EA042429B1 - COMPOSITIONS AND METHODS FOR DIAGNOSTICS AND TREATMENT OF PROSTATE CANCER USING PROGASTRIN BINDING MOLECULES - Google Patents

Description

Introduction

The present invention relates to the prevention and treatment of cancer, more specifically it relates to methods and compositions for the prevention or treatment of prostate cancer. The compositions of the invention comprise a progastrin binding molecule, in particular an anti-hPG (human progastrin) antibody, while the methods of the invention comprise the use of a progastrin binding molecule, and in particular an anti-hPG antibody.

According to the International Agency for Research on Cancer, prostate cancer (PC) is the second most common cancer in men and the fifth leading cause of cancer-related death in men. In 2012, it affected 1.1 million men and caused 307,000 deaths. It is the most common non-skin cancer in men in the United States. It is estimated that one in six white men and one in five black men will be diagnosed with prostate cancer in their lifetime, with an increased likelihood with increasing age.

Most prostate cancers (95%) are adenocarcinoma or glandular cancers that begin when normal prostate cells that secrete seminal fluid mutate into cancerous cells. Approximately 4% of prostate cancers have a transitional cell morphology and are believed to arise from the urothelial lining of the prostatic urethra. It is believed that a small number of cases that have a neuroendocrine morphology arise from neuroendocrine stem cells normally present in the prostate gland or from impaired differentiation programs during cell transformation. Squamous cell carcinomas account for less than 1% of all prostate carcinomas. Prostate cancer most commonly metastasizes to the bones, lymph nodes, and may invade the rectum, bladder, and lower ureters after local progression.

Treatments typically include surgery, chemotherapy, radiation therapy, and targeted therapy alone or in combination. However, outcomes are often poor with less than 10% 5-year survival globally. This is mainly because most people are only diagnosed with advanced disease, with direct implications for survival rates. In some Asian countries, screening efforts have been shown to be associated with higher survival rates.

The five-year survival rate of the entire population with prostate cancer is very high (about 99%). However, this ratio drops significantly with the metastasis of this cancer (about 28.5%). Fortunately, approximately 80% of patients are diagnosed with localized disease (Surveillance E; End Results Program (SEER). Surveillance, Epidemiology, and Ends Results Program. Fast Stats; 2016 [cited September 12, 2016]. Available at: http://seer .cancer.gov/faststats/selections.php). This is largely due to improvements in screening methods. However, the most commonly used biomarker, prostate specific antigen (PCA), has proven controversial as a diagnostic test due to its limitations.

Therefore, there is still a need for new compositions and methods for the prevention or treatment of prostate cancer.

This is the purpose of the present invention.

Description of the invention

The present invention now provides an antibody that specifically binds to progastrin for use in the prevention or treatment of prostate cancer. The present invention also provides a composition for use in the prevention or treatment of prostate cancer, wherein said composition comprises a progastrin-binding antibody, and methods for the prevention or treatment of prostate cancer, comprising the use of a composition comprising a progastrin-binding antibody, alone or in combination. with any other known prophylactic or therapeutic methods against prostate cancer.

The anti-hPG antibodies described herein, in particular the neutralizing anti-hPG antibodies, inhibit PG-dependent prostate tumor cell proliferation, making them useful therapeutic agents for the treatment of prostate cancer. Accordingly, pharmaceutical compositions comprising an anti-hPG antibody and methods of using anti-hPG antibodies and/or pharmaceutical compositions for the treatment of prostate cancer are also provided. Pharmaceutical compositions may be formulated for any convenient route of administration, including, for example, parenteral, subcutaneous, or intravenous injection, and will typically include an anti-hPG antibody and one or more acceptable carriers, excipients, and/or diluents suitable for the desired route of administration, and may include other possible components, as will be further described below. For therapeutic applications, these compositions may be packaged in unit dosage forms for ease of administration.

Methods of treatment typically involve administering to a subject in need of treatment, such as a subject

- 1 042429 one diagnosed with prostate cancer, an effective amount of an anti-PG antibody and/or a pharmaceutical composition thereof to obtain a therapeutic benefit. The therapeutic benefit, described in more detail below, includes any reduction in the intensity of prostate cancer, such as slowing or stopping the progression of prostate cancer, reducing the severity of prostate cancer, inhibiting the growth of prostate tumors or prostate cancer cell proliferation, reducing the size of prostate tumors, and /or a decrease in serum levels of PG in patients with prostate cancer. The subject may or may not be a human, including a pet (eg, cat, dog, cow, pig, horse) or non-pet. Preferably, the subject to be treated is a human. Subjects in whom anti-hPG antibody therapy is beneficial may include: patients at any stage of disease progression (eg, with stage 0, I, II, III, or IV prostate cancer), patients who have received anti-prostate cancer therapy ( eg chemotherapy, radiation therapy, surgical resection), or patients who are receiving other therapy for prostate cancer.

Also provided are methods for inhibiting prostate cancer stem cell growth in a patient by administering to a patient in need of inhibiting prostate cancer stem cell growth an anti-PG antibody and/or a pharmaceutical composition thereof in an effective amount to inhibit said prostate cancer stem cell.

In a number of embodiments, anti-PG antibodies are effective in reducing the proliferation or increasing the differentiation or rate of cell death of prostate cancer stem cells, or in decreasing the blood concentration of progastrin in treated patients. In other embodiments, the anti-PG antibodies and/or pharmaceutical composition thereof can be administered concomitantly with or after a second effective therapeutic agent for inhibiting the growth of colorectal cancer stem cells, for example, an antibody having a specificity different from that of progastrin.

Treatment with antibodies against hPG, as described herein, can be combined with or supplemented by other therapy. Non-limiting examples of other therapies for prostate cancer include chemotherapy, radiation therapy, surgical resection, and antibody therapy, as described herein. In a specific example, anti-hPG antibodies are administered in combination with chemotherapeutic agents. In another specific example, anti-hPG antibodies are administered in addition to surgical resection.

The present invention also relates to pharmaceutical compositions containing antibodies against PG, preferably with a pharmaceutically acceptable carrier and/or excipient. In some embodiments, the pharmaceutical composition further comprises a second therapeutic agent. In some embodiment, the second therapeutic agent is a biological agent or a chemotherapeutic agent. Examples of biological agents include monoclonal antibodies against EGFR (epidermal growth factor receptor) and monoclonal antibodies against VEGF (vascular endothelial growth factor), while chemotherapeutic agents contain compounds such as, for example, alkylating agents, antimetabolites, antitumor antibiotics, mitosis inhibitors, function inhibitors. chromatin, anti-angiogenesis agents, antiestrogen agents, antiandrogen agents and immunomodulators.

Human pre-progastrin, a 101 amino acid peptide (reference amino acid sequence: AAB19304.1), is the primary translation product of the gastrin gene. Progastrin is formed by cleavage of the first 21 amino acids (signal peptide) from preprogastrin. The 80 amino acid chain of progastrin is further processed through cleavage and modification by enzymes to several biologically active hormonal forms of gastrin: gastrin 34 (G34) and gastrin 34 with glycine extension (G34-Gly), containing amino acids 38-71 of progastrin, gastrin 17 (G17) and gastrin 17 with glycine extension (G17-Gly) containing progastrin amino acids 55-71.

Monoclonal antibodies against human progastrin (anti-hPG antibodies) and their use for diagnosis or therapy have been described in the following documents: WO 2011/083088 for colorectal cancer, WO 2011/083090 for breast cancer, WO 2011/083091 for pancreatic cancer, WO 2011/116954 for colorectal and gastrointestinal cancers, and WO 2012/013609 and WO 2011/083089 for liver pathologies.

The present invention will become clearer from the detailed description provided herein and from the accompanying drawings, which are provided by way of illustration only and do not limit the intended scope of the present invention.

In a first aspect, the present invention relates to a progastrin binding molecule for use in the prevention or treatment of prostate cancer. The present disclosure also provides a composition for use in the prevention or treatment of prostate cancer, wherein said composition comprises an antibody that binds to progastrin, or an antigen-binding fragment thereof.

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A progastrin-binding molecule is defined herein as any molecule that binds to progastrin but does not bind to gastrin-17 (G17), gastrin-34 (G34), glycine-extended gastrin 17 (G17-Gly) or glycine-extended gastrin 34 (G34-Gly). The progastrin-binding molecule of the present invention may be any progastrin-binding molecule, such as, for example, an antibody molecule or a receptor molecule. Preferably, the progastrin binding molecule is an anti-progastrin antibody (anti-PG antibody) or an antigen-binding fragment thereof.

The term progastrin refers to the peptide of mammalian progastrin and in particular human progastrin. For the avoidance of doubt, without any precise definition, the expression human progastrin or hPG refers to the human PG of the sequence SEQ ID NO: 1. Namely, human progastrin contains N-terminal and C-terminal domains that are not biologically present in the forms mentioned above. the active hormone gastrin. Preferably, the sequence of said N-terminal domain is represented by SEQ ID NO: 2. In another preferred embodiment, the sequence of said C-terminal domain is represented by SEQ ID NO: 3.

Thus, in a first embodiment, this invention provides an antibody that binds to progastrin but does not bind to any of the other products derived from the gastrin gene for use in the treatment of prostate cancer.

By binding, binding, or the like, is meant that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Methods for determining whether two molecules bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. In a specific embodiment, said antibody, or antigen-binding fragment thereof, binds to progastrin with an affinity that is at least two times greater than its binding affinity for a non-specific molecule such as BSA (bovine serum albumin) or casein. In a more specific embodiment, said antibody, or antigen-binding fragment thereof, binds only to progastrin.

The expression prostate cancer refers to any type of cancer that occurs in the prostate gland. Prostate cancer includes, in particular, adenocarcinoma of the prostate, but also sarcomas, small cell carcinomas, neuroendocrine tumors, transitional cell carcinomas, which can also develop within the prostate. The expression prostate cancer also includes prostate cancer associated with metastasis, in particular metastasis to bone, lymph nodes, but also to the rectum, bladder and lower ureters.

In a specific embodiment, the invention provides an anti-PG antibody for use in the prevention or treatment of prostate cancer, said antibody recognizing an epitope comprising an amino acid sequence corresponding to the amino acid sequence of progastrin.

In a more specific embodiment, said anti-PG antibody for use in the prevention or treatment of prostate cancer recognizes a progastrin epitope, wherein said epitope comprises an amino acid sequence corresponding to the amino acid sequence of the N-terminal portion of progastrin, wherein said amino acid sequence may include hPG residues 10-14, residues 9- 14 hPG, hPG residues 4-10, hPG residues 2-10, or hPG residues 2-14, wherein the hPG amino acid sequence is SEQ ID NO: 1.

In a more specific embodiment, an anti-PG antibody for use in the prevention or treatment of prostate cancer recognizes a progastrin epitope, wherein said epitope comprises an amino acid sequence corresponding to the amino acid sequence of the C-terminal portion of progastrin, wherein said amino acid sequence may include hPG residues 71-74, residues 69 -73 hPG, hPG residues 71-80 (SEQ ID NO: 40), hPG residues 76-80, or hPG residues 67-74, wherein the hPG amino acid sequence is SEQ ID NO: 1.

In a more specific embodiment, an anti-PG antibody for use in the prevention or treatment of prostate cancer has an affinity for progastrin of at least 5000 nM, of at least 500, 100, 80, 60, 50, 4o, 30, 20, 10, 7, 5, 4, 3, 2, 1, 0.5, 0.1 nM, 50, 10, 5, 1 pM, or at least 0.1 pM when determined in a manner such as the above method.

Preferably, the anti-PG antibody for use in the prevention or treatment of prostate cancer is a neutralizing anti-PG antibody.

The expression "neutralizing antibody against PG" means an antibody that binds to PG and blocks PG-dependent signaling, resulting in the inhibition of PG-induced responses in tumor cells and in particular in prostate tumor cells. Inhibition of PG-induced responses of prostate cancer cells can be mediated by repression of cell differentiation, repression of cell death and/or stimulation of cell proliferation.

The term antibody, as used herein, is intended to include polyclonal and monoclonal antibodies. An antibody (or immunoglobulin)

- 3 042429 um from a glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (or domain) (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains: CH1, CH2 and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain: CL. The VH and VL regions can be further subdivided into regions of hypervariability, referred to as complementarity determining regions (CDRs) or hypervariable regions, which are primarily responsible for binding to an antigen epitope, and interspersed with regions that are more conserved, referred to as framework regions. (FR). A method for identifying CDRs within the light and heavy chains of an antibody and determining their sequence is well known to those skilled in the art. For the avoidance of doubt, in the absence of any indication to the contrary in the text, the expression CDR means the hypervariable regions of the heavy and light chains of an antibody, as defined by IMGT, where the unique numbering of the IMGT provides a standardized delimitation of framework regions and complementarity determining regions, CDR1-IMGT: 27-38 , CDR2.

A unique IMGT numbering has been defined to compare variable domains, whatever the antigen receptor, chain type, or species [Lefranc M.-P., Immunology Today 18, 509 (1997)/Lefranc M.-P., The Immunologist, 7, 132-136 (1999) / Lefranc, M.-P., Pommie, C, Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L, Thouvenin-Contet, V. and Lefranc, Dev. Associate Immunol., 27, 55-77 (2003)]. In the unique IMGT numbering, conserved amino acids always have the same position, e.g. cysteine 23 (1st CYS), tryptophan 41 (CONSERVATIVE TRP), hydrophobic amino acid 89, cysteine 104 (2nd CYS), phenylalanine or tryptophan 118 (J -PHE or J-TRP). The unique IMGT numbering provides a standardized delimitation of the framework regions (FR1-IMGT: positions 1-26, FR2-IMGT: 39-55, FR3IMGT: 66-104 and FR4-IMGT: 118-128) and complementarity-determining regions: CDR1-IMGT: 27-38, CDR2-IMGT: 56-65 and CDR3-IMGT: 105-117. Since spaces represent unoccupied positions, the CDR-IMGT lengths (shown between brackets and separated by dots, eg [8.8.13]) become key information. Unique IMGT numbering is used in 2D (two-dimensional) graphical representations, designated as IMGT Colliers de Perles [Ruiz, M. and Lefranc, M.-P., Immunogenetics, 53, 857-883 (2002) / Kaas, Q. and Lefranc, M.-P., Current Bioinformatics, 2, 21-30 (2007)], and in 3D structures in IMGT/3Dstructure-DB [Kaas, Q., Ruiz, M. and Lefranc, M.-P. , T cell receptor and MHC structural data. Nucl. Acids. Res., 32, D208-D210 (2004)].

Each VH and VL consists of three CDRs and four FRs, organized from amino to carboxy in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (CIq) of the classical complement system. Antibodies can be of different isotypes (namely: IgA, IgD, IgE, IgG or IgM).

In a particular embodiment, said progastrin-binding antibody or antigen-binding fragment thereof is selected from the group consisting of: polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, camelized antibodies, IgA1 antibodies, IgA2 antibodies, IgD antibodies, IgE antibodies, IgG1 antibodies , IgG2 antibodies, IgG3 antibodies, IgG4 antibodies and IgM antibodies.

A polyclonal antibody is an antibody that has been produced among or in the presence of one or more other non-identical antibodies. In general, polyclonal antibodies are produced from B lymphocytes in the presence of several other B lymphocytes producing non-identical antibodies. Typically, polyclonal antibodies are obtained directly from the immunized animal.

The term monoclonal antibody refers to an antibody arising from a nearly homogeneous population of antibodies, where this population contains identical antibodies, except for a few possible naturally occurring mutations, which may be in minimal proportions. A monoclonal antibody arises from the growth of a single cell clone, such as a hybridoma, and is distinguished by one class and subclass of heavy chains and one type of light chains.

The expression antigen-binding fragment of an antibody is meant to indicate any peptide, polypeptide or protein that retains the ability to bind to the target (also commonly referred to as an antigen) of said antibody, usually with the same epitope, and contains an amino acid sequence of at least 5 contiguous amino acid residues, at least 10 contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least at least 60 contiguous amino acid residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least 90 contiguous amino acid residues, at least 100 contiguous amino acid residues, at least 125 contiguous amino acid residues, at least pe 150 contiguous amino acid residues, at least 175 contiguous amino acid residues, or at least 200 contiguous amino acid residues of an antibody amino acid sequence.

In a specific embodiment, said antigen-binding fragment contains at least one CDR of the antibody from which it is derived. In addition, in a preferred embodiment, said antigen binding fragment contains 2, 3, 4 or 5 CDRs, more preferably 6 CDRs of the antibody from which they are derived.

Antigen binding fragments may be selected, without limitation, from the group consisting of Fv, scFv (sc stands for single chain), Fab, F(ab')2, Fab', scFv-Fc, or diabodies, or fusion proteins with random peptides such as as XTEN (extended recombinant polypeptide) or PAS motifs, or any fragment whose half-life would be increased by chemical modification such as the addition of a poly(alkylene) glycol such as poly(ethylene) glycol (RESylation) (pegylated fragments are called Fv-PEG, scFv-PEG, Fab-PEG, F(ab') ₂ -PEG or Fab'-PEG) (PEG is polyethylene glycol), or by incorporation into a liposome, said fragments having at least one of the characteristic CDRs of an antibody of the invention. Preferably, said antigen-binding fragments will be or will consist of a partial sequence of the heavy or light chain variable region of the antibody from which they are derived, said partial sequence being sufficient to retain the same binding specificity as the antibody from which it is derived and sufficient affinity, preferably at least equal to 1/100, more preferably at least 1/10 of the affinity of the antibody from which it is derived, in relation to the target.

In another specific embodiment, in the method for diagnosing prostate cancer according to the invention, a biological sample from a subject is brought into contact with an antibody that binds to progastrin, where the specified antibody was obtained by an immunization method known to a person skilled in the art, where a peptide is used as an immunogen, the amino acid sequence of which is contains all or part of the progastrin amino acid sequence. More specifically, said immunogen contains a peptide selected from:

a peptide, the amino acid sequence of which contains or consists of the amino acid sequence of full-length progastrin and, in particular, full-length human progastrin SEQ ID NO: 1;

a peptide whose amino acid sequence corresponds to part of the amino acid sequence of progastrin and, in particular, full-length human progastrin SEQ ID NO: 1;

a peptide whose amino acid sequence corresponds to part or all of the amino acid sequence of the N-terminal portion of progastrin, and in particular to peptides containing or consisting of the amino acid sequence

SWKPRSQQPDAPLG (SEQ ID NO 2), and a peptide whose amino acid sequence corresponds to part or all of the amino acid sequence of the C-terminal portion of progastrin, and in particular peptides containing or consisting of the amino acid sequence

QGPWLEEEEEAYGWMDFGRRSAEDEN (SEQ ID NO: 3), a peptide whose amino acid sequence corresponds to part of the amino acid sequence of the C-terminal part of progastrin and, in particular, peptides containing the amino acid sequence FGRRSAEDEN (SEQ ID NO: 40) corresponding to amino acids 71-80 of progastrin.

The specialist will understand that such immunization can be used to obtain either polyclonal or monoclonal antibodies, depending on what is desired. Methods for obtaining each of these types of antibodies are well known in this field. Thus, one skilled in the art will readily select and use a method for producing polyclonal and/or monoclonal antibodies against any given antigen.

Examples of monoclonal antibodies that have been generated using an immunogen containing the amino acid sequence “SWKPRSQQPDAPLG” corresponding to the amino acid sequence 1-14 of human progastrin (N-terminal) include the monoclonal antibodies designated as mAb3, mAb4, mAb16, mAb19, and mAb20 as described in the following tab. 1-4, but are not limited to them. Other monoclonal antibodies have been described, although it is not clear whether these antibodies actually bind to progastrin (WO 2006/032980). Experimental epitope mapping results show that mAb3, mAb4, mAb16, mAb19, and mAb20 indeed bind specifically to an epitope within the indicated N-terminal amino acid sequence of hPG. Polyclonal antibodies specifically recognizing an epitope within the N-terminus of progastrin as represented by SEQ ID NO: 2 have been described in the art (see, for example, WO 2011/083088).

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Table 1

Deposition of hybridoma mAb Amino acid sequences SEQID NO 6V5V11S10 tal3 CDR 1 VH GYIFTSYW SEQID NO 4 CDR 2 VH FYPGNSDS SEQID NO 5 CDR 3 VH TRRDSPQY SEQ ID NO 6 CDR 1 VL QSIVHSNGNTY SEQ ID NO 7 CDR2 VL KVS SEQID NO 8 CDR 3 VL FQGSHVPFT SEQ ID NO 9 mVH 3 EVQLQQSGTVLARPGASVKMS CKASGYIFTSYWVHWVKQRPG QGLEWIGGFYPGNSDSRYNQ KFKGKATLTAVTSASTAYMDLS SLTNEDSAVYFCTRRDSPQYW GQGTTLTVSS SEQ ID NO 41 mvl 3 huVH 3 huVL 3 DVLMTQTPLSLPVSLGDQASIS CRSSQSIVHSNGNTYLEWYLQ KPGQSPKLLIYKVSNRFSGVPD RFSGSGSGTDFTLKISRLEAED LGVYYCFQGSHVPFTFGGGTK QVQLVQSGAEVKKPGASVKVS CKASGYI FTSYWVHWVRQAPG QRLEWMGGFYPGNSDSRYSQ KFQGRVTITRDTSASTAYMELS SLRSEDTAVYYCTRRDSPQYW GQGTLVTVSS DWMTQSPLSLPVTLGQPASIS CRSSQSIVHSNGNTYLEWFQQ RPGQSPRRLIYKVSNRFSGVP DRFSGSGSGTDFTLKISRVEAE DVGVYYCFQGSHVPFTFGGGT KVEIK SEQ ID NO 42 SEQ ID NO 53 SEQ ID NO 54

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table 2

Deposition of hybridoma mAb Amino acid sequences SEQID NO 20D2C3G2 rnAb4 CDR 1 VH GYTFSSW SEQID NO 10 CDR 2 VH FLPGSGST SEQ ID NO 11 CDR 3 VH ATDGNYDWFAY SEQ ID NO 12 CDR 1 VL QSLVHSSGVTY SEQ ID NO 13 CDR2 VL KVS SEQ ID NO 14 CDR 3 VL SQSTHVPPT SEQ ID NO 15 mvh 4 QVQ LQQSGAELM KPGASVKIS CKATGYTFSSSWIEWLKQRPG HGLEWIGEFLPGSGSTDYNEK FKGKATFTADTSSDTAYMLLSS LTSEDSAVYYCATDGNYDWFA YWGQGTLVTVSA SEQ ID NO 43 mvl 4 huVH 4 huVL4 DLVMTQTPLSLPVSLGDQASIS CRSSQSLVHSSGVTYLHWYLQ KPGQSPKLLIYKVSNRFSGVPD RFSGSGSGTDFTLKISRVEAED LGVYFCSQSTHVPPTFGSGTK LEIK QVQLVQSGAEVKKPGASVKVS CKASGYTFSSSWMHWVRQAP GQGLEWMGIFLPGSGSTDYAQ KFQGRVTMTRDTSTSTVYMEL SSLRSEDTAVYYCATDGNYDW FAYWGQGTLVTVSS DIVMTQTPLSLSVTPGQPASIS CKSSQSLVHSSGVTYLYWYLQ KPGQSPQLLIYKVSNRFSGVP DRFSGSGSGTDFTLKISRVEAE DVGVYYCSQSTHVPPTFGQGT KLEIK SEQ ID NO 44 SEQ ID NO 55 SEQ ID NO 56

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Table 3

Deposition of hybridoma mAb Amino acid sequences SEQID NO 1E9D9B6 taL16 CDR 1 VH GYTFTSYY SEQ ID NO 16 CDR 2 VH INPSNGGT SEQ ID NO 17 CDR 3 VH TRGGYYPFDY SEQ ID NO 18 CDR 1 VL QSLLDSDGKTY SEQ ID NO 19 CDR2 VL LVS SEQID NO 20 CDR 3 VL WQGTHSPYT SEQ ID NO 21 mvh 16 QVQLQQSGAELVKPGASVKLS C KASG YTFTSYYM YWVKQ R P GQGLEWIGEINPSNGGTNFNE KFKSKATLTVDKSSSTAYMQLS SLTSEDSAVYYCTRGGYYPFD YWGQGTTLTVSS SEQ ID NO 45 mvl 16 DWMTQTPLTLSVTIGRPASIS SEQ ID NO 46

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CKSSQSLLDSDGKTYLYWLLQ RPGQSPKRLIYLVSELDSGVPD RITGSGSGTDFTLKISRVEAED LGVYYCWQGTHSPYTFGGGT KLEIK huVH 16a QVQLVQSGAEVKKPGASVKVS CKASGYTFTSYYMYWVRQAP GQGLEWMGIINPSNGGTSYAQ KFQGRVTMTRDTSTSTVYMEL SSLRSEDTAVYYCTRGGYYPF DYWGQGTTVTVSS SEQ ID NO 57 huVH 16b QVQLVQSGAEVKKPGASVKVS CKASGYTFTSYYMHWVRQAP GQGLEWMGIINPSNGGTSYAQ KFQGRVTMTRDTSTSTVYMEL SSLRSEDTAVYYCTRGGYYPF DYWGQGTTVTVSS SEQ ID NO 58 huVH 16c QVQLVQSGAEVKKPGASVKVS CKASGYTFTSYYMYWVRQAP GQGLEWMGEINPSNGGTNYA QKFQGRVTMTRDTSTSTVYME LSSLRSEDTAVYYCTRGGYYP FDYWGQGTTVTVSS SEQ ID NO 59 huVL 16a DWMTQSPLSLPVTLGQPASIS CRSSQSLLDSDGKTYLYWFQQ RPGQSPRRLIYLVSNRDSGVP DRFSGSGTDFTLKISRVEAE DVGVYYCWQGTHSPYTFGQG TKLEIK SEQ ID NO 60 huVL 16b DWMTQSPLSLPVTLGQPASIS CRSSQSLLDSDGKTYLNWFQQ RPGQSPRRLIYLVSNRDSGVP DRFSGSGSGTDFTLKISRVEAE DVGVYYCWQGTHSPYTFGQG TKLEIK SEQ ID NO 61 huVL 16c DWMTQSPLSLPVTLGQPASIS CRSSQSLLDSDGKTYLYWFQQ RPGQSPRRLIYLVSERDSGVP DRFSGSGSGTDFTLKISRVEAE DVGVYYCWQGTHSPYTFGQG TKLEIK SEQ ID NO 62

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Table 4

Deposition of hybridoma mAb Amino acid sequences SEQID NO 1B3B4F11 taL19 CDR 1 VH GYSITSDYA SEQ ID NO 22 CDR 2 VH ISFSGYT SEQ ID NO 23 CDR 3 VH AREVNYGDSYHFDY SEQ ID NO 24 CDR 1 VL SQHRTYT SEQ ID NO 25 CDR2 VL VKKDGSH SEQ ID NO 26 CDR 3 VL GVGDAIKGQSVFV SEQ ID NO 27 mvh 19 DVQLQESGPGLVKPSQSLSLT CTVTGYSITSDYAWNWIRQFP GNKLEWMGYISFSGYTSYNPS LKSRISVTRDTSRNQFFLQLTS VTTEDTATYYCAREVNYGDSY HFDYWGQGTIVTVSS SEQ ID NO 47 mvl 19 QLALTQSSSASFSLGASAKLTC TLSSQHRTYTIEWYQQQSLKP PKYVMEVKKDGSHSTGHGIPD RFSGSSSGADRYLSISNIQPED EAIYICGVGDAIKGQSVFVFGG GTKVTVL SEQ ID NO 48 huVH 19a QVQLQESGPGLVKPSQTLSLT CTVSGYSITSDYAWNWIRQHP GKGLEWIGYISFSGYTYYNPSL KSRVTISVDTSKNQFSLKLSSV TAADTAVYYCAREVNYGDSYH SEQ ID NO 63

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FDYWGQGTLVTVSS huVH 19b QVQLQESGPGLVKPSQTLSLT CTVSGYSITSDYAWSWIRQHP GKGLEWIGYISFSGYTYYNPSL KSRVTISVDTSKNQFSLKLSSV TAADTAVYYCAREVNYGDSYH FDYWGQGTLVTVSS SEQ ID NO 64 huVH 19c QVQLQESGPGLVKPSQTLSLT CTVSGYSITSDYAWNWIRQHP GKGLEWIGYISFSGYTSYNPSL KSRVTISVDTSKNQFSLKLSSV TAADTAVYYCAREVNYGDSYH FDYWGQGTLVTVSS SEQ ID NO 65 huVL 19a QLVLTQSPSASASLGASVKLTC TLSSQHRTYTIEWHQQQPEKG PRYLMKVKKDGSHSKGDGIPD RFSGSSSGAERYLTISSLQSED EADYYCGVGDAIKGQSVFVFG GGTKVEIK SEQ ID NO 66 huVL 19b QLVLTQSPSASASLGASVKLTC TLSSQHRTYTIAWHQQQPEKG PRYLMKVKKDGSHSKGDGIPD RFSGSSSGAERYLTISSLQSED EADYYCGVGDAIKGQSVFVFG GGTKVEIK SEQ ID NO 67 huVL 19c QLVLTQSPSASASLGASVKLTC TLSSQHRTYTIEWHQQQPEKG PRYLMEVKKDGSHSKGDGIPD RFSGSSSGAERYLTISSLQSED EADYYCGVGDAIKGQSVFVFG GGTKVEIK SEQ ID NO 68

Examples of monoclonal antibodies that can be obtained using an immunogen containing the amino acid sequence "QGPWLEEEEEAYGWMDFGRRSAEDEN" (C-terminal portion of progastrin) corresponding to the amino acid sequence 55-80 of human progastrin include the antibodies indicated in the following table. 5 and 6 as but not limited to mAb8 and mAb13. The experimental results of epitope mapping show that mAb13 indeed specifically binds to an epitope within the indicated C-terminal amino acid sequence of hPG.

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Table 5

Deposition of hybridoma mAb Amino acid sequences SEQID NO 1C10D3B9 ta8 CDR 1 VH GFTFTTYA SEQ ID NO 28 CDR 2 VH ISSGGTYT SEQ ID NO 29 CDR 3 VH ATQGNYSLDF SEQID NO 30 CDR 1 VL KSLRHTKGITF SEQ ID NO 31 CDR2 VL QMS SEQ ID NO 32 CDR 3 VL AQNLELPLT SEQ ID NO 33 mVH 8 EVQLVESGGGLVKPGGSLRL SCAASGFTFTTYAMSWVRQA PGKGLEWVATISSGGTYTYY ADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCATQGN YSLDFWGQGTTVTVSS SEQ ID NO 49 mvl 8 DIVMTQSPLSLPVTPGEPASI SCRSSKSLRHTKGITFLYWYL QKPGQSPQLLIYQMSNLASG VPDRFSSSGSGTDFTLKISRV EAEDVGVYYCAQNLELPLTF GGGTKVEIK SEQ ID NO 50 VH hZ8CV1 EVQLVESGGGLVKPGGSLRL SCAASGFTFTTYAMSWVRQA PGKGLEWVSSISSGGTYTYY ADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCATQGN YSLDFWGQGTTVTVSS SEQ ID NO 69

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VL hZ8CV1 DIVMTQSPLSLPVTPGEPASI SCRSSKSLRHTKGITFLYWYL QKPGQSPQLLIYQMSNRASG VPDRFSGSGSGTDFTLKISRV EAEDVGVYYCAQNLELPLTF GGGTKVEIK SEQ ID NO 70 VH hZ8CV2 EVQLVESGGGLVKPGGSLRL SCAASGFTFTTYAMSWVRQA PGKGLEWVATISSGGTYTYY ADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCATQGN YSLDFWGQGTTVTVSS SEQ ID NO 71 VL hZ8CV2 DIVMTQSPLSLPVTPGEPASI SCRSSKSLRHTKGITFLYWYL QKPGQSPQLLIYQMSNLASG VPDRFSSSGSGTDFTLKISRV EAEDVGVYYCAQNLELPLTF GGGTKVEIK SEQ ID NO 72 CH hZ8CV2 EVQLVESGGGLVKPGGSLRL SCAASGFTFTTYAMSWVRQA PGKGLEWVATISSGGTYTYY ADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCATQGN YSLDFWGQGTTVTVSSASTK GPSVFPLAPSSKSTSGGTAA LGCLVKDYFPEPVTVSWNSG ALTSGVHTFPAVLQSSGLYSL SSVVTVPSSSLGTQTYICNVN HKPSNTKVDKRVEPKSCDKT HTCPPCPAPELLGGPSVFLFP PKPKDTLMISRTPEVTCWVD VSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNSTYRWS VLTVLHQDWLNGKEYKCKVS NKALPAPIEKTISKAKGQPRE SEQ ID NO 73

- 13 042429

PQVYTLPPSREEMTKNQVSL TCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSC SVMHEALHNHYTQKSLSLSP GK CL hZ8CV2 DIVMTQSPLSLPVTPGEPASI SCRSSKSLRHTKGITFLYWYL QKPGQSPQLLIYQMSNLASG VPDRFSSSGSGTDFTLKISRV EAEDVGVYYCAQNLELPLTF GGGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASWCLLNNF YPREAKVQWKVDNALQSGN SQESVTEQDSKDSTYSLSST LVGLSSYACEVEKHK SEQ ID NO 74

Table 6

Deposition of hybridoma mAb Amino acid sequences SEQID NO 2C6C3C7 TAYZ CDR 1 VH GFIFSSYG SEQ ID NO 34 CDR 2 VH INTFGDRT SEQ ID NO 35 CDR 3 VH ARGTGTY SEQ ID NO 36 CDR 1 VL QSLLDSDGKTY SEQ ID NO 37 CDR2 VL LVS SEQ ID NO 38 CDR 3 VL WQGTHFPQT SEQ ID NO 39 mvh 13 EVQLVESGGGLVQPGGSLKL SCAASGFIFSSYGMSWVRQS PDRRLELVASINTFGDRTYYP DSVKGRFTISRDNAKNTLYLQ MTSLKSEDTAIYYCARGTGTY WGQGTTLTVSS SEQ ID NO 51

- 14 042429

mvl 13 DVVLTQTPLTLSVTIGQPASIS CKSSQSLLDSDGKTYLNWLL QRPGQSPKRLIYLVSKLDSGV PDRFTGSGSGTDFTLKISRVE AEDLGVYYCWQGTHFPQTF GGGTKLEIK SEQ ID NO 52 huVH 13a EVQLVESGGGLVQPGGSLRL SCAASGFI FSSYGMSWVRQA PGKGLEWVANINTFGDRTYY VDSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGTG TYWGQGTLVTVSS SEQ ID NO 75 huVH 13b EVQLVESGGGLVQPGGSLRL SCAASGFI FSSYGMSWVRQA PGKGLEWVASINTFGDRTYY VDSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCARGTG TYWGQGTLVTVSS SEQ ID NO 76 huVL 13a DVVMTQSPLSLPVTLGQPASI SCRSSQSLLDSDGKTYLNWF QQRPGQSPRRLIYLVSNRDS GVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCWQGTHFP QTFGGGTKVEIK SEQ ID NO 77 huVL 13b DVVMTQSPLSLPVTLGQPASI SCRSSQSLLDSDGKTYLNWF QQRPGQSPRRLIYLVSKRDS GVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCWQGTHFP QTFGGGTKVEIK SEQ ID NO 78

Other examples include monoclonal and/or polyclonal antibodies against hPG obtained using an immunogen containing the amino acid sequence of SEQ ID NO: 40.

The terms anti-hPG N-terminal antibodies and anti-hPG C-terminal antibodies refer to antibodies that bind to an epitope containing amino acids located at the N-terminal portion of hPG or an epitope containing amino acids located at the C-terminal portion of hPG, respectively. Preferably, the term antibodies against the N-terminus of hPG refers to antibodies that bind to an epitope located in the progastrin domain, the sequence of which is shown by SEQ ID NO: 2. In another preferred embodiment, the term antibodies against the C-terminus of hPG refers to antibodies that bind to an epitope located in the progastrin domain, the sequence of which is presented by SEQ ID NO: 3.

The term epitope refers to the region of an antigen to which an antibody binds. Epitopes can be defined as structural or functional. Functional epitopes are usually a subset of structural epitopes, and they contain those amino acids that directly contribute to the affinity of the interaction. Epitopes can also be conformational. In some embodiments, epitopes may include determinants that are reactive surface groupings of molecules, such as amino acids, side sugar chains, phosphoryl groups, or sulfonyl groups, and in some embodiments they may have specific three-dimensional structural characteristics and/or specific charge characteristics. Determination of an epitope bound by an antibody can be performed by any epitope mapping technique known to one of skill in the art. An epitope may contain different amino acids that are consecutive within the amino acid sequence of a protein. An epitope may also contain amino acids that are not sequential within the amino acid sequence of the protein.

In a specific embodiment, said antibody is a monoclonal antibody selected from the group consisting of the following:

- 15 042429 a monoclonal antibody containing a heavy chain containing at least one, preferably at least two, preferably three of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NOs: 4, 5 and 6, respectively; and a light chain comprising at least one, preferably at least two, preferably three of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 7, 8 and 9, respectively, or sequences with at least 80%, preferably 85, 90, 95 and 98% identity after optimal alignment with the sequences of SEQ ID NO: 7, 8 and 9, respectively, a monoclonal antibody containing a heavy chain containing at least one, preferably at least at least two, preferably three of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, or sequences with at least 80%, preferably 85, 90, 95 and 98 % identity after optimal alignment with SEQ ID NOs: 10, 11 and 12, respectively, and a light chain containing at least one, preferably at least two, preferably three of CDR-L1, CDR-L2 and CDR- L3 amino acid sequences of SEQ ID NOs: 13, 14, and 15, respectively, or sequences with at least 80%, preferably 85, 90, 95, and 98% identity after optimal alignment with SEQ ID NOs: 13, 14, and 15 , respectively, a monoclonal antibody containing a heavy chain containing at least one, preferably at least two, preferably three of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOS: 16, 17 and 18, respectively, or sequences with at least 80%, preferably 85, 90, 95 and 98% identity after optimal alignment with the sequences of SEQ ID NO: 16, 17 and 18, respectively, and a light chain containing at least one, preferably at least two, preferably three of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 19, 20 and 21, respectively, or sequences with at least 80%, preferably 85, 90, 95 and 98% identity after optimal linear alignment with the sequences of SEQ ID NO: 19, 20 and 21, respectively, a monoclonal antibody containing a heavy chain containing at least one, preferably at least two, preferably three of CDR-H1, CDR-H2 and CDR-H3 amino acids sequences of SEQ ID NOS: 22, 23 and 24, respectively, or sequences with at least 80%, preferably 85, 90, 95 and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 22, 23 and 24 , respectively, and a light chain containing at least one, preferably at least two, preferably three of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 25, 26 and 27, respectively, or sequences with at least 80%, preferably 85, 90, 95 and 98% identity after optimal alignment with the sequences of SEQ ID NO: 25, 26 and 27, respectively, a monoclonal antibody containing a heavy chain containing at least m here one, preferably at least two, preferably at least three of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 28, 29 and 30, respectively, or sequences with at least 80% , preferably 85, 90, 95 and 98% identity after optimal alignment with the sequences of SEQ ID NO: 28, 29 and 30, respectively, and a light chain containing at least one, preferably at least two, preferably three of the CDRs -L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 31, 32 and 33, respectively, or sequences with at least 80%, preferably 85, 90, 95 and 98% identity after optimal alignment with the sequences of SEQ ID NO: 31, 32 and 33, respectively, and a monoclonal antibody containing a heavy chain containing at least one, preferably at least two, preferably three of CDR-H1, CDR-H2 and CDR-H3 amino acid sequences SEQID NO: 34, 35 and 36, respectively; or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 34, 35 and 36, respectively, and a light chain containing at least one, preferably at least two, preferably three of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 37, 38 and 39, respectively, or sequences with at least 80%, preferably 85, 90, 95 and 98% identity after optimal alignment with the sequences of SEQ ID NO: 37, 38 and 39, respectively. In the meaning of the present invention, percentage identity or % identity between two nucleic acid or amino acid sequences means the percentage of identical nucleotides or amino acid residues between two sequences to be compared, obtained after optimal alignment, this percentage being purely statistical and the difference between the two data sequences are randomly distributed along their length. Comparison of two nucleic acid or amino acid sequences is traditionally carried out by comparing the sequences after they have been optimally aligned, which comparison can be done by segments or by using an alignment window. Optimal alignment of sequences for comparison can be carried out, in addition to manual comparison, by methods known to the person skilled in the art.

For an amino acid sequence showing at least 80%, preferably 85%, 90%, 95% and 98% identity with the control amino acid sequence, preferred examples include sequences containing a reference sequence, certain modifications, namely deletion, addition or substitution at least one amino acid, truncation or extension. In the case of substitution of one or more consecutive or non-sequential amino acids, substitutions are preferred in which the replaced amino acids are replaced with equivalent amino acids. It is understood here that the expression equivalent amino acids indicates any amino acids that are likely to be replaced by one of the structural amino acids, however, without modifying the biological activities of the respective antibodies, and these specific examples are defined below.

Equivalent amino acids can be determined either by their structural homology with the amino acids they replace, or by comparisons of biological activity between different antibodies that are likely to be generated.

In a more specific embodiment, said antibody is a monoclonal antibody selected from the group consisting of the following: a monoclonal antibody comprising the heavy chain of the amino acid sequence of SEQ ID NO: 41 and the light chain of the amino acid sequence of SEQ ID NO: 42; a monoclonal antibody containing the heavy chain of the amino acid sequence of SEQ ID NO: 43 and the light chain of the amino acid sequence of SEQ ID NO: 44; a monoclonal antibody containing the heavy chain of the amino acid sequence of SEQ ID NO: 45 and the light chain of the amino acid sequence of SEQ ID NO: 46; a monoclonal antibody containing the heavy chain of the amino acid sequence of SEQ ID NO: 47 and the light chain of the amino acid sequence of SEQ ID NO: 48; a monoclonal antibody containing the heavy chain of the amino acid sequence of SEQ ID NO: 49 and the light chain of the amino acid sequence of SEQ ID NO: 50; and a monoclonal antibody comprising the heavy chain of the amino acid sequence of SEQ ID NO: 51 and the light chain of the amino acid sequence of SEQ ID NO: 52.

In another specific embodiment, the antibody used in the method of the invention is a humanized antibody.

The expression humanized antibody, as used herein, means an antibody that contains CDR regions derived from a non-human antibody, with other portions of that antibody molecule derived from one or more human antibodies. In addition, some of the residues of the skeletal segments (referred to as FR, which stands for frame) can be modified to maintain binding affinity according to techniques known to the person skilled in the art (Jones et al., Nature, 321:522-525, 1986). The goal of humanization is to reduce the immunogenicity of a xenogeneic antibody, such as a mouse antibody, for administration to a human while maintaining the full antigen binding affinity and specificity of the antibody.

Humanized antibodies of the invention, or fragments thereof, can be prepared by techniques known to those skilled in the art (such as, for example, those described in Singer et al., J. Immun., 150:2844-2857, 1992). Such humanized antibodies are preferred for use in methods involving in vitro diagnostics or prophylactic and/or therapeutic treatment in vivo. Other humanization techniques are also known to the person skilled in the art. Indeed, antibodies can be humanized using a variety of techniques, including CDR grafting (EP 0451261; EP 0682040; EP 0939127; EP 0566647; US 5530101; US 6180370; US 5585089; US 5693761; US 5639641; US 6054297; and US 5877293), masking of surface residues (venation) or surface modification (EP 0592106; EP 0519596; Padlan E. A., 1991, Molecular Immunology 28(4/5): 489-498; Studnicka G. M. et al., 1994, Protein Engineering 7(6): 805814; Roguska M.A. et al., 1994, Proc. Natl. Acad. ScL U.S.A., 91:969-973) and strand shuffling (US Pat. No. 5,565,332). Human antibodies can be generated by a variety of methods known in the art, including phage display methods. See also US patent No. 4444887, 4716111, 5545806 and 5814318; and international patent applications with publication numbers WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735 and WO 91/10741.

In a more specific embodiment, said antibody is a humanized antibody selected from the group consisting of the following:

a humanized antibody comprising a heavy chain containing at least one, preferably at least two, preferably three of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NOs: 4, 5 and 6, respectively, and a light chain containing at least one,

- 17 042429 preferably at least two, preferably three of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 7, 8 and 9, respectively, or sequences with at least 80%, preferably 85 , 90, 95 and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 7, 8 and 9, respectively, a humanized antibody containing a heavy chain containing at least one, preferably at least two, preferably three of the CDRs -H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 10, 11 and 12, respectively, or sequences with at least 80%, preferably 85, 90, 95 and 98% identity after optimal alignment with the sequences of SEQ ID NO: 10, 11 and 12, respectively, and a light chain containing at least one, preferably at least two, preferably three of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NO: 13, 14 and 15, respectively directly, or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NO: 13, 14 and 15, respectively, a humanized antibody containing a heavy chain containing at least one, preferably at least two, preferably three of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 16, 17 and 18, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NOs: 16, 17 and 18, respectively, and a light chain containing at least one, preferably at least two, preferably three of the CDR- L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 19, 20 and 21, respectively, or sequences with at least 80%, preferably 85, 90, 95 and 98% identity after optimal alignment with follow SEQ ID NOs: 19, 20 and 21, respectively, a humanized heavy chain antibody comprising at least one, preferably at least two, preferably three of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NO: 22, 23 and 24, respectively, or sequences with at least 80%, preferably 85, 90, 95 and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 22, 23 and 24, respectively, and a light chain comprising at least one, preferably at least two, preferably three of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 25, 26 and 27, respectively, or sequences with at least 80%, preferably 85, 90, 95 and 98% identity after optimal alignment with the sequences of SEQ ID NO: 25, 26 and 27, respectively, a humanized antibody containing a heavy chain containing at least one, preferably at least two, preferably three of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 28, 29 and 30, respectively, or sequences with at least 80%, preferably 85, 90, 95 and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 28, 29 and 30, respectively, and a light chain containing at least one, preferably at least two, preferably three of CDR-L1, CDR-L2 and CDR -L3 amino acid sequences of SEQ ID NO: 31, 32 and 33, respectively, or sequences with at least 80%, preferably 85, 90, 95 and 98% identity after optimal alignment with the sequences of SEQ ID NO: 31, 32 and 33, respectively, and a humanized heavy chain antibody comprising at least one, preferably at least two, preferably three of the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 34, 35 and 36, respectively, or afterbirth of at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with SEQ ID NOs: 34, 35 and 36, respectively, and a light chain containing at least one, preferably at least two, preferably three of the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 37, 38 and 39, respectively, or sequences with at least 80%, preferably 85, 90, 95 and 98% identity after optimal alignment with the sequences of SEQ ID NOS: 37, 38 and 39, respectively, where the specified antibody also contains constant regions of the light chain and heavy chain derived from a human antibody.

In another more specific embodiment, said antibody is a humanized antibody selected from the group consisting of the following: a humanized antibody comprising the heavy chain variable region of the amino acid sequence of SEQ ID NO: 53 and the light chain variable region of the amino acid sequence of SEQ ID NO: 54;

a humanized antibody comprising the heavy chain variable region of the amino acid sequence of SEQ ID NO: 55 and the light chain variable region of the amino acid sequence of SEQ ID NO: 56;

a humanized antibody comprising a heavy chain variable region of an amino acid sequence selected between SEQ ID NOs: 57, 58 and 59 and a light chain variable region of an amino acid sequence selected between SEQ ID NOs: 60, 61 and 62;

a humanized antibody comprising a heavy chain variable region of an amino acid sequence selected between SEQ ID NOs: 63, 64, and 65 and a light chain variable region of an amino acid sequence selected between SEQ ID NOs: 66, 67, and 68;

a humanized antibody comprising a heavy chain variable region of an amino acid sequence selected between SEQ ID NOs: 69 and 71 and a light chain variable region of an amino acid sequence selected between SEQ ID NOs: 70 and 72; and a humanized antibody comprising a heavy chain variable region of an amino acid sequence selected between SEQ ID NOs: 75 and 76, and a light chain variable region of an amino acid sequence selected between SEQ ID NOs: 77 and 78;

wherein said antibody also contains light chain and heavy chain constant regions derived from a human antibody.

More preferably, said antibody comprises the heavy chain variable region of the amino acid sequence of SEQ ID NO: 71 and the light chain variable region of the amino acid sequence of SEQ ID NO: 72, said antibody also comprising light chain and heavy chain constant regions derived from a human antibody.

Even more preferably, said antibody comprises the heavy chain of the amino acid sequence of SEQ ID NO: 73 and the light chain of the amino acid sequence of SEQ ID NO: 74.

In another aspect, the invention also provides immunoconjugates (interchangeably referred to as antibody-drug conjugates or ADCs) comprising an antibody that binds to progastrin as described herein, said antibody being conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent. an agent, a drug, a growth inhibitory agent, a toxin (eg, a protein toxin, an enzymatically active bacterial, fungal, plant or animal toxin, or fragments thereof), or a radioactive isotope (ie, a radioconjugate).

Immunoconjugates have been used for local delivery of cytotoxic agents, ie. drugs that kill or inhibit cell growth or proliferation in the treatment of cancer (Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549; Wu et al. (2005) Nature Biotechnology 23(9): 1137- 1146; Payne, G. (2003) i 3:207-212; Syrigos and Epenetos (1999) Anticancer Research 19:605-614; Niculescu-Duvaz and Springer (1997) Adv. Drug Deliv. Rev. 26:151-172 ; US patent No. 4975278). Immunoconjugates provide targeted delivery of a drug moiety to and intracellular accumulation within a tumor, where systemic administration of unconjugated drugs can result in unacceptable levels of toxicity to normal cells as well as tumor cells that are being eliminated (Baldwin et al., Lancet (Mar. 15 , 1986) pp. 603-05 Thorpe (1985) Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review, in Monoclonal Antibodies '84: Biological And Clinical Applications (A. Pinchera et al., eds) pp. 475-506 Both polyclonal antibodies and monoclonal antibodies have been described as useful in these strategies (Rowland et al. (1986) Cancer Immunol. Immunother. 21:183-87) Drugs used in these methods include daunomycin, doxorubicin, methotrexate and vindesine (Rowland et al. (1986) supra) Toxins used in antibody-toxin conjugates include bacterial toxins such as diphtheria toxin, plant toxins, such as ricin, small molecule toxins such as geldanamycin (Mandler et al. (2000) J. Nat. Cancer Inst. 92(19):1573-1581; Mandler et al. (2000) Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al. (2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al. (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623) and calicheamicin (Lode et al. (1998) Cancer Res. 58:2928 Hinman et al (1993) Cancer Res 53:3336-3342). These toxins may exert their cytotoxic effects through mechanisms including tubulin binding, DNA binding, or topoisomerase inhibition. Some cytotoxic drugs tend to be inactive or less active when conjugated to large antibodies or protein receptor ligands.

In some embodiments, the immunoconjugate contains an antibody and a chemotherapeutic agent or other toxin. Useful chemotherapeutic agents in the preparation of immunoconjugates are described herein (eg, above). Enzymatically active toxins and their fragments that can be used include diphtheria toxin A chain, non-binding diphtheria toxin active fragments, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites proteins fordii, dianthine proteins, Phytolaca americana proteins (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, fenomycin, enomycin and tricothecenes. See, for example, WO 93/21232, published October 28, 1993. A variety of radioisotopes are available for the production of radioconjugated antibodies. Examples include ²¹² Bi, ¹³¹ I, ¹³¹ In, ⁹⁰ Y and ¹⁸⁶ Re. Antibody-cytotoxic agent conjugates are prepared using a variety of bifunctional protein-binding agents such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyladipimidate HCl) , active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazonium benzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate); and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, the immunotoxin ricin can be

- 19 042429 receive as described in Vitetta et al., Science, 238: 1098 (1987). Labeled with carbon 14

1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is a typical chelator for conjugating a radioisotope to an antibody. See WO94/11026.

This document also discusses conjugates of an antibody and one or more small molecular weight toxins such as calicheamicin, maytansinoids, dolastatins, aurostatins, trichothecene and CC 1065, and derivatives of these toxins that have toxin activity.

The immunoconjugate of the invention may further comprise a linker.

The terms linker, linker unit or ligament means a chemical moiety containing a covalent bond or chain of atoms that covalently attaches a binding protein to at least one cytotoxic agent.

Linkers can be prepared using a variety of bifunctional protein-binding agents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(Mmaleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane ( IT), bifunctional imidoester derivatives (such as dimethyladipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis-(pasidobenzoyl)hexanediamine), bis-diazonium derivatives (such as as bis-(p-diazonium benzoyl)ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate) and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). Carbon-14 labeled 1-isothiocyanatobenzyl-3methyldiethylenetriaminepentaacetic acid (MX-DTPA) is a typical chelator for conjugating cytotoxic agents with a targeting system. Other crosslinkers can be BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo -SIAB, sulfo-SMCC, sulfo-SMPB and SVSB (succinimidyl-(4-vinylsulfone)benzoate), which are commercially available (for example, Pierce Biotechnology, Inc., Rockford, III., USA).

The linker may be non-cleavable or cleavable.

In another aspect, the invention provides a composition for use in the prevention or treatment of prostate cancer, said composition comprising an antibody recognizing an epitope comprising an amino acid sequence corresponding to the amino acid sequence of progastrin.

In a more specific embodiment, said composition for use in the prevention or treatment of prostate cancer comprises an antibody recognizing an epitope of progastrin, wherein said epitope comprises an amino acid sequence corresponding to the amino acid sequence of the N-terminal portion of progastrin, wherein said amino acid sequence may include residues 10-14 hPG, hPG residues 9-14, hPG residues 4-10, hPG residues 2-10, or hPG residues 2-14, wherein the hPG amino acid sequence is SEQ ID NO: 1.

In a more specific embodiment, a composition for use in the prevention or treatment of prostate cancer contains an antibody that recognizes a progastrin epitope, where said epitope includes an amino acid sequence corresponding to the amino acid sequence of the C-terminal portion of progastrin, where said amino acid sequence may include hPG residues 71-74, residues hPG residues 69-73, hPG residues 71-80 (SEQ ID NO: 40), hPG residues 76-80, or hPG residues 67-74, wherein the hPG amino acid sequence is SEQ ID NO: 1.

In a more specific embodiment, a composition for use in the prevention or treatment of prostate cancer comprises a progastrin-binding antibody, or antigen-binding fragment thereof, that has an affinity for progastrin of at least 5000 nM, at least 500, 100, 80, 60, 50 , 4o, 30, 20, 10, 7, 5, 4, 3, 2, 1, 0.5, 0.1 nM, 50, 10, 5, 1 pM, or at least 0.1 pM when determined in this way as described above.

In an even more specific embodiment, a composition for use in the prevention or treatment of prostate cancer comprises a progastrin-binding antibody, wherein said progastrin-binding molecule or antigen-binding fragment thereof is a neutralizing antibody.

In another specific embodiment, a composition for use in the prevention or treatment of prostate cancer comprises a progastrin-binding antibody, wherein said progastrin-binding molecule or antigen-binding fragment thereof is a humanized antibody.

In a particular embodiment, a composition for use in the prevention or treatment of prostate cancer comprises a progastrin-binding antibody, wherein said progastrin-binding molecule or antigen-binding fragment thereof is conjugated to one or more cytotoxic agents, such as a chemotherapeutic agent, a drug, a growth inhibitory agent, a toxin (eg, a protein toxin, an enzymatically active toxin of bacterial, fungal, plant or animal origin, or fragments thereof), or a radioactive isotope (ie, a radioconjugate), as described above.

In another specific embodiment, a composition for use in the prevention or treatment of prostate cancer for a patient comprises an antibody that binds to progastrin, where said patient has been diagnosed with prostate cancer, where the concentration of progastrin is higher in a biological sample from said patient than in control sample. Preferably, said patient has been diagnosed with prostate cancer by contacting an anti-PG antibody with a biological sample of said patient.

A biological sample, as used herein, is a biological tissue or fluid sample that contains nucleic acids or polypeptides, such as a protein, polynucleotide, or prostate cancer transcript. Such a sample should provide for the determination of progastrin expression levels. It is known that progastrin is a secreted protein. Preferred biological samples for determining progastrin protein levels thus include body fluids. The term biological fluid as used herein means any fluid that includes a substance of biological origin. Preferred biological fluids for use in the present invention include body fluids of an animal, eg a mammal, preferably a human subject. The body fluid can be any body fluid, including but not limited to blood, plasma, serum, lymph, cerebrospinal fluid (CSF), saliva, sweat, and urine. Preferably, said preferred liquid biological samples include samples such as a blood sample, a plasma sample, or a serum sample. More preferably, the biological sample is a blood sample. Indeed, such a blood sample can be obtained by completely harmless blood sampling from a patient and thus provides a non-invasive assessment of the risks that the subject will develop a tumor.

The term biological sample, as used herein, also includes the patient's solid cancer sample to be analyzed when the cancer is a solid cancer. Such a sample of solid cancer allows a specialist to perform any type of measurement of the level of the biomarker according to the invention. In some instances, the methods of the invention may further include the preliminary step of collecting a solid cancer sample from the patient. A sample of solid cancer is a sample of tumor tissue. Even in a cancer patient, the tissue that is the site of the tumor still contains non-tumor healthy tissue. The cancer sample should therefore be limited to tumor tissue taken from the patient. Said cancer sample may be a biopsy sample or a sample taken as a result of surgical resection therapy.

The biological sample is typically obtained from a eukaryotic organism, most preferably a mammal or bird, reptile or fish. Indeed, the subject that can be subjected to the method described herein can be any mammalian animal, including human, dog, cat, cattle, goat, pig, boar, sheep and monkey; or a bird; reptile or fish. Preferably the subject is a human; the human subject may be known as a patient.

By obtaining a biological sample in this document means obtaining a biological sample for use in the methods described in this invention. This will most often be done by removing a sample of cells from the animal, but can also be done by using previously isolated cells (eg, isolated by another person, at a different time and/or for a different purpose) or by performing the methods of the invention in vivo. Archival tissues with a history of treatment or outcome will be especially useful.

This sample can be obtained and, if necessary, prepared according to methods known to the person skilled in the art. In particular, it is well known in the art that the sample should be taken from the subject on an empty stomach.

In a more specific aspect, the present invention relates to a composition for use in the prevention or treatment of prostate cancer according to the invention, wherein said progastrin-binding antibody or antigen-binding fragment thereof is selected from antibodies against the N-terminus of progastrin and antibodies against the C-terminus of progastrin.

Antibody compositions for use in the methods of the invention may be formulated in a variety of formulations, including, but not limited to, an aqueous suspension, for administration by a variety of routes including, but not limited to, parenteral, intrathecal, subcutaneous, intravenous, intramuscular, intraperitoneal, intraperitoneal, infusion, or bolus administration. limited by them. In some embodiments, this composition is prepared for parenteral administration and in some specific embodiments, intravenous injection by infusion.

In a specific embodiment, a composition for use in the prevention or treatment of prostate cancer according to this invention contains an effective dose of antibodies against progastrin according to the invention in the range from 0.001 mg/kg to about 250 mg/kg, which can be given in a single administration or in many, separate administrations .

In a specific embodiment, the composition for use in the prevention or treatment of prostate cancer according to this invention contains an antibody that binds to progastrin,

- 21 042429 or antigen-binding fragment selected from among polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single-chain antibodies, camelized antibodies, IgA1 antibodies, antibodies

IgA2, IgD antibodies, IgE antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies and IgM antibodies.

Preferably, said antibodies are those described above. More preferably, said antibodies are humanized antibodies.

Preferably the present invention relates to a pharmaceutical composition comprising a composition for use in the prevention or treatment of prostate cancer according to the invention and a pharmaceutically acceptable carrier. More specifically, a pharmaceutical composition for use in the prevention or treatment of prostate cancer according to the invention comprises an antibody as described above and a pharmaceutically acceptable carrier.

In a more specific aspect, the present invention relates to a pharmaceutical composition comprising a composition for use in the prevention or treatment of prostate cancer according to the invention and a pharmaceutically acceptable carrier, wherein said anti-progastrin antibody is administered at a dose of 0.001 mg/kg to 250 mg/kg and preferably in dose of at least 0.005 mg/kg, at least 0.01 mg/kg, at least 0.05 mg/kg, at least 0.1 mg/kg, at least 0.5 mg/kg, at least 1 mg/kg, at least 5 mg/kg, at least 10 mg/kg, at least 50 mg/kg, or at least 100 mg/kg. In another aspect, the present invention relates to a kit of parts containing a composition for use in the prevention or treatment of prostate cancer according to the invention and an anti-cancer therapeutic molecule.

Indeed, anti-PG monoclinal antibody treatment as described herein can be combined with or used as an adjunct to other therapy. Non-limiting examples of other therapies include chemotherapy, radiation therapy, surgical resection, and antibody therapy.

In another aspect, the present invention relates to a set of parts containing a composition for use in the prevention or treatment of prostate cancer according to the invention and an anti-cancer therapeutic molecule selected from the following: a chemotherapeutic molecule, a targeted therapy molecule.

In a specific embodiment, the present invention relates to kits of parts containing a composition for the treatment of prostate cancer according to the invention and a chemotherapeutic molecule for simultaneous, sequential or separate administration. Useful chemotherapeutic molecules for this purpose include folate antagonists, purine antagonists, pyrimidine antagonists, DNA alkylating molecules, DNA crosslinking drugs, antibiotics, platinum complexes, proteasome inhibitors, mitotic spindle poisons, topoisomerase inhibitors, tyrosine kinse inhibitors, and others, but not limited to them.

In another specific embodiment, the present invention relates to kits of parts containing a composition according to this invention and a composition containing another targeted therapy molecule for simultaneous, sequential or separate administration. Such a targeted therapy molecule includes antibodies that target EGFR (epidermal growth factor receptor) such as cetuximab or panitumumab, antibodies that target VEGF (vascular endothelial growth factor) such as bevacizumab, antibodies that target HER2 (human EGF). -like receptor 2), such as trastuzumab or pertuzumab, antibodies that target PD-1 and PDL-1, such as pembrolizumab, antibodies that target CTLA-4 (cytotoxic T-lymphocyte antigen-4), such as ipilimumab , small molecule drugs that target EGFR such as erlotinib, small molecule drugs that target BRAF such as vemurafenib or dabrafenib, a recombinant fusion protein that targets VEGF such as but not limited to aflibercept.

In another specific aspect, the present invention relates to the use of a progastrin-binding antibody, or antigen-binding fragment thereof, for the diagnosis of prostate cancer.

In another specific aspect, the present invention relates to the use of a progastrin-binding antibody, or antigen-binding fragment thereof, for the prevention or treatment of prostate cancer.

In a more specific aspect, the present invention relates to the use of a progastrin-binding antibody, or antigen-binding fragment thereof, for the prevention or treatment of prostate cancer in a patient, where the concentration of progastrin in a biological sample of said patient has been determined and is higher than the concentration of progastrin in a control biological sample. .

In another specific aspect, this invention relates to the use of a pharmaceutical composition according to the invention for the prevention of recurrence of prostate cancer. Accordingly, the present disclosure provides useful methods and compositions for treating prostate cancer and preventing recurrence of prostate cancer in animals, including humans. These methods of treatment include administering to a subject diagnosed with prostate cancer an effective amount of an antibody that specifically binds to progastrin to provide a therapeutic benefit. The anti-PG antibody may be administered alone, as monotherapy, or in combination with or in addition to other treatments such as tumor resection, radiation therapy, chemotherapy, another antibody therapy, etc.

Solid tumors are not necessarily homogeneous tissues. Rather, some tumors contain a range of damaged cell types that have distinct phenotypic and functional properties. In this respect, such tumors are analogous to abnormal organs. The cells that make up solid tumors differ in the extent to which they are able to initiate new tumor formation when transplanted to a new site in the same host or a new host of the same or different species. Cells having this property are known as tumor or cancer initiating cells or, alternatively, tumor or cancer stem cells. See, for example, Hardavella et al., 2016, Prostate cancer stem cells-characteristics, phenotype, Transl Prostate Cancer Res. 2016, 5(3):272-279. These cells are highly oncogenic.

In general, cancer stem cells are defined by two properties: the ability to self-renew and the ability to produce daughter cells that differentiate into non-stem cells. Self-renewal is the ability to undergo cell division, while one or both daughter cells remain undifferentiated, retaining the ability to give rise to another cancer stem cell with a similar ability to proliferate as the parent cell. This property allows cancer stem cells to eventually produce a large number of cells that make up a growing tumor. Cancer stem cells also have the ability to produce daughter cells that differentiate to give rise to a spectrum of more differentiated non-stem or bulk tumor cells found in many solid tumors. Thus, when transplanted, cancer stem cells can restore the type of tumor from which they originated, even after multiple serial transplants. In addition, it is believed that cancer stem cells have genetic mutations and/or epigenetic changes that lead to altered proliferation patterns and/or low rates of apoptosis.

Cancer stem cells can be identified according to a range of phenotypic characteristics that distinguish them from the bulk of tumor cells. Useful methods for assessing whether a tumor or cell line contains cancer stem cells are familiar to those skilled in the art. Such methods are described, for example, in Hardavella et al., 2016, Prostate cancer stem cells-characteristics, phenotype, Transl Prostate Cancer Res. 2016, 5(3):272-279 and also in WO 2012/013609. Specific examples of these methods are also described in the examples of this application.

In a more specific aspect, the present invention relates to the use of a progastrin-binding antibody or antigen-binding fragment thereof for the prevention or treatment of prostate cancer in a patient where said patient has a metastasis.

In an even more specific aspect, the present invention relates to the use of a progastrin-binding antibody, or antigen-binding fragment thereof, for the prevention or treatment of prostate cancer in a patient, where said patient has metastasis, and where the concentration of progastrin in a biological sample of said patient has been determined, and it is higher than the concentration of progastrin in the control biological sample.

The components that make up the combination can be administered simultaneously, separately or sequentially, so as to obtain the maximum effectiveness of this combination; for each injection, its duration may vary from rapid administration to continuous perfusion.

The term simultaneous administration, as used herein, refers to the administration of two compounds of a composition of the invention in a single and unique pharmaceutical form. The term separate administration, as used herein, refers to the administration, at the same time, of two compounds of the composition of the invention in different pharmaceutical forms. The term sequential administration, as used herein, refers to the sequential administration of two compounds of the composition of the invention, each in a different pharmaceutical form.

The term therapeutically effective amount, as used herein, refers to the minimum concentration or amount of a compound (or compounds) that is effective in preventing, alleviating, ameliorating, or lessening the symptoms of a disease, or in prolonging the life of the patient being treated. .

In another aspect, the present disclosure provides a method for preventing recurrence of prostate cancer, comprising administering an effective amount of an anti-PG antibody to a subject in need of prevention. The methods for preventing liver cancer recurrence according to the present disclosure are carried out by administering one or more anti-PG antibodies capable of neutralizing PG described above to individuals at risk for prostate cancer recurrence.

Subjects in need of prevention of recurrence of prostate cancer are

- 23 042429 are individuals previously treated for prostate cancer who are at risk but have not been newly diagnosed with prostate cancer. Suitable subjects include individuals who have previously been treated for prostate cancer by any means, including surgical resection, chemotherapy, or any other therapy.

Effective prevention of recurrence of prostate cancer includes, but is not limited to, complete and ongoing absence of recurrence of prostate cancer. In some embodiments, effective prevention is measured by the absence of prostate cancer tumors or prostate cancer stem cells obtained from a subject at risk for prostate cancer recurrence. In some embodiments, effective prevention is determined by the absence of an increase in the blood concentration of PG in a subject at risk for recurrence of prostate cancer.

The anti-PG treatment may be administered alone as monotherapy or in combination or in addition to one or more other treatments. Other treatments include, without limitation, surgical resection and treatment with a second therapeutic agent, such as a chemotherapeutic agent or an antibody, as described above. Combination treatment as provided herein comprises administering to a patient at least two treatments, one of which is an anti-PG treatment using at least one anti-PG antibody, and the other of which is a treatment with a therapeutic agent. or procedure.

The anti-PG antibody and the second agent may be administered simultaneously, sequentially, or separately. It is stated that the anti-PG antibody and the second agent, as used herein, are administered sequentially if they are administered to the patient on the same day, for example, during the same patient visit. Sequential administration can occur at intervals of 1, 2, 3, 4, 5, 6, 7, or 8 hours. In contrast, it is indicated that the anti-PG antibody and the second agent are administered separately if they are administered to the patient on different days, for example , the anti-PG antibody, and the second therapeutic agent may be administered at intervals of 1 day, 2 days, or 3 days, one week, 2 weeks, or one month. In the methods of the present disclosure, administration of the anti-PG antibody of the disclosure may precede or follow administration of the second agent. As a non-limiting example, the anti-PG antibody and the second agent may be administered concomitantly for a period of time, followed by a second period of time in which administration of the anti-PG antibody and the second agent alternate.

The characteristics of the embodiments of the present invention will become even clearer from the following detailed description of the examples below.

Graphics Legends

Fig. 1 - DU145 cell proliferation assay: Cells were treated with either control antibody or anti-hPG 8CV2 Hz (PG Hz), a humanized anti-hPG C-terminal antibody.

Fig. 2 - effect of treatment with Hz against hPG 8CV2 (PG Hz) - a humanized anti-hPG C-terminal antibody or a humanized anti-hPG N-terminal antibody - on sphere formation by LNCaP cells.

Examples

Example 1 Neutralizing activity of anti-hPG antibodies against cancer cell lines.

1.1. Neutralizing activity of monoclonal antibodies against hPG.

Monoclonal antibodies to PG are being tested for their ability to inhibit the proliferation of several cell lines commonly used in prostate cancer research that produce and secrete progastrin. Cell survival from each of these cell lines is analyzed using different anti-hPG monoclonal antibodies.

For each experiment, 50,000 cells are seeded in 6-well plates in medium containing fetal calf serum and incubated for ₈ h. quadruple every 12 hours for 48 hours, in the absence of fetal bovine serum, 1-20 μg/ml monoclonal control antibodies (anti-puromycin monoclonal antibody) (CT mAb) or 1-20 μg/ml anti-hPG mAb (monoclonal antibody) where said mAb is an anti-hPG C-terminal monoclonal antibody or an anti-hPG N-terminal monoclonal antibody.

Said mAb is an anti-hPG C-terminal antibody selected from the following:

an antibody containing a heavy chain containing CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequences of SEQ ID NOs: 28, 29 and 30, and a light chain containing CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequences of SEQ ID NO: 31, 32 and 33;

an antibody containing a heavy chain containing CDR-H1, CDR-H2 and CDR-H3 of the amino acid sequences of SEQ ID NOs: 34, 35 and 36, and a light chain containing CDR-L1, CDR-L2 and CDR-L3 of the amino acid sequences of SEQ ID NO: 37, 38 and 39;

or an anti-hPG N-terminal antibody selected from the following:

monoclonal antibody containing a heavy chain containing CDR-H1, CDR-H2 and CDR-H3

- 24 042429 amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively, and a light chain containing

CDR-L1, CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 7, 8 and 9;

an antibody containing a heavy chain containing CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NO: 10, 11 and 12, respectively, and a light chain containing CDR-L1,

CDR-L2 and CDR-L3 amino acid sequences of SEQ ID NOs: 13, 14 and 15, respectively;

an antibody containing a heavy chain containing the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 16, 17 and 18, respectively, and a light chain containing the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences SEQ ID NOs: 19, 20 and 21, respectively;

an antibody containing a heavy chain containing the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 22, 23 and 24, respectively, and a light chain containing the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences SEQ ID NO: 25, 26 and 27, respectively.

The number of cells in T ₀ is counted in the control well for each experiment.

Specifically, the number of live cells in both control and mAb-treated anti-hPG wells is counted at 48 hours, then the difference between each cell number and the number of cells determined in T ₀ is calculated. The resulting number of cells treated with the anti-hPG mAb is then expressed as a percentage of the number of cells treated with the control mAb.

Treatment with monoclinal antibodies against hPG reduces the number of cells compared to the control antibody. Statistical significance is determined using one-way ANOVA with Tukey's post hoc test: * - p less than 0.05; ** - p less than 0.01 and *** p less than 0.001. In every cell line, anti-hPG antibodies decrease cell survival.

1.2. Neutralizing activity of humanized anti-hPG antibodies on cell survival.

Humanized PG antibodies are being tested for their ability to inhibit the proliferation of several cell lines commonly used in prostate cancer research that produce and secrete progastrin. Cell survival from each of these cell lines is analyzed using different anti-hPG monoclonal antibodies.

For each experiment, 50,000 cells are seeded in 6-well plates in medium containing fetal calf serum and incubated for ₈ h. quadruple every 12 h for 48 h, in the absence of fetal calf serum, 1-20 μg/mL humanized control antibodies (anti-human FcG1 antibody, from BioXCell) (CT Hz) or 1-20 μg/mL Hz (humanized antibody ) against hPG, wherein said Hz is a humanized anti-hPG C-terminal antibody or a humanized anti-hPG N-terminal antibody. The number of cells in T ₀ is counted in the control well for each experiment.

In particular, the number of live cells in both control and Hz anti-hPG treated wells is counted at 48 hours, then the difference between each cell number and the number of cells defined in T ₀ is calculated. The resulting number of cells treated with Hz against hPG is then expressed as a percentage of the number of cells treated with control mAb.

Treatment of Hz with anti-hPG antibodies reduced the number of cells compared to treatment with control antibody. Statistical significance is determined using one-way ANOVA with Tukey's post hoc test: * - p less than 0.05; ** - p less than 0.01 and *** - p less than 0.001. In every cell line, anti-hPG antibodies decrease cell survival.

1.3. Neutralizing activity of monoclonal antibodies against hPG in relation to the frequency of cancer stem cells.

Monoclonal antibodies to PG are tested for their ability to reduce the frequency of cancer stem cells (CSC) using limiting dilution assay (ELDA) in several cell lines commonly used for prostate cancer research that produce and secrete progastrin. The frequency of CSCs from each of these cell lines is analyzed using different anti-hPG monoclonal antibodies.

For each experiment, cells were seeded in P96 ultra-low attachment (ULA) (96-well plates) at fixed cell concentrations per well using a FACS Aria flow cytometer, and a concentration range of one to 500 cells per well was used. Cells are cultured for up to 11 days in ULA plates with M11 media (Macari et al., Oncogene, 2015) and treated every 3 or 4 days with 1-20 μg/ml of a monoclonal control antibody (anti-puromycin monoclonal antibody) (CT mAb) or 1-20 μg/ml of an anti-hPG mAb, wherein said mAb is an anti-hPG C-terminal monoclonal antibody or an anti-hPG N-terminal monoclonal antibody.

In particular, at the end of the incubation phase, the plates are observed using a phase contrast microscope and the number of positive wells per cell concentration is estimated. Finally, the web-based ELDA software tool (http://www.bioinf.wehi.edu.au/software/elda/) is used to calculate the CSC frequencies of each treatment group and analyze any statistical difference between

- 25 042429 groups (modified chi-square test).

Treatment with anti-hPG monoclinal antibodies reduced the incidence of CSCs compared with control antibody treatment.

1.4. Neutralizing activity of humanized anti-hPG antibodies in relation to the frequency of cancer stem cells.

Analysis of the formation of spheres.

Humanized anti-PG antibodies are being tested for their ability to reduce the incidence of cancer stem cells (CSC) using a sphere formation assay in several cell lines commonly used for prostate cancer research that produce and secrete progastrin.

For each experiment, 700 cells are seeded in 24-well ultra-low-attachment (ULA) plates. Cells are cultured for up to 7 days in ULA plates with M11 media (Macari et al., Oncogene, 2015) and treated every 3 or 4 days with 20 μg/ml humanized control antibodies (anti-human FcG1, from BioXCell) (CT Hz) or 20 μg/ml Hz against hPG (PG Hz), where said Hz is a humanized anti-hPG C-terminal antibody or a humanized anti-hPG N-terminal antibody.

In particular, at the end of the incubation phase, the wells are photographed by bright field microscopy, the images are analyzed, and spheres with an average diameter greater than 25 μm are counted.

Treatment with humanized anti-hPG antibodies reduced the incidence of CSC compared to treatment with control antibody. Limit dilution analysis

Humanized anti-PG antibodies are being tested for their ability to reduce the incidence of cancer stem cells (CSC) using limiting dilution assay (ELDA) in several cell lines commonly used for prostate cancer research that produce and secrete progastrin. The frequency of CSCs from each of these cell lines is analyzed using different humanized anti-hPG antibodies.

For each experiment, cells were seeded in P96 ultra-low attachment (ULA) (96-well plates) at fixed cell concentrations per well using a FACS Aria flow cytometer, and a concentration range of one to 500 cells per well was used. Cells are cultured for up to 11 days in ULA plates with M11 medium (Macari et al., Oncogene, 2015) and treated every 3 or 4 days with 1-20 μg/ml humanized control antibodies (anti-human FcG1 antibody, from BioXCell) ( CT Hz) or 1-20 μg/ml Hz against hPG, wherein said Hz is a humanized anti-hPG C-terminal antibody or a humanized anti-hPG N-terminal antibody.

In particular, at the end of the incubation phase, the plates are observed using a phase contrast microscope and the number of positive wells per cell concentration is estimated. Finally, the web-based ELDA software tool (http://www.bioinf.wehi.edu.au/software/elda/) is used to calculate the CSC frequencies of each treatment group and analyze any statistical difference between groups (modified chi-square test). ).

Treatment with humanized anti-hPG antibodies reduced the incidence of CSC compared to treatment with control antibody.

1.5. Neutralizing activity of anti-hPG monoclonal antibodies against the WNT/βcatenin pathway.

Anti-PG monoclonal antibodies are being tested for their ability to inhibit the WNT/β-catenin pathway in several cell lines commonly used in prostate cancer research that produce and secrete progastrin using the expression of the protein survivin, a well-known target gene for the WNT/β-catenin pathway. catenin - as an indicator. Survivin expression from each of these cell lines is analyzed using different anti-hPG monoclonal antibodies.

For each experiment, 50,000 cells are plated in 6-well plates in medium containing fetal calf serum and incubated for 8 h. h for 72 h, in the absence of fetal calf serum, 1-20 μg/ml monoclonal control antibodies (monoclonal antibody against puromycin) (CT mAb) or 1-20 μg/ml mAb against hPG, where the specified mAb is a monoclonal antibody against hPG C-terminus or anti-hPG N-terminal monoclonal antibody.

Specifically, after 72 hours of treatment, the cells are harvested and the total protein is extracted using a RIPA buffer (radioimmunoprecipitation assay). An equal amount of protein from cells treated with a CT (control) mAb or anti-hPG mAb was then Western blotted using an anti-survivin antibody (monoclonal antibody, #2802 from Cell Signaling) and an anti-actin antibody as loading control (monoclonal antibody, # A4700 from SIGMA). Quantitative measurement is carried out using the GBOX chemisystem from Syngene.

Treatment with anti-hPG monoclinal antibodies reduced survivin expression compared to control antibody treatment. Statistical significance is determined using unpaired Student's t-test: * - p less than 0.05; ** - p less than 0.01 and *** - p less than 0.001.

1.6. Pathway Neutralizing Activity of Humanized Anti-hPG Antibodies

WNT/b-catenin.

Humanized anti-PG antibodies are being tested for their ability to inhibit the WNT/β-catenin pathway in several cell lines commonly used for prostate cancer research that produce and secrete progastrin using the expression of the survivin protein of the well-known target gene of the WNT/β-catenin pathway - in as an indicator. Survivin expression from each of these cell lines is analyzed using different humanized anti-hPG antibodies.

For each experiment, 50,000 cells are plated in 6-well plates in medium containing fetal calf serum and incubated for 8 h. h for 72 h, in the absence of fetal bovine serum, 1-20 μg/ml humanized control antibodies (anti-human FcG1 antibody, from BioXCell) (CT Hz) or 1-20 μg/ml Hz against hPG, where the indicated Hz is a humanized antibody against the C-terminus of hPG; or a humanized antibody against the N-terminus of hPG.

Specifically, after 72 hours of treatment, cells are harvested and total protein is extracted using RIPA buffer. An equal amount of protein from cells treated with CT Hz or Hz anti-hPG was then Western blotted using an anti-survivin antibody (monoclonal antibody, #2802 from Cell Signaling) and an anti-actin antibody as loading control (monoclonal antibody, #A4700 from SIGMA ). Quantitative measurement is carried out using the GBOX chemisystem from Syngene.

Treatment with humanized anti-hPG antibodies reduced survivin expression compared to treatment with a control antibody.

Statistical significance is determined using unpaired Student's t-test: * - p less than 0.05; ** - p less than 0.01 and *** - p less than 0.001.

Example 2 Neutralizing activity of anti-hPG antibodies against cancer cell lines.

2.1. Neutralizing activity of humanized anti-hPG antibodies on cell survival.

Humanized anti-PG antibodies are tested for their ability to inhibit the proliferation of several cell lines commonly used in prostate cancer research (ie, PC3, LNCAP, DU145, etc.) that produce and secrete progastrin. Cell survival from each of these cell lines was analyzed using different anti-hPG monoclonal antibodies.

125,000 DU145 cells were seeded in 6-well plates _in medium containing fetal calf serum and incubated for 8 h. h for 48 h, in the absence of fetal bovine serum, 20 μg/ml humanized control antibodies (anti-human FcG1 antibody, from BioXCell) (CT Hz) or 20 μg/ml Hz against hPG 8CV2 (PG Hz), where the specified Hz represents is a humanized antibody against the C-terminus of PG. The number of cells in T ₀ was counted in the control well for each experiment.

In particular, the number of live cells in both the control and Hz anti-hPG treated wells was counted at 48 hours. The difference between each cell number and the number of cells determined in T ₀ was then calculated.

Treatment of Hz with anti-hPG antibodies reduced the number of cells compared to treatment with control antibody. Statistical significance was determined using a t-test: ** - p less than 0.01.

2.2. Neutralizing activity of humanized anti-hPG antibodies in relation to the formation of spheres.

150 LNCaP cells were seeded in 24-well ultra-low-attachment (ULA) plates. These cells were cultured for 11 days in ULA plates with M11 medium (Macari et al., Oncogene, 2015) and treated every 3 or 4 days with 10 μg/ml humanized control antibodies (anti-human FcG1, from BioXCell) (CT Hz) or 10 μg/ml Hz against hPG 8CV2 (PG Hz), where said Hz is a humanized anti-hPG C-terminal antibody or a humanized anti-hPG N-terminal antibody.

In particular, at the end of the incubation phase, the wells were photographed by bright field microscopy, the images were analyzed, and spheres with an average diameter greater than 20 μm were counted.

The results shown in FIG. 2 clearly show that treatment of prostate cancer cells with anti-hPG monoclinal antibodies significantly reduced the number of spheroids that formed during growth under low attachment culture conditions compared to

-

Claims (15)

control monoclonal antibody. Statistical significance was determined using the t test: * - p less than 0.05. CLAIM 1. The use of a progastrin-binding antibody or antigen-binding fragment thereof for the manufacture of a medicament for the prevention or treatment of prostate cancer. 2. Use according to claim 1, wherein said progastrin-binding antibody or antigen-binding fragment thereof is selected from single chain antibodies, camelized antibodies, IgA1 antibodies, IgA2 antibodies, IgD antibodies, IgE antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, antibodies IgG4 and IgM antibodies. 3. Use according to any one of claims 1 or 2, wherein said progastrin-binding antibody or antigen-binding fragment thereof is selected from antibodies against the N-terminus of progastrin and antibodies against the C-terminus of progastrin. 4. Use according to any one of claims 1 to 3, wherein said progastrin-binding antibody or antigen-binding fragment thereof is a neutralizing antibody. 5. Use according to any one of claims 1 to 4, wherein said antibody is selected from the group consisting of the following: an antibody containing a heavy chain containing the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 4, 5 and 6, respectively, and a light chain containing the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences SEQ ID NO: 7, 8 and 9, respectively; an antibody containing a heavy chain containing CDR-H1, CDR-H2 and CDR-H3 amino acid sequences SEQ ID NO: 10, 11 and 12, respectively, and a light chain containing CDR-L1, CDR-L2 and CDR-L3 amino acid sequences SEQ ID NO: 13, 14 and 15, respectively; an antibody containing a heavy chain containing the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 16, 17 and 18, respectively, and a light chain containing the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences SEQ ID NOs: 19, 20 and 21, respectively; an antibody containing a heavy chain containing the CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NOs: 22, 23 and 24, respectively, and a light chain containing the CDR-L1, CDR-L2 and CDR-L3 amino acid sequences SEQ ID NOs: 25, 26 and 27, respectively; an antibody containing a heavy chain containing CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NO: 28, 29 and 30, respectively, and a light chain containing CDR-L1, CDR-L2 and CDR-L3 amino acid sequences SEQ ID NOs: 31, 32 and 33, respectively; and an antibody containing a heavy chain containing CDR-H1, CDR-H2 and CDR-H3 amino acid sequences of SEQ ID NO: 34, 35 and 36, respectively, and a light chain containing CDR-L1, CDR-L2 and CDR-L3 amino acid sequences SEQ ID NO: 37, 38 and 39, respectively. 6. Use according to any one of claims 1 to 5, wherein said antibody is selected from the group consisting of the following: a monoclonal antibody comprising the heavy chain of the amino acid sequence of SEQ ID NO: 41 and the light chain of the amino acid sequence of SEQ ID NO: 42; a monoclonal antibody comprising the heavy chain of the amino acid sequence of SEQ ID NO: 43 and the light chain of the amino acid sequence of SEQ ID NO: 44; a monoclonal antibody comprising the heavy chain of the amino acid sequence of SEQ ID NO: 45 and the light chain of the amino acid sequence of SEQ ID NO: 46; a monoclonal antibody comprising the heavy chain of the amino acid sequence of SEQ ID NO: 47 and the light chain of the amino acid sequence of SEQ ID NO: 48; a monoclonal antibody containing the heavy chain of the amino acid sequence of SEQ ID NO: 49 and the light chain of the amino acid sequence of SEQ ID NO: 50; and a monoclonal antibody comprising the heavy chain of the amino acid sequence of SEQ ID NO: 51 and the light chain of the amino acid sequence of SEQ ID NO: 52. 7. Use according to any one of claims 1 to 5, wherein said antibody is a humanized antibody. 8. Use according to claim 7, wherein said antibody is selected from the group consisting of the following: a humanized antibody comprising the heavy chain variable region of the amino acid sequence of SEQ ID NO: 53 and the light chain variable region of the amino acid sequence of SEQ ID NO: 54; a humanized antibody comprising the heavy chain variable region of the amino acid sequence of SEQ ID NO: 55 and the light chain variable region of the amino acid sequence of SEQ ID NO: 56; a humanized antibody comprising a heavy chain variable region of an amino acid sequence selected between SEQ ID NOs: 57, 58 and 59 and a light chain variable region of an amino acid sequence selected between SEQ ID NOs: 60, 61 and 62; a humanized antibody comprising a heavy chain variable region of an amino acid sequence selected between SEQ ID NOs: 63, 64 and 65 and a light chain variable region of an amino acid sequence selected between SEQ ID NOs: 66, 67 and 68; a humanized antibody comprising a heavy chain variable region of an amino acid sequence selected between SEQ ID NOs: 69 and 71 and a light chain variable region of an amino acid sequence selected between SEQ ID NOs: 70 and 72; and a humanized antibody comprising a heavy chain variable region of an amino acid sequence selected between SEQ ID NOs: 75 and 76 and a light chain variable region of an amino acid sequence selected between SEQ ID NOs: 77 and 78; wherein said antibody also contains light chain and heavy chain constant regions derived from a human antibody. 9. Use according to any one of claims 7 or 8, wherein said antibody comprises the heavy chain variable region of the amino acid sequence of SEQ ID NO: 71 and the light chain variable region of the amino acid sequence of SEQ ID NO: 72, said antibody also comprising light chain constant regions and heavy chain derived from a human antibody. 10. Use according to any one of claims 7-9, wherein said antibody comprises the heavy chain of the amino acid sequence of SEQ ID NO: 73 and the light chain of the amino acid sequence of SEQ ID NO: 74. 11. The use of a pharmaceutical composition comprising a progastrin-binding antibody or antigen-binding fragment thereof, as defined in any one of claims 1 to 10, and a pharmaceutically acceptable carrier and/or excipient for the manufacture of a medicament for the prevention or treatment of prostate cancer. 12. Use according to claim 11, wherein said pharmaceutical composition further comprises a second therapeutic agent. 13. Use according to claim 12, wherein said second therapeutic agent is a biological agent or a chemotherapeutic agent. 14. Use according to claim 13, wherein said biological agent is an anti-EGFR (epidermal growth factor receptor) monoclonal antibody or an anti-VEGF (vascular endothelial growth factor) monoclonal antibody. 15. Use according to claim 13, wherein said chemotherapeutic agent is selected from the group of alkylating agents, antimetabolites, antitumor antibiotics, mitosis inhibitors, chromatin function inhibitors, anti-angiogenesis agents, antiestrogen agents, antiandrogenic agents and immunomodulators. -