RU2784604C2 - Combination therapy of antibody to progastrin and immunotherapy for cancer treatment - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: combination for the treatment of cancer is proposed, containing a monoclonal antibody to progastrin and an inhibitor of an immune control point. The inhibitor of an immune control point is an antibody to PD1. A pharmaceutical composition containing the specified combination is also proposed.

EFFECT: invention provides increased therapeutic efficiency against cancer.

14 cl, 2 dwg, 6 tbl, 2 ex

Description

Immunotherapy has been a game-changing factor in the field of cancer therapy. Human cancers carry many somatic gene mutations and epigenetically altered genes whose products are potentially recognized as foreign antigens. Tumor cells escape the endogenous immune response by inducing tolerance among tumor-specific T cells. To ensure that the inflammatory immune response is not permanently activated after tumor antigens have stimulated the response, many controls or "checkpoints" are operated or activated. These immune checkpoints are primarily represented by the binding of T cell receptors to ligands on cells in the tumor microenvironment, forming immunological synapses that then regulate T cell function.

One approach to trigger anti-tumor immune responses has been called "checkpoint blockade", which refers to the blockade of immuno-inhibitory pathways activated by cancer cells. Developments in immune checkpoint therapies are progressing at a breathtaking pace. A major milestone has been overcome with the discovery of new molecules that act on the immune system (the body's defense system against a pathogen) and are effective in certain cancers. The use of these new molecules has made it possible to observe tumor regression in some patients with skin melanoma. Recent approvals of several blockers of the cytotoxic T-lymphocyte (CTL)-associated antigen-4-CD80/CD86 pathway (ipilimumab/ervoy) and the PD-1-PD-L1/PD-L2 pathway, such as nivolumab (opdivo), pembrolizumab (keytruda ) or atezolizumab (Tecentriq), has proclaimed immune checkpoint inhibitors (IC) - (ICI) - as a key component of advanced cancer treatment.

However, even with melanoma, most patients show only a limited or temporary response, while common cancers such as breast, prostate, or colon cancer respond only sporadically. In addition, pancreatic adenocarcinoma and colorectal adenocarcinoma generally remain resistant to these treatments, especially single agent blockade, PD-1. Despite the observation of robust responses to checkpoint-blocking antibodies, therefore, it is clear that not all patients, even within these subsets of cancers responding to immunotherapy, show tumor regression.

Therefore, there is still a need to identify additional pathways that provide either agonism or additional inhibition of existing immune checkpoint inhibitor pathways.

DESCRIPTION OF THE INVENTION

The present inventors have shown that the combination of a progastrin binding molecule and an immune checkpoint inhibitor results in greater therapeutic efficacy against cancer. Namely, the present inventors have shown that administration of an anti-progastrin monoclonal antibody in combination with an immune checkpoint inhibitor, such as an anti-PD-1 antibody, significantly increases the survival of xenograft mice with colorectal cancer cell lines. This is illustrated by the median survival time, which is significantly increased compared to one of each therapy. In addition, the combination of said anti-progastrin monoclonal antibody and an immune checkpoint inhibitor results in more than twice the level of interferon-γ expression.

In a first aspect, the present invention relates to a combination comprising a progastrin binding molecule and an immune checkpoint inhibitor. Preferably, the invention relates to a combination of a progastrin binding molecule and an immune checkpoint inhibitor.

Immune checkpoint inhibitors

The term "checkpoint inhibitor", as used herein, refers to a molecule, such as, for example, a small molecule, a soluble receptor, or an antibody, that targets an immune checkpoint and blocks the function of said immune checkpoint. More specifically, a "checkpoint inhibitor" as used herein is a molecule, such as, for example, a small molecule, a soluble receptor, or an antibody, that blocks certain proteins produced by certain cell types of the immune system, such as T cells, and some cancer cells. Such proteins, "immune checkpoints" or "immune checkpoint proteins" as used herein, regulate the function of T cells in the immune system. Namely, they help keep immune responses under control and can prevent T cells from killing cancer cells. These immune checkpoint proteins achieve this by interacting with specific ligands that send a signal to the T cell and essentially turn off or inhibit the function of the T cell. Inhibition of these proteins leads to the restoration of T-cell function and the immune response to cancer cells. Examples of checkpoint proteins include CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4 (belongs to the CD2 family of molecules and is expressed on all NK cells). (natural killer), γ and T memory cells CD8+ (αβ)), CD 160 (also referred to as BY55), CGEN-15049, CHK1 and CHK2 kinases, IDO1, A2aR, and various B-7 family ligands, but are not limited to them.

In a first embodiment, the immune checkpoint inhibitor is an inhibitor of any of CTLA-4, PDL1, PDL2, PD1, B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4 (belongs to the family of molecules CD2 and is expressed on all NK cells, γδ and CD8+ (αβ) memory T cells), CD 160 (also referred to as BY55), CGEN-15049, CHK1 and CHK2 kinases, IDO1, A2aR, and any of the various B-7 family ligands.

The term "inhibitor" or "antagonist", as used herein, refers to a molecule that is capable of inhibiting or otherwise reducing one or more biological activities of a target protein, such as any one of the immune control proteins. points described above. In some embodiments, an immune checkpoint protein inhibitor (e.g., an antagonist antibody as provided herein) may, for example, act by inhibiting or otherwise reducing the activation and/or cell signaling pathways of a cell expressing said immune checkpoint protein (e.g., T cells ), thereby inhibiting the biological activity of the cell relative to the biological activity in the absence of the antagonist.

Exemplary immune checkpoint inhibitors include an anti-CTLA-4 antibody (eg, ipilimumab), an anti-LAG-3 antibody (eg, BMS-986016), an anti-B7-H3 antibody, an anti-B7-H4 antibody, an anti-Tim3 antibody (eg, TSR- 022, MBG453), anti-BTLA antibody, anti-KIR antibody, anti-A2aR antibody, anti-CD200 antibody, anti-PD-1 antibody (e.g. pembrolizumab, nivolumab, cemiplimab, pidilizumab), anti-PD-L1 antibody (e.g. atezolizumab, avelumab, durvalumab, BMS 936559), VISTA antibody (e.g., JNJ 61610588), CD28 antibody, CD80 or CD86 antibody, B7RP1 antibody, B7-H3 antibody, HVEM antibody, CD137 antibody (e.g., urelumab), anti-CD137L, anti-OX40 antibody (e.g., 9 B12, PF-04518600, MEDI6469), anti-OX40L antibody, anti-CD40 or CD40L antibody, anti-GAL9 antibody, anti-IL-10 antibody, PD-1 ligand extracellular domain fusion protein, e.g., PDL-1 or PD-L2 and IgG1 (e.g. AMP-224), OX40 ligand extracellular domain fusion protein, e.g. OX40L and IgG1 (e.g. MEDI6368), drug I DO1 (e.g. epacadostat) and drug A2aR. A number of immune checkpoint inhibitors have been approved or are currently in clinical trials. Such inhibitors include ipilimumab, pembrolizumab, nivolumab, cemiplimab, pidilizumab, atezolizumab, avelumab, durvalumab, BMS 936559, JNJ 61610588, urelumab, 9B12, PF-04518600, BMS-986016, TSR-022, MEDI69,836453

Examples of immune checkpoint inhibitors are listed, for example, in Marin-Acevedo et al., Journal of Hematology & Oncology 11:8, 2018; Kavecansky and Pavlick, AJHO 13(2): 9-20, 2017; Wei et al., Cancer Discov 8(9): 1069-86, 2018.

Preferably, the immune checkpoint inhibitor is a CTLA-4, LAG-3, Tim3, PD-1, PD-L1, VISTA, CD1 37, OX40, or IDO1 inhibitor.

In some embodiments, the inhibitor is a small molecule drug. In some embodiments, the inhibitor is a soluble receptor. In some embodiments, the inhibitor is an antibody.

The term "small molecular weight drug" is used herein in a broad sense to refer to an organic, inorganic, or organometallic compound typically having a molecular weight of less than about 1000. The small molecular weight drugs of the invention encompass oligopeptides and other biomolecules having a molecular weight of less than approximately 1000.

"Soluble receptor" in this document refers to a peptide or polypeptide containing the extracellular domain of the receptor, but not its transmembrane or cytoplasmic domains.

The term "antibody" as used herein is intended to include polyclonal and monoclonal antibodies. An antibody (or "immunoglobulin") consists of a glycoprotein containing at least two heavy (H) chains and two light (L) chains linked 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 termed "complementarity determining regions" (CDRs) or "hypervariable regions", which are primarily responsible for antigen epitope binding, and which are interspersed with regions that are more conserved, referred to as frame regions (FR). The method of 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 denotes the hypervariable regions of the heavy and light chains of an antibody when defined by IMGT, where the unique numbering of the IMGT gives a standardized limitation of framework regions and complementarity determining regions, CDR1-IMGT: 27 -38, CDR2.

, C, Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. При уникальной нумерации IMGT консервативные аминокислоты всегда имеют то же самое положение, например, цистеин 23 (1-ый CYS), триптофан 41 (КОНСЕРВАТИВНЫЙ-TRP), гидрофобная аминокислота 89, цистеин 104 (2-ой CYS), фенилаланин или триптофан 118 (J-PHE или J-TRP). Уникальная нумерация IMGT дает стандартизированное отграничение каркасных областей (FR1-IMGT:положения 1-26, FR2-IMGT: 39-55, FR3-IMGT: 66-104 и FR4-IMGT: 118-128) и областей, определяющих комплементарность: CDR1-IMGT: 27-38, CDR2-IMGT: 56-65 и CDR3-IMGT: 105-117. Поскольку пробелы представляют незанятые положения, длины CDR-IMGT (показанные между скобками и разделенные точками, например [8.8.13]) становятся критически важной информацией. Уникальная нумерация IMGT используется в 2D (двумерных) графических представлениях, обозначенных 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)] и в 3D (трехмерных) структурах в 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)].A unique IMGT numbering has been defined to compare variable domains, whatever the antigen receptor, chain type or species [Lefranc M.-R., Immunology Today 18, 509 (1997) / Lefranc M.-R., The Immunologist, 7, 132-136 (1999) / Lefranc, M.-P.,

, C, Ruiz, M., Giudicelli, V., Foulquier, E., Truong, L., Thouvenin-Contet, V. and Lefranc, Dev. Comp. Immunol., 27, 55-77 (2003)]. With unique IMGT numbering, conserved amino acids always have the same position, such as 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 numbering of the IMGT gives a standardized delimitation of the framework regions (FR1-IMGT: positions 1-26, FR2-IMGT: 39-55, FR3-IMGT: 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 critical information. Unique IMGT numbering is used in 2D (two-dimensional) graphical representations, designated IMGT by 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 arranged 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 the 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 (Clq) of the classical complement system. Antibodies can be of different isotypes (namely: IgA, IgD, IgE, IgG or IgM).

A "polyclonal antibody" is an antibody that is produced among or in the presence of one or more other non-identical antibodies. In general, polyclonal antibodies are produced from a B lymphocyte 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" means an antibody derived from an almost 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 chain.

In some embodiment, the inhibitor is an antagonistic antibody, i. an antibody that inhibits or reduces one or more biological activities of an antigen, such as one of the immune checkpoint proteins described herein. Some antagonistic antibodies substantially or completely inhibit one or more biological activities of said antigen. The term "inhibit" or its grammatical equivalent, when used in the context of an antibody, refers to an antibody that suppresses, limits, or reduces the biological activity of the antigen to which the antibody binds. The inhibitory effect of an antibody may be an effect that results in a measurable change in the biological activity of the antigen.

In one embodiment, the immune checkpoint inhibitor is selected from the group consisting of ipilimumab, pembrolizumab, nivolumab, cemiplimab, pidilizumab, atezolizumab, avelumab, durvalumab, BMS 936559, JNJ 61610588, urelumab, 9B12, PF-04518600, BMS-986016, TSR-986016 022, MBG453, MEDI6469, MEDI6383 and epacadostat.

In one embodiment, the immune checkpoint inhibitor is a CTLA-4, PD-1, or PD-L1 inhibitor. In a preferred embodiment, said immune checkpoint inhibitor is an antibody to any of CTLA-4, PD-1, or PD-L1. More preferably, said antibody is an antagonist antibody. Even more preferably, said antagonist antibody is selected from among ipilimumab, pembrolizumab, nivolumab, cemiplimab, pidilizumab, atezolizumab, avelumab and durvalumab.

In one embodiment, the immune checkpoint inhibitor is a PD-1 inhibitor. In a preferred embodiment, said immune checkpoint inhibitor is an anti-PD-1 antibody. More preferably, said antibody is an antagonistic antibody. Even more preferably, the immune checkpoint inhibitor is pembrolizumab, nivolumab, cemiplimab, or pidilizumab.

Anti-hPG antibodies

Progastrin (PG) is produced by colorectal tumor cells and is thought to stimulate the proliferation of these cells by triggering a signal transduction pathway that blocks normal cell differentiation processes, including processes that lead to cell death. Depletion of the transcript of the gastrin gene, which codes for progastrin, induces cell differentiation and programmed cell death in tumor cells in in vitro and in vivo CRC (colorectal cancer) models, reducing tumor cell proliferation. Without intending to be bound by any theory of action, it is believed that antibodies to hPG, by binding to PG, block or inhibit its ability to interact with its signaling partner(s). This in turn inhibits the signal transduction pathway in colorectal tumor cells that would otherwise lead to proliferation.

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

The term "progastrin" refers to a mammalian progastrin peptide, and in particular human progastrin. For the avoidance of doubt, without any particular definition, the expression "human progastrin" or "hPG" refers to the human PG of the sequence SEQ ID NO 1. Namely, human progastrin contains an N-terminal domain and a C-terminal domain that are not present in biologically active hormonal forms of gastrin mentioned above. 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.

A "progastrin-binding molecule" as used herein refers to any molecule that binds to progastrin but does not bind to gastrin-17 (G17), gastrin-34 (G34), gastrin 17 with a glycine extension (G17-Gly), or gastrin 34c glycine extension (G34-Gly) and C-terminal flanking peptide (CTFP). 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-hPG antibody) or an antigen-binding fragment thereof.

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.

In a more specific embodiment, the present hPG antibody 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, hPG residues 9-14, residues 4-10 hPG, 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, the hPG antibody 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, 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, the hPG antibody has an affinity for progastrin of at least 5000 nM, at least 500 nM, 100 nM, 80 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 7 nM , 5 nM, 4 nM, 3 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 50 pM, 10 pM, 5 pM, 1 pM, or at least 0.1 pM when determined in this way, as described in this document.

Preferably, the anti-hPG antibody is a neutralizing anti-hPG antibody.

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

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

In another specific embodiment, a progastrin-binding antibody has been prepared by an immunization method known to the person skilled in the art, wherein the immunogen is a peptide whose amino acid sequence contains all or part of the amino acid sequence of progastrin. More specifically, said immunogen contains a peptide selected from the following:

- 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, the amino acid sequence of which 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 part of progastrin and, in particular, to 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. The person skilled in the art will thus easily select and apply a method for producing polyclonal and/or monoclonal antibodies against any given antigen.

Examples of monoclonal antibodies that have been produced using an immunogen containing the amino acid sequence "SWKPRSQQPDAPLG" corresponding to 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 Table 1 - Table 4, but 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). The experimental results of epitope mapping show that mAb3, mAb4, mAb16, mAb19 and mAb20 indeed specifically bind 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 represented by SEQ ID NO 2 have been described in the art (see, for example, WO 2011/083088).

Examples of monoclonal antibodies that can be obtained by using an immunogen containing the amino acid sequence "QGPWLEEEEEAYGWMDFGRRSAEDEN", (C-terminal part of progastrin) corresponding to the amino acid sequence 55-80 of human progastrin, include antibodies designated as mAb8 and mAb13 in the following Tables 5 and 6 , but are not limited to them. 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.

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

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

The term "epitope" refers to the region of an antigen that is bound by an antibody. Epitopes can be defined as structural or functional. Functional epitopes, in general, are a subset of structural epitopes, and they have 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, sugar side 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 sequentially located 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 a protein.

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

- 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 NO 4, 5 and 6, respectively, or sequences with at least 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, 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%- noah, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NOS 7, 8 and 9, 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 NO 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 NOS 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 NOs 13, 14 and 15, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of 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 NO 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 the sequences of SEQ ID NOS 19, 20 and 21, 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 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 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 NOS 25, 26 and 27, 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 NO 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 the CDR-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 NOS 31, 32 and 33, respectively, and

- 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 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 NOS 37, 38 and 39, respectively. In the context of the present invention, "percent identity" or "% identity" between two nucleic acid or amino acid sequences means the percentage of identical nucleotides or amino acid residues between the two sequences to be compared, obtained after optimal alignment, this percentage being purely statistical, and the differences between two given sequences are distributed randomly over 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 may be by cut 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 a reference amino acid sequence, preferred examples include those containing the reference sequence, certain modifications , namely: deletion, addition or substitution of at least one amino acid, truncation or extension. In the case of a substitution of one or more consecutive or non-consecutive amino acids, substitutions are preferred in which the substituted amino acids are replaced by "equivalent" amino acids. It is meant 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 those specific examples 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 containing 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 containing 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 a specific embodiment, the antibody of the present combination is a chimeric antibody.

The term "chimeric antibody", as used herein, is an antibody in which a constant region, or portion thereof, is altered, replaced, or exchanged such that the variable region binds to a constant region of a different species or class, or antibody subclass. The term "chimeric antibody" also refers to an antibody in which the variable region, or a portion thereof, is altered, replaced, or exchanged such that the constant region binds to a variable region of a different species or belonging to a different class or subclass of the antibody.

In another specific embodiment, the antibody of the present combination 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 the antibody molecule derived from one or more human antibodies. In addition, certain skeletal segment residues (referred to as Caracas FRs) can be modified to retain binding affinity according to techniques known to one of skill 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 full antigen binding affinity and antibody specificity.

Humanized antibodies of the invention, or fragments thereof, can be prepared by methods known to those skilled in the art (such as those described in Singer et al., J. Immun., 150:2844-2857, 1992). Such humanized antibodies are preferred for use in methods involving in vitro diagnosis or in vivo preventive and/or therapeutic treatment. The person skilled in the art is also aware of other humanization techniques. 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;15 US 58867; US 5877293), veneering 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) : 805-814; Roguska M.A. et al., 1994, Proc. Natl. Acad. Sci. U.S.A., 91:969-973) and chain 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 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 NO 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, 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 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 the sequences of SEQ ID NOS 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 NOs 13, 14 and 15, respectively, or sequences with at least 80%, preferably 85%, 90%, 95% and 98% identity after optimal alignment with the sequences of SEQ ID NOS 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 the sequences of SEQ ID NOS 19, 20 and 21, 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 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 NOS 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 the CDR-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 NOS 31, 32 and 33, respectively, and

- 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 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 NOS 37, 38 and 39, respectively, where the specified antibody also contains constant regions of the light chain and heavy chain derived from human antibody.

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

- humanized antibody containing the variable region of the heavy chain of the amino acid sequence of SEQ ID NO 53 and the variable region of the light chain of the amino acid sequence of SEQ ID NO 54;

- humanized antibody containing the variable region of the heavy chain of the amino acid sequence of SEQ ID NO 55 and the variable region of the light chain 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 a heavy chain variable region of the amino acid sequence of SEQ ID NO 71 and a 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.

Antibody fragments

Fragments of an antibody, namely its antigen-binding fragments, are also covered by this invention.

"Antibody fragments" comprise a portion of a full-length antibody, typically its antigen-binding or variable region. Examples of antibody fragments include Fab, Fab', F(ab')2, Fv and Scfv fragments; diabody; linear antibodies; minibodies (Olafsen et al. (2004) Protein Eng. Design & Sel. 17(4):315-323), Fab expression library-produced fragments, anti-idiotypic (anti-Id) antibodies, CDR (complementarity determining region) and epitope-binding fragments of any described herein that immunospecifically bind to cancer cell antigens, viral antigens, or microbial antigens; single chain antibody molecules; and multispecific antibodies formed from antibody fragments.

The term "antigen binding domain" of an antibody (or "antigen binding fragment") refers to one or more antibody fragments that retain the ability to specifically bind to an antigen (eg, hPG). It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full length antibody. Examples of binding fragments encompassed within the term "antigen binding domain" of an antibody include: (i) a Fab fragment, a monovalent fragment consisting of the V _L , V _H , CL and CH1 domains; (ii) an F(ab')2 fragment, a divalent fragment containing two Fab fragments linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of V _H and CH1 domains; (iv) an Fv fragment consisting of the V _L and V _H domains of one antibody arm, (v) a single domain fragment or dAb (Ward et al., (1989) Nature 341:544-546) which consists of a V _H domain; and (vi) a dedicated complementarity determining region (CDR), or (vii) a combination of two or more dedicated CDRs that can optionally be joined by a synthetic linker. In addition, although the two domains of the Fv fragment, V _L and V _H , are encoded by separate genes, they can be linked using genetic engineering methods with a synthetic linker that allows them to be made into a single protein chain in which the V _L and V H regions V _H pair to form monovalent molecules (known as single-stranded Fvs (scFvs); see, for example, Bird et al. (1988) Science 242:423-426 and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85:5879-5883). It is also intended that such single chain antibodies are encompassed within the term "antigen binding domain" of an antibody.

These antigen-binding fragments are prepared using conventional techniques known to those skilled in the art, and these fragments are screened for utility in the same manner as intact antibodies. Antigen-binding moieties can be obtained by genetic engineering techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

Antibody derivatives

The anti-hPG antibodies of the present invention may be further modified to contain additional non-protein moieties that are known in the art and readily available. In particular, this document includes monoclonal antibodies to hPG, which are derivatized, covalently modified or conjugated with other molecules for use in diagnostic and therapeutic applications. For example, and not by way of limitation, derivatized antibodies include antibodies that have been modified, for example, by glycosylation, acetylation, PEGylation, phosphorylation, amidation, derivatization with known protecting/blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein, etc. d. Any of the numerous chemical modifications can be carried out by known methods, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, this derivative may contain one or more non-classical amino acids.

Preferably, water-soluble polymers are suitable moieties for antibody derivatization. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleic anhydride copolymer, polyamino acids ( or random copolymers) and dextran or poly(n-vinylpyrrolidone)polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (eg, glycerol), polyvinyl alcohol, and mixtures thereof. This polymer may be of any molecular weight and may be branched or unbranched. The number of polymers attached to an antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the number and/or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the specific properties or functions of the antibody to be improved, whether the antibody derivative will be used in therapy under certain conditions, etc. .

In a specific example, the hPG antibodies of the present disclosure may be attached to poly(ethylene glycol) (PEG) moieties. In a specific example, the antibody is an antibody fragment and the PEG moieties are attached via any available amino acid side chain or terminal amino acid functional group located on the antibody fragment, e.g., any free amino, imino, thiol, hydroxyl, or carboxyl group. Such amino acids may occur naturally in the antibody fragment or may be engineered into the fragment using genetic engineering techniques. See, for example, US Pat. No. 5,219,996. You can use many sites to attach two or more than two PEG molecules. The PEG moieties may be covalently linked through the thiol group of at least one cysteine residue located in a given antibody fragment. When a thiol group is used as the point of attachment, suitably activated effector moieties may be used, for example thiol-selective derivatives such as maleimide and cysteine derivatives.

In a specific example, the anti-hPG antibody conjugate is a modified Fab' fragment that is PEGylated, ie. has PEG (poly(ethylene glycol)) covalently attached to it, for example according to the method disclosed in EP0948544. See also Poly(ethyleneglycol) Chemistry, Biotechnical and Biomedical Applications, (J. Milton Harris (ed.), Plenum Press, New York, 1992); Poly(ethyleneglycol) Chemistry and Biological Applications, (J. Milton Harris and S. Zalipsky, eds., American Chemical Society, Washington D.C., 1997); Bioconjugation Protein Coupling Techniques for the Biomedical Sciences, (M. Aslam and A. Dent, eds., Grove Publishers, New York, 1998) and Chapman, 2002, Advanced Drug Delivery Reviews 54:531-545. The PEG may be attached to the cysteine at the hinge region. In one example, a PEG-modified Fab' fragment has a maleimide group covalently linked to one thiol group in the modified hinge region. The lysine moiety may be covalently linked to the maleimide group, and a methoxy poly(ethylene glycol) polymer having a molecular weight of about 20,000 Da can be attached to each of the amino groups on the lysine moiety. The total molecular weight of the PEG attached to the Fab' fragment may therefore be approximately 40,000 Da.

In another embodiment, conjugates of an antibody and a non-protein moiety are provided that can be selectively heated by exposure to radiation. In one embodiment, this non-protein moiety is a carbon nanotube (Kam et al, Proc. Natl. Acad. Sci. USA 102: 11600-11605 (2005)). The radiation can be of any wavelength and includes, but is not limited to, wavelengths that do not harm normal cells but that heat the non-protein moiety to a temperature that kills nearby cells to the antibody-non-protein moiety.

Immunoconjugates

In another aspect, the invention also provides immunoconjugates (interchangeably referred to as "antibody-drug conjugates" or "ADCs") comprising an anti-hPG antibody as described herein, said antibody being conjugated to one or more cytotoxic agents, such as 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).

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 a tumor and intracellular accumulation within it, where systemic administration of unconjugated drugs can lead to unacceptable levels of toxicity to normal cells as well as to cancer 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 reported to be 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, low molecular weight 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 in obtaining immunoconjugates chemotherapeutic agents are described in this document (for example, 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, modesin 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 preparation of radioconjugated antibodies. Examples include 212Bi, 131I, 131In, 90Y and 186Re. 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 HCI), 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-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene-2,6-diisocyanate); and bis-active fluorine compounds (such as 1,5-difluoro-2,4-dinitrobenzene). For example, the ricin immunotoxin can be prepared as described in Vitetta et ah, Science, 238:1098 (1987).

Carbon-14 labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is a typical chelator for conjugating a radioisotope to an antibody. See W094/11026.

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 "bundle" 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-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional imidoester derivatives (such as dimethyl adipimidate HCI), 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-diazoniumbenzoyl)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-3-methyldiethylenetriaminepentaacetic 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".

Nucleic acids and expression systems

The present disclosure encompasses polynucleotides encoding immunoglobulin light and heavy chain genes for antibodies, namely, anti-hPG antibodies, vectors containing such nucleic acids, and host cells capable of producing the antibodies of this disclosure.

In a first aspect, the present invention relates to one or more polynucleotides encoding an antibody, namely: an antibody capable of specific binding to progastrin as described above.

In a first embodiment, a polynucleotide encoding the heavy chain of an anti-hPG antibody described above is provided. Preferably, said heavy chain comprises three heavy chain CDRs of SEQ ID NO 4, 5, and 6. More preferably, said heavy chain comprises a heavy chain containing the variable region of SEQ ID NO 14. Even more preferably, said heavy chain has the full sequence of SEQ ID NO 16 .

In another embodiment, the polynucleotide encodes for the light chain of the anti-hPG antibody described above. Preferably, said light chain comprises three light chain CDRs of SEQ ID NO 7, 8 and 9. More preferably, said light chain comprises a light chain containing the variable region of SEQ ID NO 15. Even more preferably, said light chain has the full sequence of SEQ ID NO 17 .

According to this invention, a variety of expression systems can be used to express an antibody of the invention. In one aspect, such expression systems are vehicles whereby coding sequences of interest can be generated and then purified, but are also cells that can express an IgG antibody in situ when transiently transfected with suitable coding nucleotide sequences.

The present invention provides vectors containing the polynucleotides described above. In one embodiment, the vector contains a polynucleotide encoding the heavy chain of an antibody of interest (eg, an anti-hPG antibody). In another embodiment, said polynucleotide encodes a light chain of an antibody of interest (eg, an anti-hPG antibody). The invention also provides vectors containing polynucleotide molecules encoding fusion proteins, modified antibodies, antibody fragments and their probes.

In order to express the heavy and/or light chain of an antibody of interest (e.g., anti-hPG antibodies), polynucleotides encoding said heavy and/or light chains are inserted into expression vectors such that the genes are linked in a functional manner to transcriptional and translational sequences.

"Operably linked" sequences include both expression control sequences that are adjacent to the gene of interest and expression control sequences that act remotely or at a distance to control the gene of interest. The term "expression control sequence" as used herein refers to polynucleotide sequences that are necessary to affect the expression and processing of the coding sequences to which they are ligated. Expression control sequences include suitable transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance the efficiency of translation (ie Kozak consensus sequence); sequences that increase protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences differs depending on the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, such control sequences typically include promoters and a transcription termination sequence. The term "control sequences" is intended to include, at a minimum, all components whose presence is essential for expression and processing, and may also include additional components whose presence is beneficial, such as leader sequences and fusion partner sequences.

The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid" - this term refers to a loop of circular double-stranded DNA into which additional DNA segments can be ligated. Another type of vector is the viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial replicator and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of a host cell when introduced into the host cell and thereby replicate along with the host genome.

Some vectors are capable of directing the expression of genes to which they are linked in a functional manner. Such vectors are referred to herein as "recombinant expression ion vectors" (or simply "expression vectors"). In general, expression vectors used in genetic engineering techniques are in the form of plasmids. In the present description of the invention, the terms "plasmid" and "vector" can be used interchangeably, since the plasmid is the most commonly used form of the vector. However, this invention is intended to include forms of expression vectors such as bacterial plasmids, YACs (artificial yeast chromosomes), cosmids, retroviruses, episomes derived from EBV (Epstein-Barr virus), and all other vectors that the person skilled in the art will know as convenient for allowing expression of the heavy and/or light chains of an antibody of interest (eg, an anti-hPG antibody). The person skilled in the art will recognize that the polynucleotides encoding the heavy and light chains can be cloned into different vectors or into the same vector. In a preferred embodiment, said polynucleotides are cloned into two vectors.

The polynucleotides of the invention and vectors containing these molecules can be used to transform a suitable host cell. The term "host cell" as used herein is meant to refer to a cell into which a recombinant expression vector has been introduced in order to express an antibody of interest (eg, an anti-hPG antibody). It is to be understood that such terms are intended to refer not only to a particular cell of a subject, but also to the progeny of such a cell. Since certain modifications may occur in successive generations, either due to mutation or environmental influences, such progeny may not actually be identical to the parent cell, but are still included within the scope of the term "host cell" in as used in this document.

Transformation can be carried out by any known method of introducing polynucleotides into the host cell. Such methods are well known to those skilled in the art and include dextran-mediated transformation, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, polynucleotide liposome encapsulation, biolistic injection, or direct microinjection of DNA into nuclei.

The host cell may be co-transfected with one or more expression vectors. For example, a host cell can be transfected with a vector encoding both the heavy chain and the light chain of an antibody of interest (eg, an anti-hPG antibody) as described above. Alternatively, the host cell can be transformed with a first vector encoding the heavy chain of the antibody of interest (eg, an anti-hPG antibody) and a second vector encoding the light chain of said antibody. Mammalian cells are commonly used to express recombinant therapeutic immunoglobulins, especially for the expression of whole recombinant antibodies. For example, mammalian cells such as HEK293 cells (human embryonic kidney 293 cells) or CHO cells (Chinese hamster ovary cells), in combination with a vector containing an expression signal, such as a signal carrying the promoter element of the major intermediate early gene from human cytomegalovirus, are is an efficient system for expressing a humanized hPG antibody of the invention (Foecking et al., 1986, Gene 45:101; Cockett et al., 1990, Bio/Technology 8:2).

In addition, a host cell may be selected that modulates the expression of the inserted sequences or modifies and processes the gene product in a particular desired manner. Such modifications (eg, glycosylation) and processing of protein products may be important for protein function. Different host cells have characteristics and specific mechanisms for post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems are selected to ensure correct modification and processing of the expressed antibody of interest. Therefore, it is possible to use eukaryotic host cells that possess the cellular machinery for proper processing of the primary transcript, glycosylation of the gene product. Such mammalian host cells include, but are not limited to, CHO, COS (monkey kidney), HEK293, NS/0, BHK (baby hamster kidney), Y2/0, 3T3, or myeloma cells (all of these cell lines are publicly available). depositories such as the National Collection of Microorganism Cultures, Paris, France, or the American Collection of Type Cell Cultures, Manassas, VA, USA).

For long-term production of recombinant proteins in high yield, stable expression is preferred. In one embodiment of the present invention, cell lines can be constructed that stably express the antibody. Rather than using expression vectors that contain viral replicators, host cells are transformed with DNA under the control of appropriate expression regulatory elements, including promoters, enhancers, transcription terminators, polyadenylation sites, and other suitable sequences known to one of skill in the art, and a selectable marker. After the introduction of foreign DNA, genetically modified cells can be allowed to grow for one to two days in an enriched medium, and then they are transferred to a selective medium. A selectable marker on a recombinant plasmid confers resistance to selection and allows cells to stably integrate the plasmid into the chromosome and propagate it into cell lines. Other methods for constructing stable cell lines are known in the art. In particular, methods for site-specific integration have been developed. According to these methods, the transformed DNA, under the control of suitable regulatory expression elements, including promoters, enhancers, transcription terminators, polyadenylation sites, and other suitable sequences, is integrated into the host cell genome at a specific target site that has previously been cleaved (Moele et al., Proc Natl Acad Sci U.S.A., 104(9): 3055-3060; US 5792632; US 5830729; US 6238924; WO 2009/054985; WO 03/025183; WO 2004/067753).

A variety of selection systems can be used according to the invention, including genes for thymidine kinase herpes simplex virus (Wigler et al., Cell 11:223, 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska et al., Proc Natl Acad Sci USA 48: 202, 1992) , glutamate synthase selection in the presence of methionine sulfoximide (Adv Drug Del Rev, 58: 671, 2006, and internet site or Lonza Group Ltd. literature) and adenine phosphoribosyltransferase genes (Lowy et al., Cell 22: 817, 1980) in tk cells, hgprt or apt respectively. You can also use antimetabolite resistance as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc Natl Acad Sci USA 77: 357, 1980); gpt, which confers resistance to mycophenolic acid (Mulligan et al., Proc Natl Acad Sci USA 78: 2072,1981); neo, which confers resistance to an aminoglycoside, G-418 (Wu et al., Biotherapy 3: 87, 1991); and hygro, which confers resistance to hygromycin (Santerre et al., Gene 30: 147, 1984). Methods known in the art of genetic engineering can be conventionally used to select the desired recombinant clone, and such methods are described, for example, in Ausubel et al., eds., Current Protocols in Molecular Biology, John Wiley & Sons (1993). The levels of antibody expression can be increased by vector amplification. When a marker in an antibody expressing vector system is amplifiable, increasing the level of inhibitor present in culture will increase the copy number of the marker gene. Since the amplified region is associated with a gene encoding an IgG antibody of the invention, production of said antibody will also be increased (Crouse et al., Mol Cell Biol 3: 257, 1983). There are alternative ways to carry out the expression of the gene according to the invention, and they are known to the person skilled in the art. For example, a modified zinc finger protein can be engineered such that it is capable of binding to expression regulatory elements upstream of the gene of the invention; expression of said engineered zinc finger (ZFN) protein in the host cell of the invention results in increased protein production (see, for example, Reik et al., Biotechnol. Bioeng., 97(5): 1180-1189, 2006). In addition, ZFN can stimulate DNA integration at a given genomic position, resulting in highly efficient site-specific gene addition (Moehle et al, Proc Natl Acad Sci USA, 104: 3055, 2007).

An antibody of interest (eg, an anti-hPG antibody) can be obtained by growing a culture of transformed host cells under the necessary culture conditions to express the desired antibody. The resulting expressed antibody can then be purified from the culture medium or cell extracts. Soluble forms of the antibody of interest (eg, anti-hPG antibodies) can be isolated from the culture supernatant. It can then be purified by any method known in the art of purification of the immunoglobulin molecule, e.g., chromatography (e.g., ion exchange, affinity, in particular based on the affinity of protein A for Fc, etc.), centrifugation, differential solubility, or any other standard purification technique. proteins. Suitable purification methods will be apparent to one of ordinary skill in the art.

Another aspect of the invention thus relates to a method for producing an antibody (eg, an anti-hPG antibody) described herein, comprising the following steps:

a) growing the above-described host cell in a culture medium under suitable culture conditions; and

b) isolating the antibody (eg, anti-hPG antibody) from the culture medium or from said cultured cells.

Pharmaceutical compositions

The combination of anti-hPG monoclonal antibodies and immune checkpoint inhibitors can be formulated. Possibly, these compositions may contain one or more additional therapeutic agents, such as the third therapeutic agents described below. These compositions will typically be supplied as part of a sterile pharmaceutical composition, which will typically include a pharmaceutically acceptable carrier and/or excipient. In another aspect, the invention thus provides a pharmaceutical composition comprising an anti-hPG antibody, an immune checkpoint inhibitor, and a pharmaceutically acceptable carrier. and/or excipient.

The composition may be in any suitable form (depending on its desired route of administration to the patient). The term "administering" as used herein means a method of providing a subject with a dosage of a compound (e.g., an anti-hPG antibody and/or an immune checkpoint inhibitor, as described above) or a composition (e.g., a pharmaceutical composition, e.g. , a pharmaceutical composition containing an anti-hPG antibody and/or an immune checkpoint inhibitor as described above). The compositions used in the methods described herein can be administered, for example, intravitreally (for example, by intravitreal injection), via eye drops, intramuscularly, intravenously, intradermally, transdermally, intra-arterially, intraperitoneally, intralesionally, intracranially, intra-articularly, into prostate gland, intrapleural, intratracheal, intrathecal, intranasal, intravaginal, intrarectal, local, intratumoral, peritoneal, subcutaneous, subconjunctival, intravesicular, mucosal, intrapericardial, intraumbilical, eye, intraorbital, oral, local, percutaneous, by inhalation, by injections, by implantation, by infusion, by continuous infusion, by localized perfusion directly bathing the target cells, by catheter, by lavage, in creams or in liquid formulations. The compositions used in the methods described herein can also be administered systemically or locally. The route of administration may vary depending on various factors (eg, the compound or composition is being administered and the severity of the condition, disease, or disorder being treated). The most appropriate route of administration in any given case will depend on the particular antibody, the subject, and the nature and severity of the disease or physical condition of the subject. The anti-hPG antibody and/or immune checkpoint inhibitor may be prepared in an aqueous solution and administered by subcutaneous injection.

Pharmaceutical compositions may conveniently be presented in unit dose forms containing a given amount of anti-hPG antibody and/or immune checkpoint inhibitor per dose. Such a unit may contain, for example, but not limited to, 5 mg to 5 g, such as 10 mg to 1 g or 20 to 50 mg. Pharmaceutically acceptable carriers for use in this disclosure may take a wide variety of forms, depending on for example, the condition being treated or the route of administration.

Pharmaceutical compositions of this disclosure may be prepared for storage as lyophilized preparations or aqueous solutions by mixing an antibody having the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers typically used in the art (which are all referred to herein as "carriers"). ”), i.e. buffering agents, stabilizers, preservatives, isotonic additives, non-ionic detergents, antioxidants and various other additives. See Remington's Pharmaceutical Sciences, 16th edition (Osol, ed. 1980). Such supplements should be non-toxic to recipients at the dosages and concentrations used.

Buffering agents help maintain the pH in a range that approximates physiological conditions. They may be present in concentrations ranging from about 2 mM to about 50 mM. Suitable buffering agents for use in the present disclosure include both organic and inorganic acids and their salts, such as citrate buffers (e.g., monosodium citrate-disodium citrate, citric acid-trisodium citrate, citric acid-monosodium citrate, etc.). .d.), succinate buffers (e.g. succinic acid-monosodium succinate mixture, succinic acid-sodium hydroxide mixture, succinic acid-disodium succinate mixture, etc.), tartrate buffers (e.g. tartaric acid-sodium tartrate mixture, tartaric acid-potassium tartrate, tartaric acid-sodium hydroxide, etc.), fumarate buffers (e.g., fumaric acid-monosodium fumarate, fumaric acid-disodium fumarate, monosodium fumarate-disodium fumarate, etc.) , gluconate buffers (e.g. gluconic acid-sodium gluconate, gluconic acid-sodium hydroxide, gluconic acid-potassium gluconate, etc.), oxalate buffers (e.g. oxalic acid -sodium oxalate, oxalic acid-sodium hydroxide, oxalic acid-potassium oxalate, etc.), lactate buffers (e.g. lactic acid-sodium lactate, lactic acid-sodium hydroxide, lactic acid-potassium lactate and etc.) and acetate buffers (eg acetic acid-sodium acetate, acetic acid-sodium hydroxide, etc.). In addition, phosphate buffers, histidine buffers and trimethylamine salts such as Tris can be used.

Preservatives may be added to retard microbial growth and may be added in amounts ranging from 0.2% to 1% (w/v). Suitable preservatives for use in the present disclosure include phenol, benzyl alcohol, meta-cresol, methylparaben, propylparaben, octadecyldimethylbenzylammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens such as methyl or propyl paraben, catechine. , resorcinol, cyclohexanol and 3-pentanol. Isotonic additives, sometimes known as "stabilizers", may be added to provide isotonicity to the liquid compositions of the present disclosure, and these include polyhydric sugar alcohols, for example, trihydric or higher sugar alcohols such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol. "stabilizers" refers to a broad category of excipients that can vary in function from a bulking agent to an additive that solubilizes the therapeutic agent or helps prevent denaturation or sticking to the container wall. Typical stabilizers can be polyhydric sugar alcohols (listed above); amino acids such as arginine, lysine, glycine, glutamine, asparagine, histidine, alanine, ornithine, L-leucine, 2-phenylalanine, glutamic acid, threonine, etc., organic sugars or sugar alcohols such as lactose, trehalose, stachyose , mannitol, sorbitol, xylitol, ribitol, myoinositol, galactitol, glycerol and the like, including cyclitols such as inositol; polyethylene glycol; amino acid polymers; sulfur reducing agents such as urea, glutathione, thioctic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thiosulfate; low molecular weight polypeptides (eg, peptides of 10 residues or less); proteins such as human serum albumin, bovine serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, monosaccharides such as xylose, mannose, fructose, glucose; disaccharides such as lactose, maltose, sucrose, trisaccharides such as raffinose, and polysaccharides such as dextran. Stabilizers may be present in the range of 0.1 to 10,000 parts by weight per part by weight of active protein.

Non-ionic surfactants or detergents (also known as "wetting agents") may be added to help solubilize the therapeutic agent as well as protect the therapeutic agent against shake-induced aggregation, which also allows surface shear to be applied to the preparation without inducing protein denaturation. Suitable non-ionic surfactants include polysorbates (20, 80, etc.), poloxamers (184, 188, etc.), pluronic polyols, polyoxyethylene sorbitan monoesters (TWEEN®-20, TWEEN®-80, etc.). d.). Non-ionic surfactants may be present in the range from about 0.05 mg/ml to about 1.0 mg/ml, for example, from about 0.07 mg/ml to about 0.2 mg/ml.

Additional miscellaneous excipients include bulking agents (eg starch), chelators (eg EDTA (ethylenediaminetetraacetic acid)), antioxidants (eg ascorbic acid, methionine, vitamin E), and co-solvents.

The present invention is further directed to a pharmaceutical composition comprising at least:

i) one anti-hPG antibody and

ii) immune checkpoint inhibitor

as combined products for simultaneous, separate or sequential use.

The term "simultaneous use" as used herein refers to the administration of two compounds of the composition according to this invention in the same and identical pharmaceutical form.

The term "separate use" as used herein refers to the administration of two compounds of the composition of this invention at the same time in different pharmaceutical forms.

The term "sequential use" as used herein refers to the sequential administration of two compounds of the composition of this invention, each in a different pharmaceutical form.

Anti-hPG antibody and immune checkpoint inhibitor compositions may be administered alone, as mixtures of one or more anti-hPG monoclonal antibodies and/or one or more immune checkpoint inhibitors, in admixture or combination with other useful cancer treatments, ie CRC, or in addition to another anti-cancer therapy, ie CRC. Examples of a suitable combination and additional therapies are given below.

The present disclosure encompasses pharmaceutical kits containing neutralizing hPG antibodies (including antibody conjugates) and immune checkpoint inhibitors described herein. This pharmaceutical kit is a package containing a neutralizing hPG antibody and/or an immune checkpoint inhibitor (for example, either in lyophilized form or as an aqueous solution) and one or more of the following:

- a third therapeutic agent, for example, as described below;

a device for administering a neutralizing antibody to hPG and/or an immune checkpoint inhibitor, such as a pen, needle and/or syringe; and

- pharmaceutical grade water or buffer for resuspension of the antibody, if the antibody and/or inhibitor is in lyophilized form.

Each unit dose of the anti-hPG antibody and/or immune checkpoint inhibitor may be individually packaged and the kit may contain one or more unit doses (e.g., two unit doses, three unit doses, four unit doses, five unit doses, eight unit doses, doses, ten single doses or more). In a specific embodiment, each one or more than one unit dose is contained in a syringe or pen.

Effective dosages

Combinations of anti-hPG antibodies and immune checkpoint inhibitors will typically be used in an effective amount to achieve the intended result, for example, in an effective amount to treat cancer in a subject in need thereof. Pharmaceutical compositions containing anti-hPG antibodies and/or immune checkpoint inhibitors may be administered to patients (eg, human subjects) at therapeutically effective dosages.

The term "therapeutically effective dosage" means the amount of active compound or conjugate that elicits the desired biological response in a subject. Such a response includes alleviating the symptoms of the disease or disorder being treated, preventing, inhibiting, or delaying the recurrence of a disease symptom or disease itself, prolonging the life of a subject compared to no treatment, or preventing, inhibiting, or delaying the progression of a disease symptom or disease itself. More specifically, a "therapeutically effective" dosage, as the term is used herein, is an amount that provides a therapeutic benefit. A therapeutically effective dosage is also one at which any toxic or deleterious effects of the agent are outweighed by the therapeutically beneficial effects. In the context of CRC therapy, the term "therapeutic benefit" means any amelioration of cancer, including any one or combination of stopping or slowing the progression of cancer (e.g., from one stage of cancer to the next), stopping or delaying the worsening or worsening of symptoms or signs of cancer, reducing the severity of cancer , inducing cancer remission, inhibiting tumor cell proliferation, tumor size or number of tumors, or decreasing serum levels of PG.

Determining this effective amount is entirely within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein. The toxicity and therapeutic efficacy of a compound or conjugate can be determined by standard pharmaceutical procedures in cell cultures and experimental animals. The effective amount of the present combination or other therapeutic agent to be administered to a subject will depend on the stage, category, and condition of the multiple myeloma, and the characteristics of the subject, such as general health, age, sex, body weight, and drug tolerance. The effective amount of the present combination or other therapeutic agent to be administered will also depend on the route of administration and dosage form. The amount and dosage interval may be adjusted individually to provide plasma levels of the active compound that are sufficient to maintain the desired therapeutic effects.

The amount of combination of anti-hPG antibody and immune checkpoint inhibitor administered will depend on a variety of factors, including the nature and stage of the CRC being treated, the form, route and site of administration, the therapeutic regimen (e.g., whether another therapeutic agent is being used), the age and condition of the individual. the subject being treated, the sensitivity of the patient being treated to anti-hPG antibodies and/or immune checkpoint inhibitors. A suitable dosage can be easily determined by a person skilled in the art. Ultimately, the appropriate dosage to be used will be determined by the attending physician. This dosage may be repeated as often as necessary. If side effects develop, the amount and/or frequency of dosage may be changed or reduced in accordance with normal clinical practice. The correct dosage and treatment regimen can be established by tracking the progress of therapy using conventional techniques known to those skilled in the art.

Effective dosages can initially be estimated from in vitro assays. For example, the initial dose for use in animals may be formulated to achieve a circulating blood or serum concentration of the humanized anti-hPG antibody that is at or above the binding affinity of the antibody to progastrin as measured in vitro. Similarly, the initial dose for use in animals may be formulated to achieve a circulating blood or serum concentration of an immune checkpoint inhibitor that is at or above the binding affinity of that inhibitor for the corresponding immune checkpoint protein as measured in vitro. Calculating dosages to achieve such circulating blood or serum concentrations, taking into account the bioavailability of a particular antibody, is entirely within the ability of those skilled in the art. For guidance, the reader is referred to Fingl & Woodbury, "General Principles" in Goodman and Gilman's The Pharmaceutical Basis of Therapeutics, Chapter 1, latest edition, Pagamonon Press, and to the references cited there.

Initial dosages can be estimated from in vivo data such as animal models. Useful animal models for analyzing the efficacy of compounds in the treatment of CRC are well known in the art. Additionally, animal models of CRC are described in the Examples below. Those of ordinary skill in the art can adapt such information to determine appropriate dosages for human administration.

An effective dose of the combination of an anti-hPG antibody and an immune checkpoint inhibitor as described herein can range from about 0.001 mg/kg to about 75 mg/kg per single (eg, bolus) administration, multiple administrations, or continuous administration, or to achieve a serum concentration of 0 .01-5000 µg/mL serum concentration per single (eg, bolus) administration, multiple administrations, or continuous administration, or any effective range or value within these limits, depending on the condition being treated, route of administration, and age, weight, and condition subject. In a specific embodiment, each dose may vary from about 0.5 μg to about 50 μg per kilogram of body weight, for example, from about 3 μg to about 30 μg per kilogram of body weight.

The amount, frequency, and duration of administration will depend on a variety of factors such as the age, weight, and disease state of the patient. The therapeutic regimen for administration may last for 2 weeks to infinity, for 2 weeks to 6 months, 3 months to 5 years, 6 months to 1 or 2 years, 8 months to 18 months, or the like. Perhaps this therapeutic regimen provides for repeated administration, for example, once a day, twice a day, every two days, three days, five days, one week, two weeks or one month. Repeated administration may be at the same or at a different dose. This introduction can be repeated once, twice, three times, four times, five times, six times, seven times, eight times, nine times, ten times or more. A therapeutically effective amount of the combination of the hPG antibody and the immune checkpoint inhibitor may be administered as a single dose or over the course of a therapeutic regimen, e.g., over a course lasting a week, two weeks, three weeks, one month, three months, six months, one a year or longer.

Therapeutic methods

The ability of the present combinations of hPG antibodies and immune checkpoint inhibitors to block PG-dependent responses, including cell proliferation, and improve response to immunotherapy makes them useful for cancer treatment. Accordingly, one aspect of the present invention thus relates to the present combination of an anti-hPG antibody and an immune checkpoint inhibitor as a drug.

In another aspect, the present disclosure provides methods for treating cancer in a patient in need thereof. Namely, cancers that can be treated with the combination of the invention are cancers that depend on progastrin for growth and/or proliferation. Preferably, the progastrin-dependent cancer is colorectal cancer (CRC). In general, these methods include administering to a patient a therapeutically effective amount of the combination of an anti-hPG antibody and an immune checkpoint inhibitor described herein. In another embodiment, the present disclosure provides the combination of an anti-hPG antibody and an immune checkpoint inhibitor described herein for use in the treatment of CRC.

The "subject" or "patient" to which the present combination of an anti-hPG antibody and an immune checkpoint inhibitor is administered is preferably a mammal, such as a non-primate (e.g., bovine, pig, horse, cat, dog, rat, etc.) or a primate (for example, a monkey or a human). The subject or patient may be a human, such as an adult patient or a pediatric patient.

Suitable patients for anti-hPG antibody/immune checkpoint inhibitor combination therapy are those diagnosed with CRC. CRC can be of any type and at any clinical stage of manifestation. Suitable subjects include patients with CRC tumors (operable or inoperable), patients whose tumors have been surgically removed or resected, patients with a CRC tumor containing cells carrying a mutation in an oncogene such as, for example, RAS or APS, patients who have received or are receiving another anti-CRC therapy in combination with or in addition to humanized anti-hPG antibody therapy. Other anti-CRC therapies include chemotherapy, radiation therapy, surgical resection, and treatment with one or more therapeutic antibodies, as detailed below.

In other embodiments, combinations of anti-hPG antibodies and immune checkpoint inhibitors as disclosed herein are administered in compositions to a subject in need of prevention of metastatic colorectal cancer in a therapeutically effective amount. Such subjects include, but are not limited to, those identified as having primary colorectal cancer but in whom the cancer is not known to have spread to distant tissues or organs. In some embodiments of these methods, the anti-hPG antibodies are humanized anti-hPG antibodies.

In other embodiments, combinations of anti-hPG antibodies and immune checkpoint inhibitors as disclosed herein are formulated in a therapeutically effective amount to a subject in need of preventing recurrence of metastatic colorectal cancer. Such subjects include, but are not limited to, subjects previously treated for primary or metastatic colorectal cancer, where such cancer appears to have disappeared after treatment. In some embodiments of these methods, the anti-hPG antibodies are humanized anti-hPG antibodies.

In other embodiments, combinations of hPG antibodies and immune checkpoint inhibitors as disclosed herein are formulated in a therapeutically effective amount to a subject in need of inhibition of colorectal cancer stem cell growth. Such subjects include, but are not limited to, those having colorectal cancer whose growth or metastasis is at least in part attributed to the presence of cancer stem cells within it. In other embodiments, methods are provided for preventing or inhibiting the growth of colorectal cancer stem cells by contacting such stem cells with an effective amount of an anti-PG antibody/immune checkpoint inhibitor composition to prevent or inhibit the growth of such cells. Such methods can be carried out in vitro or in vivo. In some embodiments of these methods, the anti-hPG antibodies are humanized anti-hPG antibodies.

Anti-hPG antibody/immune checkpoint inhibitor combination therapy can be combined with or supplemented with one or more other treatments. Other treatments include, without limitation, chemotherapy, radiation therapy, surgical resection, and antibody therapy, as described herein.

Combination therapy with an anti-hPG antibody/immune checkpoint inhibitor may be adjunctive to other treatments, including surgical resection.

Combination therapy, as provided herein, involves administering to a patient at least two agents, the first of which is an anti-hPG antibody/immune checkpoint inhibitor combination, and the second of which is another therapeutic agent. According to this embodiment, the invention relates to an anti-hPG antibody/immune checkpoint inhibitor combination as described above for the treatment of CRC, wherein said combination is administered with said other therapeutic agent. The combination of anti-hPG antibody/immune checkpoint inhibitor and other therapeutic agent may be administered simultaneously, sequentially or separately.

The term "therapeutic agent" embraces biological agents such as antibody, peptide, protein, enzyme, and chemotherapeutic agents. Therapeutic also encompasses cell-binding agent (CBA) immunoconjugates and chemical compounds such as antibody-drug conjugates (ADCs). The drug in the conjugates may be a cytotoxic agent, such as the cytotoxic agent described herein.

The combination of an hPG antibody/immune checkpoint inhibitor and another therapeutic agent is said to be administered sequentially, as the term is used herein, if they are administered to the patient on the same day, e.g., during the same visit. patient. Sequential administration may occur with a time separation of 1, 2, 3, 4, 5, 6, 7 or 8 hours. In contrast, the combination of this disclosure and the other therapeutic agent are said to be administered separately if they are administered to a patient on different days, for example, the combination of this disclosure and the other therapeutic agent may be administered with 1-day, 2-day or 3- daily, one-weekly, 2-weekly or monthly intervals. In the methods of the present disclosure, administration of the combination of this disclosure may precede or follow administration of the other therapeutic agent.

As a non-limiting example, the present combination and another therapeutic agent may be administered concomitantly over a period of time, followed by a second period of time in which administration of the humanized anti-hPG antibody of this disclosure and the other therapeutic agent alternates.

Combination therapies of the present disclosure may result in more than an additive or synergistic effect conferring a therapeutic benefit when neither the anti-hPG antibody/immune checkpoint inhibitor combination nor the other therapeutic agent is administered in an amount that alone is therapeutically effective. Thus, such agents may be administered in smaller amounts, reducing the possibility and/or severity of adverse effects.

In a preferred embodiment, the other therapeutic agent is a chemotherapeutic agent. The term "chemotherapeutic agent", as used herein, refers to a substance that, when administered to a subject, treats or prevents the development of cancer in that subject's body.

Chemotherapeutic agents include, but are not limited to, alkylating agents, antimetabolites, antitumor antibiotics, mitosis inhibitors, chromatin function inhibitors, antiangiogenic agents, antiestrogens, antiandrogens, or immunomodulators.

The term "alkylating agent" refers to any substance that can crosslink or alkylate any molecule, preferably nucleic acid (eg DNA) in a cell. Examples of alkylating agents include nitrogen mustard such as mexporethamine, chlorambucol, melphalen, hydrochloride, pipobromen, prednimustine, disodium phosphate or estramustine; oxazophorins such as cyclophosphamide, altretamine, trofosfamide, sulfophosphamide or ifosfamide; aziridines or imine-ethylenes such as thiotepa, triethyleneamine or altetramine; a nitrosourea such as carmustine, streptozocin, fotemustine or lomustine; alkylsulfonates such as busulfan, treosulfan or improsulfan; triazenes such as dicarbazine; or platinum complexes such as cisplatin, oxaliplatin and carboplatin.

The term "antimetabolites" refers to substances that block the growth and/or metabolism of cells by interfering with certain activities, usually DNA synthesis. Examples of antimetabolites include methotrexate, 5-fluorouracil, floxuridine, 5-fluorodeoxyuridine, capecitabine, cytarabine, fludarabine, cytosine arabinoside, 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), chlordeoxyadenosine, 5-azacytidine, gemcitabine, cladribine , deoxycoformycin and pentostatin.

The term "antineoplastic antibiotics" refers to compounds that can prevent or inhibit the synthesis of DNA, RNA and/or protein. Examples of anticancer antibiotics include doxorubicin, daunorubicin, idarubicin, valrubicin, mitoxantrone, dactinomycin, mithramycin, plicamycin, mitomycin C, bleomycin, and procarbazine.

"Mitosis inhibitors" prevent the normal development of the cell cycle and mitosis. In general, microtubule inhibitors or taxoids such as paclitaxel and docetaxel are able to inhibit mitosis. Vinca alkaloids such as vinblastine, vincristine, vindesine, and vinorelbine are also able to inhibit mitosis.

The term "inhibitors of chromatin function" or "topoisomerase inhibitors" refers to substances that inhibit the normal function of proteins that model chromatin, such as topoisomerase I or topoisomerase II. Examples of inhibitors of chromatin function include, for topoisomerase I, camptothecin and its derivatives such as topotecan or irinotecan, and, for topoisomerase II, etoposide, etoposide phosphate and teniposide.

The term "anti-angiogenic agent" refers to any drug, compound, substance or agent that inhibits the growth of blood vessels. Representative anti-angiogenic agents include, but are by no means limited to, the following: razoxin, marimastat, batimastat, prinomastat, tanomastat, ilomastat, CGS-27023A, halofuginone, COL-3, neovastat, BMS-275291, thalidomide, CDC 501, DMXAA, L -651582, squalamine, endostatin, SU5416, SU6668, interferon-alpha, EMD121974, interleukin-12, IM862, angiostatin and vitaxin.

"Antiestrogen" or "anti-estrogen" refers to any substance that reduces, antagonizes or inhibits the action of estrogen. Examples of anti-extrogen agents are tamoxifen, toremifene, raloxifene, droloxifene, iodoxifene, anastrozole, letrozole and exemestane.

"Antiandrogens" or "antiandrogens" refers to any substance that reduces, antagonizes or inhibits the action of an androgen. Examples of antiandrogens are flutamide, nilutamide, bicalutamide, spironolactone, cyproterone acetate, finasteride and cimitidine.

"Immunomodulators" are substances that stimulate the immune system.

Examples of immunomodulators include interferon, interleukin such as aldesleukin, OST-43, denileukin diphlitox and interleukin-2, tumor necrosis factors such as tazonermine or other immunomodulators such as lentinan, sisofiran, roquinimex, pidotimod, pegademase, thymopentin, poly I: C or levamisole in combination with 5-fluorouracil.

des Enseignants de Chimie

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antitumouraux et perspectives dans le traitement des cancers, edition TEC & DOC, 2003.For further details, one skilled in the art could refer to the manual edited by "Association

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vol. 6,

antitumouraux et perspectives dans le traitement des cancers, edition TEC & DOC, 2003.

As chemical agents or cytotoxic agents, all kinase inhibitors can also be mentioned, such as, for example, gefitinib or erlotinib.

In general, examples of suitable chemotherapeutic agents include the following: 1-dehydrotestosterone, 5-fluorouracil decarbazine, 6-mercaptopurine, 6-thioguanine, actinomycin D, adriamycin, aldesleukin, alkylating agents, sodium allopurinol, altretamine, amifostine, anastrozole, anthramycin (AMC) , antimitotics, cis-dichlorodiamine platinum (II) (DDP) cisplatin), diaminodichloroplatinum, anthracyclines, antibiotics, antimetabolites, live BCG (intravesical), betamethasone sodium phosphate and betamethasone acetate, bicalutamide, bleomycin sulfate, busulfan, calcium leucovorin, calicheamicin, capecitabine, carboplatin, lomustine (CCNU), carmustine (BSNU), chlorambucil, cisplatin, cladribine, colchicine, conjugated estrogens, cyclophosphamide, cyclotosfamide, cytarabine, cytochalasin B, cytoxan, dacarbazine, dactinomycin, dactinomycin (formerly actinomycin), daunirubicin HCI, daunorubicin citrate, denileukin-diftitox, dexrazoxane, dibromomannite, dihydroxyanthracindione, docetaxel, dolasetron mesylate, dox orubicin HCI, dronabinol, L-asparaginase E, coli, emetine, epoetin-a, Erwinia L-asparaginase, esterified estrogens, estradiol, estramustine sodium phosphate, ethidium bromide, ethinyl estradiol, etidronate, etoposide citrororum factor, etoposide phosphate, filgrastim, floxuridine, fluconazole, fludarabine phosphate, fluorouracil, flutamide, folinic acid, gemcitabine HCI, glucocorticoids, goserelin acetate, gramicidin D, granisetron HCI, hydroxyurea, idarubicin HCI, ifosfamide, interferon alfa-2a, irinotecan HCI, letrozole, leucovorin calcium, leuprolide acetate, levamisole HCI, lidocaine, lomustine, maytansinoid, meclorethamine HCI, medroxyprogesterone acetate, megestrol acetate, melphalan HCI, mercaptipurine, mesna, methotrexate, methyltestosterone, mithramycin, mitomycin C, mitotane, mitoxantrone, nilutamide, octreotide acetate, ondansetron HCI, oxalitaxaliplatin, paclitiatriplatin pamindronate, pentostatin, pilocarbine HCI, plimycin, polyfeprosan 20 with carmustine implant, sodium porfimer, pro caine, procarbazine HCI, propranolol, rituximab, sargramostin, streptozocin, tamoxifen, taxol, tegafur, teniposide, tenoposide, testolactone, tetracaine, thiotepa, chlorambucil, thioguanine, thiotepa, topotecan HCI, toremifene citrate, trastuzumab, tretinoin, valrubicin, vinblastine sulfate, vincristine sulfate and vinorelbine tartrate, but not limited to.

The anti-hPG antibody/immune checkpoint inhibitor combinations disclosed herein may be administered to a patient in need of colorectal cancer treatment receiving a combination of chemotherapeutic agents. Typical combinations of chemotherapeutic agents include 5-fluorouracil (5FU) in combination with leucovorin (folinic acid or LV); capecitabine in combination with uracil (UFT) and leucovorin; tegafur in combination with uracil (UFT) and leucovorin; oxaliplatin in combination with 5FU or in combination with capecitabine; irinotecan in combination with capecitabine; mitomycin C in combination with 5FU, irinotecan, or capecitabine. It is also possible to use other combinations of chemotherapeutic agents disclosed in this document.

As is known in the relevant field, chemotherapeutic regimens against colorectal cancer have been standardized in clinical trials using combinations of different chemotherapeutic agents. Such regimens are commonly known by abbreviations and include 5FU Mayo, 5FU Roswell Park, LVFU2, FOLFOX, FOLFOX4, FOLFOX6, bFOL, FUFOX, FOLFIRI, IFL, XELOX, CAPOX, XELIRI, CAPIRI, FOLFOXIRI. See, for example, Chau, I., et al., 2009, Br. J. Cancer 100:1704-19 and Field, K., et al., 2007, World J. Gastroenterol. 13:3806-15, both of which are incorporated by reference.

Anti-hPG antibody/immune checkpoint inhibitor combinations can also be combined with other therapeutic antibodies. Accordingly, anti-hPG antibody/immune checkpoint inhibitor combination therapy can be combined with or administered in addition to another monoclonal antibody, such as, for example, but not limited to, an anti-EGFR (EGF receptor (epidermal growth factor)) monoclonal antibody or a monoclonal antibody to VEGF (vascular endothelial growth factor). Specific examples of anti-EGFR antibodies include cetuximab and panitumumab. A specific example of an anti-VEGF antibody is bevacizumab.

According to this embodiment, the invention relates to an anti-hPG antibody/immune checkpoint inhibitor combination as described above for the treatment of CRC, wherein said combination is administered with a chemotherapeutic agent. The combination of anti-hPG antibody/immune checkpoint inhibitor and chemotherapeutic agent may be administered simultaneously, sequentially or separately.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as is generally understood by a person skilled in the art to which this invention belongs.

DESCRIPTION OF GRAPHICS

Fig. 1: BALB/cAnNRj mice were implanted with a colorectal carcinoma cell line - CT26.WT - and treated with one of a control antibody, an anti-PD-1 antibody, an anti-PG antibody, or a combination of an anti-PD-1 antibody and an anti-PG antibody: (A) Kaplan survival plot -Meyer; (B) median survival time (in days).

Fig. 2: qPCR (quantitative polymerase chain reaction) analysis of IFNγ expression in BALB/cAnNRj mice xenotransplanted with a colorectal carcinoma cell line - CT26.WT - and treated with one of a control antibody, an anti-PD-1 antibody, an anti-PG antibody, or a combination of anti- PD-1 and anti-PG antibodies.

EXAMPLES

Example 1

BALB/cAnNRj mice were implanted subcutaneously in the flank with 0.5 M CT26.WT cells (ATCC-CRL-2638) per mouse on day 1. Mice were randomized into individual treatment groups (n equal to 15 mice per group) as indicated:

Control group: 15 mice injected intraperitoneally with NaCI (5 ml/kg) plus rat IgG2A (10 mg/kg - 5 ml/kg) twice a week.

Anti-PG group: 15 mice injected intraperitoneally with NaCl (5 ml/kg) plus anti-PG antibody (30 mg/kg - 5 ml/kg) twice a week.

Anti-PP-1 group: 15 mice injected intraperitoneally with NaCI (5 ml/kg) plus anti-PD1 antibody (10 mg/kg - 5 ml/kg) twice a week.

Group 4 anti-PG+ anti-PP1: 15 mice injected intraperitoneally with anti-PG antibody (30 mg/kg-5 ml/kg) plus anti-PD1 antibody (10 mg/kg-5 ml/kg) twice a week.

The anti-PG antibody is Mab8, whose CDR, V _H and V _L are described in Table 5.

The anti-PD-1 antibody is the monoclonal antibody 29F.1A12 (obtained from BioXCell, 10 Technology Dr, Suite 2B West Lebanon, NH 03784, USA).

Animals were observed and weighed twice a week at the same time that tumor volumes were measured using a digital caliper. Tumor size was calculated using the following formula: V=length×width ^2/2 where length represents the largest diameter of the tumor and width represents the perpendicular diameter of the tumor. Animals were sacrificed either at the end of the study or when tumors reached a volume of 1500 mm ³ , if tumor ulceration was observed, if body weight loss exceeded 20%, or if a significant deterioration in the health of the mouse was observed. Sacrifice by displacement of the cervical vertebrae was performed after gas anesthesia (isoflurane).

On FIG. 1 shows Kaplan-Meier survival for each group. The log-rank test (Mantel-Cox) demonstrates a statistical increase in survival between the combination group (anti-PG + anti-PDI) and single treatment (anti-PG or anti-PDI) with an increase in median survival from 15 to 20 days (plus 33%, p less than 0.0001), confirming the superior activity of this combination compared to each of the monotherapies.

Example 2

BALB/cAnNRj mice were implanted subcutaneously in the flank with 0.5 M CT26.WT cells (ATCC-CRL-2638) per mouse on day 1. Mice were randomized into individual treatment groups (n equal to 15 mice per group) as indicated:

Control group: 15 mice injected intraperitoneally with NaCI (5 ml/kg) plus rat IgG2A (10 mg/kg - 5 ml/kg) twice a week.

Anti-PG group: 15 mice injected intraperitoneally with NaCl (5 ml/kg) plus anti-PG antibody (30 mg/kg - 5 ml/kg) twice a week.

Anti-PP-1 group: 15 mice injected intraperitoneally with NaCI (5 ml/kg) plus anti-PD1 antibody (10 mg/kg - 5 ml/kg) twice a week.

Group 4 anti-PG + anti-PD1: 15 mice injected intraperitoneally with anti-PG antibody (30 mg/kg-5 ml/kg) plus anti-PD1 antibody (10 mg/kg-5 ml/kg) twice a week.

One week after the start of treatment, 4 mice per group were sacrificed and the tumor was isolated for RNA extraction and qPCR to measure interferon-gamma (IFNγ) expression. Indeed, several publications have shown that CTLA-4 and PD-1 inhibitors, as well as other immune checkpoint blockade therapies, lead to an increase in IFNγ production. On FIG. 2 shows that the anti-PD1 antibody induces IFNγ production in the present mouse model as expected (3.2 magnification). More interestingly, a greater increase in IFNγ production was observed using anti-PG plus anti-PD1 antibody combination therapy (fold increase of 6), confirming the superior activity of this combination compared to either of the monotherapies.

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SEQUENCE LIST

<110> PROGASTRINE ET CANCERS S.a r.l.

<120> ANTI-PROGASTRIN ANTIBODY COMBINATION THERAPY

AND IMMUNOTHERAPIES FOR CANCER TREATMENT

<130> B375304PCTD37823

<150> US 62/594,755

<151> 20171205

<160> 78

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Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

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Gly Glu Ile Asn Pro Ser Asn Gly Gly Thr Asn Phe Asn Glu Lys Phe

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Lys Ser Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Thr Ala Tyr

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Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 53

<211> 115

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 53

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Ser Tyr

20 25 30

Trp Val His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Glu Trp Met

35 40 45

Gly Gly Phe Tyr Pro Gly Asn Ser Asp Ser Arg Tyr Ser Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Arg Asp Ser Pro Gln Tyr Trp Gly Gln Gly Thr Leu Val Thr

100 105 110

Val Ser Ser

115

<210> 54

<211> 112

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 54

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Ile Val His Ser

20 25 30

Asn Gly Asn Thr Tyr Leu Glu Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Gly

85 90 95

Ser His Val Pro Phe Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 55

<211> 118

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 55

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Ser Ser Ser

20 25 30

Page 17seq listing

Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Phe Leu Pro Gly Ser Gly Ser Thr Asp Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Asp Gly Asn Tyr Asp Trp Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 56

<211> 112

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 56

Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Val His Ser

20 25 30

Ser Gly Val Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Lys Val Ser Asn Arg Phe Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ser Gln Ser

85 90 95

Thr His Val Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 57

<211> 117

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 57

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Asn Gly Gly Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Gly Tyr Tyr Pro Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 58

<211> 117

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 58

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Glu Trp Met

35 40 45

Gly Ile Ile Asn Pro Ser Asn Gly Gly Thr Ser Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Gly Tyr Tyr Pro Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 59

<211> 117

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 59

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr

20 25 30

Tyr Met Tyr Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Glu Ile Asn Pro Ser Asn Gly Gly Thr Asn Tyr Ala Gln Lys Phe

50 55 60

Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Arg Gly Gly Tyr Tyr Pro Phe Asp Tyr Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 60

<211> 112

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 60

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Tyr Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Leu Val Ser Asn Arg Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Ser Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 61

<211> 112

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 61

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Leu Val Ser Asn Arg Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Ser Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 62

<211> 112

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 62

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Tyr Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Leu Val Ser Glu Arg Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Ser Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

<210> 63

<211> 121

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 63

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Phe Ser Gly Tyr Thr Tyr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Val Asn Tyr Gly Asp Ser Tyr His Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 64

<211> 121

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 64

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Phe Ser Gly Tyr Thr Tyr Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Val Asn Tyr Gly Asp Ser Tyr His Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 65

<211> 121

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 65

Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln

1 5 10 15

Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Ser Ile Thr Ser Asp

20 25 30

Tyr Ala Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp

35 40 45

Ile Gly Tyr Ile Ser Phe Ser Gly Tyr Thr Ser Tyr Asn Pro Ser Leu

50 55 60

Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser

65 70 75 80

Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Glu Val Asn Tyr Gly Asp Ser Tyr His Phe Asp Tyr Trp Gly

100 105 110

Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 66

<211> 115

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 66

Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala

1 5 10 15

Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Arg Thr Tyr Thr

20 25 30

Ile Glu Trp His Gln Gln Gln Pro Glu Lys Gly Pro Arg Tyr Leu Met

35 40 45

Lys Val Lys Lys Asp Gly Ser His Ser Lys Gly Asp Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp

85 90 95

Ala Ile Lys Gly Gln Ser Val Phe Val Phe Gly Gly Gly Thr Lys Val

100 105 110

Glu Ile Lys

115

<210> 67

<211> 115

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 67

Page 24seq listing

Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala

1 5 10 15

Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Arg Thr Tyr Thr

20 25 30

Ile Ala Trp His Gln Gln Gln Pro Glu Lys Gly Pro Arg Tyr Leu Met

35 40 45

Lys Val Lys Lys Asp Gly Ser His Ser Lys Gly Asp Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp

85 90 95

Ala Ile Lys Gly Gln Ser Val Phe Val Phe Gly Gly Gly Thr Lys Val

100 105 110

Glu Ile Lys

115

<210> 68

<211> 115

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 68

Gln Leu Val Leu Thr Gln Ser Pro Ser Ala Ser Ala Ser Leu Gly Ala

1 5 10 15

Ser Val Lys Leu Thr Cys Thr Leu Ser Ser Gln His Arg Thr Tyr Thr

20 25 30

Ile Glu Trp His Gln Gln Gln Pro Glu Lys Gly Pro Arg Tyr Leu Met

35 40 45

Glu Val Lys Lys Asp Gly Ser His Ser Lys Gly Asp Gly Ile Pro Asp

50 55 60

Arg Phe Ser Gly Ser Ser Ser Gly Ala Glu Arg Tyr Leu Thr Ile Ser

65 70 75 80

Ser Leu Gln Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Gly Val Gly Asp

85 90 95

Ala Ile Lys Gly Gln Ser Val Phe Val Phe Gly Gly Gly Thr Lys Val

100 105 110

Glu Ile Lys

115

<210> 69

<211> 117

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 69

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Thr Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ser Ser Ile Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Gln Gly Asn Tyr Ser Leu Asp Phe Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 70

<211> 112

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 70

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Arg His Thr

20 25 30

Lys Gly Ile Thr Phe Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 71

<211> 117

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 71

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Thr Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Gln Gly Asn Tyr Ser Leu Asp Phe Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser

115

<210> 72

<211> 112

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 72

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Arg His Thr

20 25 30

Lys Gly Ile Thr Phe Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 73

<211> 447

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 73

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Thr Tyr

20 25 30

Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Thr Ile Ser Ser Gly Gly Thr Tyr Thr Tyr Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Thr Gln Gly Asn Tyr Ser Leu Asp Phe Trp Gly Gln Gly Thr Thr

100 105 110

Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu

115 120 125

Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys

130 135 140

Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser

145 150 155 160

Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser

165 170 175

Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser

180 185 190

Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn

195 200 205

Thr Lys Val Asp Lys Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His

210 215 220

Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val

225 230 235 240

Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr

245 250 255

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu

260 265 270

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys

275 280 285

Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser

290 295 300

Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys

305 310 315 320

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile

325 330 335

Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro

340 345 350

Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu

355 360 365

Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn

370 375 380

Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser

385 390 395 400

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg

405 410 415

Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu

420 425 430

His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 74

<211> 219

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 74

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Lys Ser Leu Arg His Thr

20 25 30

Lys Gly Ile Thr Phe Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Gln Met Ser Asn Leu Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Ala Gln Asn

85 90 95

Leu Glu Leu Pro Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210> 75

<211> 114

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 75

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asn Ile Asn Thr Phe Gly Asp Arg Thr Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Gly Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser

<210> 76

<211> 114

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 76

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Tyr

20 25 30

Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Ser Ile Asn Thr Phe Gly Asp Arg Thr Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Gly Thr Gly Thr Tyr Trp Gly Gln Gly Thr Leu Val Thr Val

100 105 110

Ser Ser

<210> 77

<211> 112

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 77

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Leu Val Ser Asn Arg Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

<210> 78

<211> 112

<212> PRT

<213> Artificial

<220>

<223> synthetic peptide

<400> 78

Asp Val Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Leu Gly

1 5 10 15

Gln Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp Ser

20 25 30

Asp Gly Lys Thr Tyr Leu Asn Trp Phe Gln Gln Arg Pro Gly Gln Ser

35 40 45

Pro Arg Arg Leu Ile Tyr Leu Val Ser Lys Arg Asp Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Trp Gln Gly

85 90 95

Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys

100 105 110

38

<---

Claims (14)

1. Combination for the treatment of cancer, containing a monoclonal antibody to progastrin (anti-hPG) and an inhibitor of the immune checkpoint, where the specified antibody to hPG contains 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 of SEQ ID NOS 31, 32 and 33, respectively, and where the specified immune checkpoint inhibitor is an antibody to PD1. 2. The combination of claim 1 wherein said anti-hPG antibody is selected from single chain antibodies, camelized antibodies, chimeric antibodies, IgA1 antibodies, IgA2 antibodies, IgD antibodies, IgE antibodies, IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, IgG4 antibodies, and antibodies IgM. 3. A combination according to claim 1 or 2, wherein said anti-hPG antibody is an antibody to the C-terminus of progastrin. 4. Combination according to any one of paragraphs. 1-3, wherein said anti-hPG antibody is a neutralizing antibody. 5. Combination according to any one of paragraphs. 1-4, wherein said anti-hPG antibody is a monoclonal antibody comprising a heavy chain variable region having the amino acid sequence of SEQ ID NO 49 and a light chain variable region having the amino acid sequence of SEQ ID NO 50. 6. Combination according to any one of paragraphs. 1-4, wherein said anti-hPG antibody is a humanized antibody. 7. The combination of claim 6 wherein said anti-hPG antibody is a humanized antibody comprising a heavy chain variable region of an amino acid sequence selected from SEQ ID NOs 69 and 71 and a light chain variable region of an amino acid sequence selected from SEQ ID NOs 70 and 72, wherein said antibody also contains light chain and heavy chain constant regions derived from a human antibody. 8. The combination according to claim 6 or 7, wherein said anti-hPG 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, wherein said antibody also comprises the light chain and heavy chain constant regions. chains derived from a human antibody. 9. Combination according to any one of paragraphs. 6-8, wherein said anti-hPG 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. 10. Combination according to any one of paragraphs. 1-9, wherein said anti-PD1 antibody is pembrolizumab, nivolumab, cemiplimab, or pidilizumab. 11. The combination according to any one of paragraphs. 1-10 for use as a medicine. 12. The combination according to any one of paragraphs. 1-10 for use in the treatment of colorectal cancer. 13. Pharmaceutical composition for the treatment of cancer, containing a combination according to any one of paragraphs. 1-10, a pharmaceutically acceptable carrier and/or excipient. 14. Pharmaceutical composition according to claim 13 for simultaneous, separate or sequential use.

Images