from the magnetic beads and transferred to a clean vial.

Immunophenotyping of CLL CD5⁺ and/or CDl9⁺ cells by FACS analysis of the cells isolated from the patient’s blood sample was performed using Streptavidin- Phycoerythrin (SAPE)- or Peridinin-chlorophyll a protein (PerCP) -conjugated anti- CD19 or anti-CD5 antibodies as described in Materials and Method. As seen in Fig. 9, about 97% of the cells thus isolated were indeed CLL cells.

EXAMPLE 8

Sequencing of anti-hTERT monoclonal antibody LU-001

The amino acid sequences of the proteins comprising the light chain of the anti-hTERT mAh LU-001 was assessed by Edman degradation, and the sequencing of the heavy chain was performed using the in-gel protein digestion and liquid chromatography-mass spectrophotometry (LC-MS) procedures, as described in Materials and Methods. The light chain and heavy chain amino acids sequences of LU-001 designated herein as SEQ ID NO:l and SEQ ID NOG, respectively, are:

DVVMTQTPLTLSVTIGQPASISCKSSQNLLYSDGKTYLNWLLQRPGQS PKRLIYLVSKVDSGVPDRFTGSGSGTDFTLKISRVEAEDLGVYFCWQG THLPYTFGGGTKLEIK (SEQ ID NO: 1); and

QVQLQQSGAELVRPGASVTLSCKASGYIFTDYEKHWVKQTPVHGLEW

IGAIDPESGSTVYNQRFKGKATLTADKSSGTAYMELRSLTSEDSAVYF CFLLRLFAYWGQGTLVTVSA (SEQ ID NO: 2)

The nucleic acid sequencing was performed using the degenerate primer-based sequencing technique described in Materials and Methods. The light chain and heavy chain nucleic acids sequences of LU-001 designated herein as SEQ ID NOG and SEQ ID NO:4, respectively, are:

GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTGACCATTG GACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAACCTCTTATA

TAGTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGC CAGTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAGTGGACTCTG GAGTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCAC ACTGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTT TGCTGGCAAGGTACTCATCTTCCGTACACGTTCGGAGGGGGGACCA

AGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTT CCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTG TGCTTCTTGAACAACTTCTACCCCAGAGACATCAATGTCAAGTGGA AGATTGATGGCAGTGAACGACAAAATGGTGTCCTGAACAGTTGGA CTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCC

TC AC ATTGACC A AGGACG AGT ATG AACGAC AT A AC AGCT AT ACCTG TGAGGCCACTCACAAGACATCAACTTCACCCATCGTCAAGAGCTTC AACAGGAATGAGTGTTAA (SEQ ID NO: 3); and

5’- caggtgCAGCTGCAGCAGTCTGGGGCTGAACTGGTGAGGCCTGGGGCT TCAGTGACGCTGTCCTGCAAGGCTTCGGGCTACATATTTACTGATT ATGAAAAGCACTGGGTGAAGCAGACACCTGTGCATGGCCTGGAGT GGATTGGAGCTATTGATCCTGAAAGTGGTAGTACTGTCTACAATCA GAGATTCAAGGGCAAGGCCACACTGACTGCAGACAAATCTTCCGG

CACAGCCTACATGGAACTCCGCAGCCTGACATCTGAGGATTCTGCC GTCT ATTT CTGCTTTTT ACT ACGGCT ATTT GCTT ACT GGGGCC A AGG GACTCTGGTCACTGTCTCTGCAGCC-3' (SEQ ID NO: 4). The variable region of the light (VL) chain comprises the nucleotide sequence of SEQ ID No:6: GATGTTGTGATGACCCAGACTCCACTCACTTTGTCGGTGACCATTGG

ACAACCAGCCTCCATCTCTTGCAAGTCAAGTCAGAACCTCTTATATA GTGATGGAAAGACATATTTGAATTGGTTGTTACAGAGGCCAGGCCA GTCTCCAAAGCGCCTAATCTATCTGGTGTCTAAAGTGGACTCTGGA GTCCCTGACAGGTTCACTGGCAGTGGATCAGGGACAGATTTCACAC TGAAAATCAGCAGAGTGGAGGCTGAGGATTTGGGAGTTTATTTTTG

CTGGCAAGGTACTCATCTTCCGTACACGTTCGGAGGGGGGACCAAG CTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCC CACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTG CTTCTTGAACAACTTC.

Although specific embodiments were described herein, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present disclosure. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

WHAT IS CLAIMED IS:

1. An antibody that specifically binds one or more telomerase reverse transcriptase (TERT) peptides, wherein the antibody comprises a heavy chain comprising the nucleic acid sequence of SEQ ID NO:4, and a light chain comprising the nucleic acid sequence of SEQ ID NO: 3, a fragment thereof or a homolog thereof.

2. The antibody according claim 1, wherein the light chain comprises a variable light region (V_L) comprising the amino acid sequence of SEQ ID NO:6.

3. The antibody according to claim 1 or 2, comprising a heavy chain comprising the amino acid sequence of SEQ ID NO:2, and a light chain comprising the amino acid sequence of SEQ ID NO:l, a fragment thereof or a homolog thereof.

4. The antibody according to any one or more of claims 1 to 3, wherein the TERT peptide is a human telomerase reverse transcriptase (hTERT) peptide.

5. The antibody according to claim 4, wherein the hTERT peptide is the peptide comprising the amino acid sequence of SEQ ID NO: 5.

6. The antibody according to any one or more of claims 1 to 5, wherein the antibody is a monoclonal antibody.

7. The antibody according to any one or more of claims 1 to 6, wherein the antibody fragment is an antigen binding fragment that specifically binds one or more telomerase reverse transcriptase (TERT) peptides, selected from the group consisting of Fab, F(ab')2, Fd, Fv, dAb, scFv, and a complementarity determining region (CDR).

8. The antibody according to claim 7, wherein the antigen binding fragment is

F(ab')2.

9. The antibody according to any one or more of claims 1 to 8, which is a chimeric antibody comprising an antigen-binding fragment that specifically binds one or more TERT peptides, fused to a human IgG or IgM constant region.

10. The antibody according to any one or more of claims 1 to 9, wherein the antibody homolog comprises a variable heavy (VH) region and a variable light (VL) region having at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99.9% identity to the variable regions of SEQ ID NOs: 1, 2, 3, 4 and 6.

11. The antibody according to any one or more of claims 1 to 10, wherein the antibody or the antigen binding part thereof specifically binds two or more different but structurally related telomerase reverse transcriptase peptides.

12. The antibody according to claim 11, wherein at least one of said two or more telomerase reverse transcriptase peptides is a human telomerase reverse transcriptase (hTERT) peptide.

13. The antibody according to claim 12, wherein the hTERT peptide is a peptide of SEQ ID NO:5.

14. The antibody according to any one or more of claims 1 to 13, having a non-specific binding level that is less than 5%, less than 3%, less than 2%, less than 1.5%, less than 1%, less than 0.5%, less than 0.2%, or less than 0.1%, and any intermediate value therebetween.

15. The antibody according to any one or more of claims 1 to 14, conjugated to one or more of: (a) a tag useful for purification and detection; (b) a chemical moiety that alters the physical properties of the antibody; (c) an immunoadhesion peptide or protein; and (d) a drug.

16. A composition comprising at least one antibody, a fragment thereof or a homolog thereof according to any one or more of claims 1 to 15, and a physiologically acceptable excipient.

17. A method for diagnosis of cancer in a subject, the method comprising:

(b) contacting the sample with an anti-telomerase reverse transcriptase (TERT) antibody, a fragment thereof or a homolog thereof that specifically recognizes one or more TERT antigens presented by cancer cells, according to any one or more of claims 1 to 15; and

(c) measuring binding of the antibody, fragment or homolog thereof to TERT- presenting cells in the sample, wherein detection of binding indicates the presence and amount of cancer cells in the biological sample.

18. A method for isolating and detecting cancer cells in a sample the method comprising:

(b) contacting the sample with an anti-telomerase reverse transcriptase (TERT) antibody, a fragment thereof or a homolog thereof according to any one or more of claims 1 to 15;

(d) isolating the cells and measuring binding of the antibody, a fragment thereof or a homolog thereof to isolated, TERT-presenting cells, wherein detection of binding indicates the presence and amount of cancer cells in the biological sample.

19. The method according to claim 18, wherein the isolation means are magnetic beads and isolation of cells attached thereto is achieved by exposing the sample to an external magnetic field.

20. The method according to any one or more of claims 17 to 19, wherein the biological sample is a tissue biopsy or a bodily fluid selected from blood, plasma, urine, and saliva.

21. The method according to any one or more of claims 17 to 20, wherein the cancer cells are circulating cancer cells (CTCs).

22. A method for diagnosing cancer and monitoring response to cancer therapy in a subject, the method comprising

(a) administering to a subject suspected of having cancer, or a subject subjected to anti-cancer treatment, a diagnostically effective amount of an anti- telomerase reverse transcriptase (TERT) antibody, a fragment thereof or a homolog thereof according to any one or more of claims 1 to 15; and

(b) measuring binding of the anti-TERT antibody, a fragment thereof or a homolog thereof in the subject, wherein detection of binding indicates the presence or level of cancer in the subject, thereby diagnosing or monitoring response to cancer therapy in the subject.

23. The method according to any one or more of claims 17 to 22, wherein the anti-TERT antibody, fragment thereof or homolog thereof is labeled with one or more detectable substances selected from the group consisting of an enzyme, a prostatic group, a fluorescent material, a luminescent material, a radioactive material, a contrast agent, an isotope and inorganic micro- or nanoparticles.

24. A method for treating cancer in a subject, the method comprising administering to a subject inflicted with cancer a therapeutically effective amount of an anti-telomerase reverse transcriptase (TERT) antibody, a fragment thereof or a homolog thereof that specifically recognizes one or more telomerase antigens presented by cancer cells according to any one or more of claims 1 to 15, wherein the anti-TERT antibody, fragment thereof or homolog thereof is conjugated to an anti cancer drug, thereby treating cancer in the subject.

25. A nucleic acid molecule selected from: (i) a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:3, a fragment thereof or a homolog thereof; (ii) a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:4, a fragment thereof or a homolog thereof; (iii) a nucleic acid molecule comprising a nucleotide sequence of SEQ ID NO:3 and a nucleotide sequence of SEQ ID NO:4, a fragment thereof or a homolog thereof.

26. A recombinant or an engineered cell or cell line comprising: (i) a nucleic acid molecule as defined in claim 25; or (ii) a vector, a plasmid, or an artificial chromosome comprising or having contained therein the nucleic acid molecule as defined in claim 25, wherein, optionally, the nucleic acid molecule or the vector, plasmid or artificial chromosome are stably integrated into the cell’s chromosome, or are stably episomally expressed.

27. The recombinant or an engineered cell or cell line according to claim 26, being a cancer cell or a cancer cell line, or an immortalized cell line.

28. A hybridoma that can produce an antibody, or a fragment thereof as defined in any one or more of claims 1 to 15, that specifically binds one or more telomerase reverse transcriptase peptides (TERT).

29. A kit at least one of: an anti-TERT antibody, an antigen-binding fragment thereof and/or a homolog thereof according to any one or more of claim 1 to 15; reagents required for the detection and quantification of the antibody; and instructions for use.

30. The kit of claim 29, for use in one or more of: diagnosis of cancer in a subject, isolating and detecting cancer cells in a sample from a subject, monitoring response to cancer therapy in a subject, and treating cancer in a subject.

31. The kit according to claim 30, wherein the cancer cells are circulating tumor cells (CTCs).