EA041823B1 - MONOCLONAL ANTIBODIES TO PROGASTRIN AND THEIR USE - Google Patents

Description

1. Cross-references to related applications

This application claims priority under 35 U.S.C. § 119(e) of Provisional Application No. 61/252625, filed October 16, 2009, the contents of which are hereby incorporated by reference in their entirety.

2. Statement regarding federally funded research

Absent.

3. Parties to a joint research agreement

None.

4. Links to sequence listings, tables or computer programs

This application includes a sequence listing.

5. Technical field to which the invention belongs

This application is directed, inter alia, to monoclonal antibodies to progastrin, compositions and methods for making such antibodies, as well as methods for using such antibodies, for example, in the diagnosis and/or treatment of colorectal cancer.

6. Background of the invention

Colorectal cancer (CRC) is a major health issue, affecting more than 1,000,000 people each year and causing over 500,000 deaths each year. CRC is the second leading cause of death from cancer. In the United States alone, 147,000 new cases and over 49,900 deaths were reported in 2009 due to CRC. There are three forms of CRC: sporadic CRC; hereditary non-polyposis colon cancer (HNPCC), which is caused by embryonic mutations in DNA error repair genes, and familial adenomatous polyposis (FAP), which is caused by embryonic mutations in the APC gene. Sporadic CRC accounts for approximately 85% of cases, while HNPCC accounts for approximately 5% and FAP accounts for approximately 1% (Heyer et al., 1999, Oncogene, 18:5325-5333).

Clinical management of CRC typically involves surgical removal of tumors, which is often followed by chemotherapy. Currently, approximately 50% of patients with CRC die within five years of being diagnosed. The lack of reliable screening tests and the ineffectiveness of currently available therapies are major causes of the high mortality rate. There is an urgent need for new clinical approaches for the diagnosis of CRC, as well as methods of treatment that are effective against colorectal cancer tumors, which have minimal side effects on other healthy tissue.

7. Summary of the Invention

This application provides compositions and methods useful for diagnosing and/or treating colorectal cancer (CRC) in animals, including humans. This application is based on a variety of inventions, in part on the inventors' discovery of monoclonal antibodies that specifically bind to progastrin (PG), such as human progastrin (hPG), a polypeptide that is produced by CRC tumor cells and that exhibits anti-proliferative properties in in vitro models of CRC.

Progastrin is produced by colorectal tumor cells and is thought to stimulate the proliferation of these cells by triggering a signal transduction pathway that blocks the normal processes of cell differentiation, including those that lead to cell death. Depletion of transcripts 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 models, reducing tumor cell proliferation. Without wishing to be bound by any theory of this process, through PG binding, anti-hPG antibodies are speculated to block or inhibit the ability of progastrin to interact with its signal partner(s). This in turn inhibits signal transduction pathways in colorectal cancer cells, which would otherwise lead to proliferation.

Accordingly, in one aspect, the present disclosure provides monoclonal antibodies that specifically bind to PG, such as hPG, but not other gastrin gene products. With reference to FIG. 1, the gastrin gene is translated into a 101 amino acid polypeptide called pre-progastrin, which contains a signal sequence (underlined) that, when cleaved off, gives rise to progastrin, an 80 amino acid polypeptide. Progastrin, in turn, is cleaved to form a 34 amino acid product corresponding to progastrin residues 38-71, which is then extended at its carboxyterminal end by a glycine residue to form glycine-extended G34 (G34-Gly). The by-product of the cleavage is a 5 amino acid peptide called the C-terminal flanking peptide, or CTFP, which includes progastrin residues 75-80. G34-Gly is then further cleaved to form a 17-residue polypeptide that corresponds in sequence to residues 55-71 of progastrin and is referred to as G17-Gly. Removal of the C-terminal glycines G34-Gly and G17-Gly followed by C-terminal amidation provides G34 and G17, respectively, both of which are C-terminally amidated. Thus, while the first 37 progastrin residues

- 1 041823 are unique to him (i.e. not present in his processing products such as G34,

G34-Gly, G17, G17-G1, or CFTP), residues 38-80 are also present in the post-translational products of the gastrin gene.

Applicants have found that while anti-PG monoclonal antibodies can be generated according to methods known to one of skill in the art, the choice of antigen is important. Not all antigens that are derived from hPG stimulate the formation of monoclonal antibodies that specifically bind to hPG under physiological conditions. As described below, various antigens used to stimulate the formation of polyclonal antibodies to hPG, such as recombinant full-length human progastrin (see, for example, Singh WO 08/076454) and peptides corresponding to the last ten amino acids at the C-terminal end of hPG (see ., for example, Hollande WO 07/135542), do not cause the production of anti-hPG monoclonal antibodies. Applicants have, however, found that antigenic N- and C-terminal sequences within the hPG sequence can be used to generate monoclonal antibodies that specifically bind to hPG. Quite surprisingly, Applicants have found that it is not necessary to limit antigenic sequences to strings of PG sequences that are unique to them in order to obtain monoclonal antibodies that specifically bind to PG and not to other products that are derived from the gastrin gene. Peptide antigens having sequences in common with other gastrin gene products, such as G17, G34 and CTFP, allow the production of monoclonal antibodies that not only bind to hPG, but also bind specifically to it.

Applicants have generated monoclonal antibodies using antigens that are derived from other regions of the hPG molecule. Monoclonal anti-PG antibodies obtained using a peptide antigen having a sequence corresponding to the N-terminal region of hPG and/or one that binds to the N-terminal region of hPG are referred to in this application as N-terminal anti-hPG monoclonal antibodies. A specific example of an antigenic region that can be used to construct an immunogen useful in making an N-terminal anti-PG monoclonal antibody is hPG residues 1-14: SWKPRSQQPDAPLG (SEQ ID NO: 25). Exemplary immunogens, including this immunogen, that are useful in making an N-terminal anti-PG monoclonal antibody are described in Table 1. 1A and in the examples section.

Monoclonal anti-PG antibodies obtained using a peptide antigen having a sequence corresponding to the C-terminal region of hPG and/or one that binds to the C-terminal region of hPG are referred to in this application as C-terminal anti-hPG monoclonal antibodies. A specific example of an antigenic site that can be used to construct an immunogen useful in making a C-terminal anti-PG monoclonal antibody corresponds to residues 55-80 hPG: QGPWLEEEEEAYGWMDFGRRSAEDEN (SEQ ID NO: 27). Exemplary immunogens, including this antigen, that are useful in making a C-terminal anti-PG monoclonal antibody are described in Table 1. 1B and in the examples section.

For some applications, it is desirable to have anti-hPG monoclonal antibodies with high affinity for hPG. For some applications, such as therapeutic applications, an affinity that is at least about 100 nM is desirable, although antibodies that have high affinities, such as affinities that are at least about 90, 80, 70, 60 , 50, 40, 30, 25, 20, 15, 10, 7, 6, 5, 4, 3, 2, 1, 0.1, 0.01 nM or even more may be desirable. Various exemplary specific anti-PG monoclonal antibodies disclosed in this application exhibit affinities that range from 10 ^-6 to 10 ^-12 M (see Table 6). An anti-PG monoclonal antibody having an affinity particularly suitable for the particular application desired can be readily selected from among these, or prepared or constructed using various immunogens, complementarity determining region (CDR) sequences, V _H variable heavy chain sequences, and variable light chain V _L and the methods described in this application. The affinity of any particular anti-PG monoclonal antibody can be determined using techniques well known in the art or described herein, such as, for example, ELISA, isothermal titration calorimetry (ITC), BIAcore, or fluorescence polarization analysis.

hPG is a relatively small polypeptide that is only 80 amino acids in length. It can be expected that any monoclonal antibody that specifically binds to hPG with a relatively high affinity (eg, one that is approximately 10 nM) will interfere with the ability of PG to interact with its signal partner(s) and as a result inhibit CRC proliferation. cells. However, applicants have found that not all anti-PG monoclonal antibodies are neutralized (ie, not all monoclonal antibodies that bind to PG interfere with or inhibit its biological signaling activity). Furthermore, as will be discussed in more detail in the Examples section, Applicants have found that certain anti-PG monoclonal antibodies, despite showing high specificity and high affinity for PG, do not neutralize PG. For example, anti-hPG Mab 14 binds hPG to a K _D that is approximately 6 pM, but does not inhibit the growth of CRC cells in vitro, as detailed in the Examples section below. While non-neutralizing monoclonal antibodies that specifically bind to hPG are useful for diagnostic purposes, those anti-hPG monoclonal antibodies that neutralize PG are particularly useful for therapeutic applications in the treatment of CRC.

As used herein, a neutralizing anti-hPG monoclonal antibody is an anti-hPG monoclonal antibody that provides a statistically significant reduction in the number of live CRC cells in a test sample treated with an anti-hPG monoclonal antibody compared to a control sample treated with using a non-specific monoclonal antibody. A specific assay to evaluate the ability of any particular anti-hPG monoclonal antibody to neutralize hPG is described in the Detailed Description section below. Those anti-hPG monoclonal antibodies that exhibit at least a 50% reduction in live cells in this assay are expected to be particularly useful in the treatment of CRC, although anti-hPG monoclonal antibodies exhibiting lower levels of neutralizing activity, e.g. a statistically significant reduction of 40%, 30%, 20%, 15% or even 10% of the number of living cells, in this analysis is assumed to provide therapeutic benefits.

Accordingly, in some embodiments, the anti-PG monoclonal antibodies are anti-PG neutralizing monoclonal antibodies. The ability of an anti-PG monoclonal antibody to neutralize PG was found to be independent of epitope. As presented in the Examples section, both N-terminal and C-terminal anti-PG antibodies have neutralizing activity. Thus, in some embodiments, the anti-PG neutralizing monoclonal antibodies are N-terminal neutralizing antibodies, in other embodiments, the anti-PG monoclonal antibodies are C-terminal neutralizing antibodies.

Epitope mapping has revealed that N-terminal anti-PG monoclonal antibodies do not all bind to the same epitope even when antibody genesis is induced by the same immunogen. The same is true for C-terminal anti-hPG monoclonal antibodies. Epitopes that bind to typical N-terminal and C-terminal anti-hPG monoclonal antibodies, as identified by alanine scanning and SPOT techniques, are provided in the Examples section of Table 1. 8 and 9.

In some embodiments, anti-hPG monoclonal antibodies bind to an epitope comprising an amino acid sequence corresponding to the N-terminal portion of hPG. In specific embodiments, N-terminal anti-hPG monoclonal antibodies bind to an epitope that includes hPG residues 10-14 (SEQ ID NO: 28), hPG residues 9-14 (SEQ ID NO: 29), hPG residues 4-10 (SEQ ID NO: 30), residues 2-10 hPG (SEQ ID NO: 31) or residues 2-14 hPG (SEQ ID NO: 32).

In some embodiments, anti-hPG monoclonal antibodies bind to an epitope comprising an amino acid sequence corresponding to a portion of the C-terminal region of hPG. In specific embodiments, C-terminal anti-hPG monoclonal antibodies bind to an epitope that includes hPG residues 71-74 (SEQ ID NO: 33), hPG residues 69-73 (SEQ ID NO: 34), hPG residues 76-80 (SEQ ID NO: 35) or residues 67-74 hPG (SEQ ID NO: 36).

It is contemplated that the corresponding CDRs and/or VH and V _L chains of anti-hPG monoclonal antibodies that bind to approximately the same epitopes can be replaced to produce anti-hPG monoclonal antibodies. For example, as shown in Table. 9, exemplary anti-hPG monoclonal antibodies MAb 5 and MAb 6 bind to the same epitope. An anti-hPG monoclonal antibody can be designed to include in its VL chain various combinations of the VL CDRs of the two antibodies and/or in its VH chain various combinations of the VH CDRs of the two antibodies. As a specific example, to illustrate various possible combinations, such an antibody may include in its V _L chain CDR 1 and 2 MAb 5 (V _L CDR 1.5 and V _L CDR 2.5, respectively) and CDR 3 MAb 6 (V _L CDR 3.6), and in its VH circuit CDR 1 MAb 6 (VH CDR 1.6) and CDR 2 and 3 MAb 5 (VH CDR 2.5 and VH CDR 3.5, respectively).

Several anti-hPG monoclonal antibodies having high specificity and affinity for hPG have been identified that exhibit good antitumor activity in in vitro assays, and in some cases their CDR sequences, their VH and VL chain sequences and/or the proposed VH and VL chains for humanized variants. Several hybridomas were also deposited. All of these exemplary anti-hPG monoclonal antibodies, as well as other specific embodiments of anti-hPG monoclonal antibodies useful in the various kits and methods described herein, such as monoclonal antibodies that compete for PG binding to a reference antibody, are described in more detail in section Detailed Description of the Invention.

Anti-hPG monoclonal antibodies as used herein include antibodies that compete with a reference anti-hPG monoclonal antibody for binding to hPG. Reference anti-hPG monoclonal antibody may be any one of the anti-hPG monoclonal antibodies described in this application. Non-limiting examples include antibodies, including three CDR V _L and three CDR VH, as described in this application;

antibodies comprising a VH chain and a V _L chain having amino acid sequences such as those presented herein;

antibodies, including humanized heavy and light chain polypeptides, as presented in this application;

antibodies that are produced by one of the hybridomas disclosed in this application;

antibodies that bind to an epitope within hPG, as disclosed in this application.

Anti-PG monoclonal antibodies described in this application may be in the form of full-length antibodies, antibodies with multiple chains or single-chain antibodies, fragments of such antibodies that selectively bind PG (including, without limitation, Fab, Fab', (Fab') ₂ , Fv and scFv), surrotels (including a light chain surrogate construct), single domain antibodies, humanized antibodies, camelized antibodies, and the like. They may also be of or derived from any isotype, including, for example, IgA (eg IgA1 or IgA2), IgD, IgE, IgG (eg IgG1, IgG2, IgG3 or IgG4) or IgM. In some embodiments, the anti-PG antibody is an IgG (eg, IgG1, IgG2, IgG3, or IgG4).

Anti-PG monoclonal antibodies can be human or non-human. Examples of anti-PG antibodies of non-human origin include, without limitation, those of mammalian origin (eg, monkeys, rodents, goats, and rabbits). Anti-PG monoclonal antibodies for therapeutic use in humans are preferably humanized.

In another aspect, the present disclosure provides nucleic acids that can be used to generate anti-PG monoclonal antibodies. Provided herein are nucleic acids encoding immunoglobulin light chain and heavy chain polypeptides for the antihPG monoclonal antibodies described herein, and vectors comprising these nucleic acids. Additionally provided herein are prokaryotic and eukaryotic host cells transformed with such vectors, as well as eukaryotic host cells, such as mammalian cells, engineered to express anti-hPG monoclonal antibody light and heavy chain polypeptides. Also provided are host cells capable of both expressing the light and heavy chains and secreting the monoclonal antibodies described herein into the medium in which the host cells are cultured. In some embodiments, the host cell is a hybridoma. Also provided are methods for producing anti-hPG monoclonal antibodies by culturing host cells.

Neutralizing anti-PG monoclonal antibodies, such as anti-hPG monoclonal antibodies, bind PG and block PG-dependent signaling, resulting in inhibition of PG-induced tumor cell CRC responses. Accordingly, methods of inhibiting PG-induced CRC cell responses are also provided, which include repression of cell differentiation, repression of cell death, and/or stimulation of cell proliferation. In general, the method comprises contacting a CRC cell or exposing a population of such cells to a neutralizing anti-PG monoclonal antibody in an amount effective to inhibit one or more PG-induced CRC cell responses. The method may be carried out in vitro or in vivo by introducing a neutralizing anti-hPG monoclonal antibody into an environment containing CRC cells, which may be in cell culture or in a tumor.

The anti-PG neutralizing monoclonal antibodies described herein inhibit PG-dependent tumor cell proliferation of CRCs, making them useful therapeutic agents for the treatment of colorectal cancer. Accordingly, pharmaceutical compositions are also provided comprising a neutralizing anti-PG monoclonal antibody and methods using a neutralizing anti-PG monoclonal antibody and/or a pharmaceutical composition for the treatment of CRC. Pharmaceutical compositions may be formulated for any acceptable route of administration, including, for example, parenteral, subcutaneous, or intravenous injection, and will typically include an anti-hPG neutralizing monoclonal antibody and one or more acceptable carriers, excipients, and/or diluents suitable for the desired route of administration. , they may also include other optional components, as will be described further in the Detailed Description of the Invention. For therapeutic use, the compositions may be packaged in unit dose form for ease of administration.

Methods of treatment generally include administering to a subject in need of such treatment, such as a subject diagnosed as having CRC, an amount of a neutralizing anti-PG monoclonal antibody and/or a pharmaceutical composition thereof that is effective to provide a therapeutic benefit. The therapeutic benefits, described in more detail below, include any amelioration of CRC, such as slowing or stopping the development of CRC, reducing the severity of CRC, inhibiting the growth of CRC tumors and proliferation of CRC cells, reducing the size of CRC tumors, and/or reducing serum PG levels in CRC patients. The subject may be a human or non-human, including a domesticated animal (eg, cat, dog, cow, pig, horse) or a non-domesticated animal. Preferably, the anti-PG monoclonal antibody is specific for the PG species being treated. For example, an anti-hPG antibody is administered to a human patient, a canine anti-PG antibody is administered to a canine patient, and the like. Subjects for whom antihPG monoclonal antibody therapy is beneficial are patients with any stage of disease progression (eg, CRC stage 0, I, II, III, or IV), patients who have received CRC therapy (eg, chemotherapy, radiation therapy, surgical removal), or patients who are receiving other therapy for the treatment of CRC.

Treatment with anti-PG monoclonal antibodies as described herein may be combined with or in addition to other therapy. Non-limiting examples of other therapies for CRC include chemotherapeutic treatment, radiation therapy, surgical removal, and antibody-based therapy, as described in this application. In a specific example, anti-hPG monoclonal antibodies are administered in combination with chemotherapeutic agents. In another specific example, anti-hPG monoclonal antibodies are administered in addition to surgical removal. Anti-PG monoclonal antibodies can also be used in combination with each other.

Individuals with CRC tumors often have elevated PG levels (eg, in serum or plasma). Accordingly, anti-hPG monoclonal antibodies can be used to determine PG levels to diagnose CRC. Additionally, in patients who have already been diagnosed as having CRC, anti-hPG monoclonal antibodies can be used to select subjects eligible for anti-PG therapy or to monitor treatment efficacy. As disclosed in this application, a method for diagnosing colorectal cancer in a subject includes determining whether the amount of progastrin in a sample obtained from the subject, for example, in a blood sample or a serum sample, measured using an anti-hPG monoclonal antibody in accordance with the present invention, is above a threshold level. In a specific embodiment, the threshold level is 50 pM. In some embodiments, two anti-PG antibodies are used: one that recognizes the C-terminal region of PG and the other that recognizes the N-terminal region of PG. In this embodiment, one or both of the N-terminal or C-terminal antibodies are anti-PG monoclonal antibodies as described herein. Preferably, N-terminal and C-terminal anti-PG monoclonal antibodies are used. Antibodies may be, but need not be, neutralizing.

For the purpose of monitoring the effectiveness of anti-PG treatment, monoclonal antibodies can be used to determine if the level of progastrin decreases over a period of time in samples obtained from a subject who has been treated or is being treated with CRC by comparing the amount of PG in samples taken at different time points. . Specific embodiments of the anti-PG antibody described in the previous paragraph may also be used in this assay.

Also provided are kits suitable for the implementation of various diagnostic methods and monitoring, as well as other methods described in this application. Such kits will typically include an anti-PG monoclonal antibody as described herein, and optionally additional anti-PG antibodies and/or reagents suitable for performing a specific assay. In some embodiments, one or more anti-PG antibodies that are included in the kit are labeled with a detectable label, such as a fluorophore. In a specific embodiment, the kit includes an anti-PG antibody that specifically binds the N-terminal region of PG, an anti-PG antibody that specifically binds the C-terminal region of PG, and optionally reagents suitable for performing a diagnostic assay, wherein the N-terminal specific antibody is is an N-terminal antibody which is an N-terminal anti-PG monoclonal antibody as described herein and/or a C-terminal specific antibody is a C-terminal anti-PG monoclonal antibody as described in this application.

The characteristics and advantages of the various inventions described in this application will also be clear from the following detailed description of typical examples of its implementation.

8. Brief description of the figures

Fig. 1 provides the amino acid sequences for pre-progastrin, progastrin, and progastrin processing products, including G34, G34-Gly, G17, G17-Gly, and the C-terminal flanking peptide, CTFP.

Fig. 2 provides the polypeptide and corresponding polynucleotide sequences of VH and V _L chains for exemplary mouse anti-hPG monoclonal antibodies: anti-hPG MAb 3 (SEQ ID NOs: 16, 12, 17 and 13, respectively, in order of appearance) (FIGS. 2A, 2B ), anti-hPG MAb 4 (SEQ ID NOs: 18, 14, 19, and 15, respectively, in order of appearance) (Fig. 2C, 2D), anti-hPG MAb 8 (SEQ ID NOs: 67, 59, 71, and 63, respectively in order of appearance) (FIGS. 2E, 2F), anti-hPG Mab 13 (SEQ ID NOs: 68, 60, 72 and 64

- 5 041823, respectively, in order of development) (Fig. 2G, 2H), anti-hPG MAb 16 (SEQ ID NOs: 69, 61, 73 and 65, respectively, in order of development) (Fig. 2I, 2J) and anti-hPG MAb 19 (SEQ ID NOs: 70, 62, 74 and 66, respectively, in order of appearance) (FIGS. 2K, 2L), in which the three CDRs of each strand are underlined.

Fig. 3A-3C provide graphs illustrating the relative binding affinities (measured as absorbance at 492 nm) at increasing antibody concentrations (μg/ml) of exemplary mouse anti-hPG monoclonal antibodies, MAbs 1-4 (FIG. 3A); MAbs 5-14 and 20-23 (FIG. 3B); and MAbs 3 and 15-19 (FIG. 3C).

Fig. 4 provides a graph illustrating the ratio of absorbance (optical density) at 280 and 330 nm for four different exemplary anti-hPG monoclonal antibodies compared to a control sample of bovine serum albumin (relative units).

Fig. 5A-5C provide a diagram illustrating the binding of 23 different exemplary mouse anti-hPG monoclonal antibodies to 25 or 50 ng hPG compared to buffer alone (negative control), 250 ng KLH (negative control), and peptides that are derived from the gastrin gene ( 50 and 250 ng CTFP, G17 or G17-Gly (referred to as G-Gly in the figure) as indicated Figure 5A shows binding of anti-hPG MAbs 1-4, Figure 5B shows binding of anti-hPG MAbs 5-14 and 21-23 and Fig. 5C shows the binding of anti-hPG MAbs 3 and 15-20.

Fig. 6 provides a diagram illustrating the binding of a polyclonal N-terminal anti-hPG antibody to hPG at increasing concentrations of anti-hPG Mab 3.

Fig. 7 provides a graph illustrating the proliferation of characteristic CRC cell lines treated with anti-hPG monoclonal antibodies such as below SW480, HCT-116, LS174T, as indicated, treated with typical anti-hPG monoclonal antibodies MAb 3 and MAb 4 ( Fig. 7A, 7B, 7C, respectively, showing the change in the number of living cells at the end of treatment in relation to the beginning of treatment (T0) with an antibody) or anti-hPG polyclonal antibody (Fig. 7D, 7E, 7F, respectively, showing the change in the number of living cells at the end of treatment with respect to the start of treatment (T0) with the antibody); proliferation of CRC cell line SW620 treated with anti-hPG MAb 5-23 (FIG. 7G showing live anti-hPG treated cells as a percentage of antibody-treated control cells at end of treatment versus start of treatment (T0 )); proliferation of LS174T cells treated with anti-hPG MAb 8, 13, 14, 16, and 19 (Fig. 7H showing live anti-hPG treated cells as a percentage of antibody-treated control cells at end of treatment vs. start processing (T0)); and proliferation of HCT-116 cells treated with anti-hPG monoclonal antibodies MAb 8, 13, 14, 16, 19 (Fig. 7I showing live anti-hPG treated cells as a percentage of antibody treated control cells at the end processing with respect to the start of processing (T0)).

Fig. 8 provides a graph illustrating the number of live LS174T cells 48 hours after 4 treatments with control monoclonal antibody, anti-hPG MAb 8 (5 μg/ml), anti-hPG MAb 8 pre-incubated with hPG, control antibody pre-incubated with hPG, or just hPG.

Fig. 9 provides graphs illustrating the number of tumors per mouse (FIG. 9A) and mean tumor length and width (FIG. 9B) in mice treated with an anti-hPG antibody versus a control polyclonal antibody.

9. Detailed description of specific embodiments

9.1. Detailed description.

Progastrin (PG) was first identified as a precursor to gastrin, a gut peptide hormone that stimulates gastric acid secretion. Gastrin exists in various molecular forms (G17, G34, glycine-extended G17, glycine-extended G34) that are derived from progastrin. See fig. 1. The gastrin gene codes for a 101 amino acid product, preprogastrin. The first cleavage removes the residual 21 amino acid signal peptide (underlined in FIG. 1) and results in PG, an 80 amino acid peptide. The known human PG (hPG) polypeptide sequence is provided in SEQ ID NO: 20. As illustrated in FIG. 1, amino acid residues of hPG are numbered from 1 to 80 with the amino terminal residue being at position 1. Sequences within the first 40 amino acids of progastrin are called N-terminal, while sequences between residues 41-80 are called C- terminal.

Recent studies have shown that progastrin levels are elevated in patients with CRC. Under normal physiological conditions, progastrin makes up less than 10% of the peptides secreted in humans. In colorectal cancer, progastrin levels are significantly elevated in both plasma and tumor tissue, possibly as a result of increased gastrin gene expression associated with incomplete processing of the gene product. One study demonstrated significantly higher serum progastrin levels in CRC patients compared to controls, but showed no difference for more processed forms of gastrin (Siddheshwar et al., 2001, Gut, 48:47-52). In the tumor CRC samples analyzed, 80-100% of the samples showed elevated levels of PG. See, for example, Ciccotosto et al., 1995, Gastroenterology, 109:1142-1153; Baldwin et al. 1998 Gut 42:581-584; Van Solinge, 1993, Gastroenterology, 104:1099-1107. The role of PG in CRC was further supported by experiments demonstrating that mice that express recombinant human PG exposed to the azoxymethane oncogene had significantly higher numbers of aberrant crypt foci, adenomas, and adenocarcinomas in the intestine compared to wild-type or mice expressing amidated gastrins (Singh et al. 2000, Gastroenterology, 119:162-171).

Recently, Hollande et al. demonstrated that progastrin stimulates the beta-catenin/Tcf4 pathway by repressing ICAT, a negative regulator of the beta-catenin/Tcf4 signaling pathway, and that blocking progastrin leads to de novo expression of ICAT. See WO 2007/135542. Without intending to be bound by any theory of operation, it is hypothesized that blocking the progastrin signaling pathway results in repression of beta-catenin/Tcf4-induced proliferation as a result of increased expression of ICAT. In the absence of continuous PG dependent signaling, cell proliferation is inhibited and cell differentiation and/or cell death (including apoptosis) is triggered.

Despite the urgent need for clinical approaches to the treatment and diagnosis of CRC, confirmation that PG stimulates the proliferation of CRC tumor cells, and despite increased attention to therapies for cancer treatment based on monoclonal antibodies, there are no reports to date that demonstrate any monoclonal an antibody capable of blocking PG-dependent tumor cell proliferation, or even PG binding. Such antibodies, presented in this application for the first time, have proved difficult to develop. The first problem was that Applicants found that recombinant human progastrin, which can be used to generate polyclonal anti-hPG antibodies, did not induce the production of monoclonal antibodies in response to an immunogen in test mice. Thus, it has been necessary to design immunogens using only PG peptide fragments to produce an antibody specific for progastrin and not other gastrin gene products. Even after clones of hybridomas producing antibodies that bind to an antigenic peptide have been obtained, it has been found that peptide binding is not predictable in terms of PG binding capacity, specifically or at all. As shown in more detail in the examples below, many hybridomas produced antibodies that bind to the PG antigenic peptide used in the immunogen, but do not bind to PG.

The present disclosure provides anti-hPG monoclonal antibodies that not only bind to the peptidic antigen they are directed against, but also specifically bind hPG. Quite surprisingly, it has been found that monoclonal antibodies that are highly specific for hPG compared to their processed product (e.g. G34, G34-Gly, G17, G17-Gly, CTFP) are produced using antigens that in some cases are not unique to hPG, but which include portions of the amino acid sequence in common with one or more progastrin processing products. In addition, it was unexpectedly found that despite the relatively small size of hPG (80 amino acids), not all anti-hPG monoclonal antibodies, and even those that show a high degree of affinity and specificity for hPG, neutralize its biological activity.

Anti-hPG monoclonal antibodies.

Applicants have discovered peptide antigens useful in the production of anti-hPG monoclonal antibodies. Peptides useful in making anti-hPG antibodies as described herein include progastrin-specific sequences not found in more truncated forms of the polypeptide, such as glycine-extended or amidated gastrins or CTFP, but also include sequences found in processed forms of hPG. In some embodiments, anti-hPG monoclonal antibodies are raised against a peptidic antigen having an amino acid sequence corresponding to the N-terminal portion of hPG, which is referred to as N-terminal anti-hPG monoclonal antibodies. An exemplary specific antigenic site that can be used to construct an immunogen useful in making an N-terminal anti-PG monoclonal antibody corresponds to hPG residues 1-14 (SWKPRSQQPDAPLG (SEQ ID NO: 25)) fused to a linker sequence. In other embodiments, the anti-hPG monoclonal antibodies are raised against a peptidic antigen that has an amino acid sequence corresponding to the C-terminal region of hPG and are referred to as C-terminal anti-hPG monoclonal antibodies. An exemplary specific antigenic site that can be used to construct an immunogen useful in making a C-terminal anti-PG monoclonal antibody corresponds to residues 55-80 hPG (SEQ ID NO: 27) fused to a linker sequence. See table. 1.

Anti-PG monoclonal antibodies of the present disclosure bind PG and are useful for its detection and isolation from complex mixtures. Additionally, the anti-PG monoclonal antibodies of this application are uniquely useful for therapeutic and/or diagnostic applications in the treatment of colorectal cancer. In various embodiments, the anti-hPG monoclonal antibodies (1) specifically bind PG in contrast to other gastrin gene products;

(2) have high affinity for hPG;

(3) inhibit the proliferation of colorectal cancer cells in vitro and in vivo;

(4) reduce the size and number of tumors in vivo;

(5) determine PG in complex mixtures that contain other peptides that are derived from the gastrin gene.

The gastrin gene is expressed and extensively processed to form protein products that play specific roles in normal homeostasis. Progastrin, on the other hand, is typically not detected in the circulatory system in healthy subjects. The monoclonal antibodies of the present disclosure are designed to target progastrin, but not other peptides that are derived from the gastrin gene. Accordingly, anti-hPG monoclonal antibodies specifically bind to human and other animal progastrin, but not to other gastrin gene products such as, but not limited to, glycine-extended or amidated gastrins, or C-terminal flanking peptide (CTFP).

The specificity of anti-hPG monoclonal antibodies can be determined using ELISA as described below. 96-well plates are incubated overnight at 4° C. with acceptable concentration(s) of the polypeptide of interest (e.g., 25 and 50 ng recombinant human PG and 50 and 250 ng CTFP or other products derived from the gastrin gene) in phosphate buffered saline (PBS), after which the wells are washed three times with washing solution (PBS and 0.1% Tween-20) and then incubated for 2 h at 22°C using 100 μl of blocking solution (PBS, 0 .1% Tween-20, 0.1% bovine serum albumin or casein hydrolysate) per well. After blocking, the wells are washed three times and the antibody to be analyzed (test antibody) is added. 100 µl of test antibody (at a concentration of 0.3-1 ng/ml) in PBS and 0.1% Tween-20 is added to each well. The plates are then incubated for 2 hours at 22°C, after which the test antibody solution is discarded and replaced, after a washing step (3x100 µl of wash solution as above), with a blocking solution containing a secondary antibody, goat anti-mouse IgG (Fc) antibody fused with horseradish peroxidase. After a 1 hour incubation with the secondary antibody, add 100 µl of a substrate solution (e.g. Fast OPD or O-phenylenediamine dihydrochloride available from Sigma-Aldrich Co. prepared according to the manufacturer's instructions) to each well and incubate in the dark for 20 min at 22°C. The reaction is stopped by adding 50 µl of 4N sulfuric acid and the amount of catalyzed substrate is determined by measuring the optical density (O.D.) at 492 nm. Substrate conversion is proportional to the amount of primary (test) antibody bound to the antigen. The experiments are carried out in duplicate and the measured OD values are plotted as a function of concentration. Test antibodies are considered specific for PG if the measured O.D. is 0.2-1.5 for hPG and there is no statistically significant signal above background when using CTFP or any other peptides that are derived from the gastrin gene, where background is the average signal from control cells that contain only PBS.

Several anti-hPG monoclonal antibodies according to the present invention have been found to be highly specific. In some embodiments, anti-hPG monoclonal antibodies exhibit 100-fold higher specificity for progastrin than other gastrin gene products, and in such embodiments, 100-fold more antigen (e.g., glycine-extended or amidated gastrin) is required to produce the same binding, which is observed when the antigen is progastrin.

Other methods for determining binding include, but are not limited to, immunofluorescence assay, ELISA, radiolabelled material immunoassay (RIA), sandwich ELISA (Monoclonal Antybody Experiment Manual (publ. Kodansha Scientific (1987), Second Series Biochemical Experiment Course, Volume 5, Immunobiochemistry Research Method, published by Tokyo Kagaku Dojin (1986)).

Anti-hPG monoclonal antibodies with high affinity for PG are desirable for both therapeutic and diagnostic applications. For some uses, such as therapeutic use, an affinity that is at least about 100 nM is desirable, although antibodies having higher affinities, such as affinities that are at least about 90, 80, 70, 60, 50, 40, 30, 25, 20, 15, 10, 7, 6, 5, 4, 3, 2, 1, 0.1, 0.01 nM, 10, 1 pM, or even more are desirable. In some embodiments, the monoclonal antibodies specifically bind to hPG with a high affinity ranging from about 1 pM to about 100 nM, or with an affinity that ranges from any of the above values.

The affinity of anti-hPG monoclonal antibodies for hPG can be determined using techniques well known in the art or described herein, such as, for example, but not limited to, ELISA, isothermal titration calorimetry (ITC) ,

BIAcore, Proteon or fluorescent light polarization analysis.

By using antigens from the N- or C-terminal regions of hPG, antibodies can be generated that recognize different hPG epitopes. The epitope that is recognized by the monoclonal antibody will depend on the specific antigen used to induce antibody genesis and can be mapped using techniques known to those skilled in the art such as alanine scanning and SPOT analysis (see the Examples section below). For example, epitope mapping reveals that anti-hPG MAb 2 and MAb 4 bind to the same epitope; anti-hPG MAb 1 and MAb 3 bind to approximately the same epitope; MAb 17, MAb 18, MAb 19 and MAb 20 bind to approximately the same epitope; MAb 15 and MAb 16 bind to approximately the same epitope; anti-hPG MAb 5, MAb 6, MAb 7, MAb 9 and MAb 12 bind to approximately the same epitope as anti-hPG MAb 10; and anti-hPG MAb 11 and MAb 14 bind to approximately the same epitope.

Whether or not an anti-hPG monoclonal antibody recognizes a particular eptope can be determined using a competitive assay, as described herein, in which the epitope bound by the reference antibody is known. In some embodiments, the anti-hPG monoclonal antibody competes with a reference antibody that binds to an epitope having an amino acid sequence corresponding to the N-terminal region of hPG. In specific embodiments, the anti-hPG monoclonal antibodies compete with a reference antibody that binds to an epitope that includes hPG residues 10-14 (SEQ ID NO: 28), hPG residues 9-14 (SEQ ID NO: 29), residues 4-10 hPG (SEQ ID NO: 30), hPG residues 2-10 (SEQ ID NO: 31) or hPG residues 2-14 (SEQ ID NO: 32). In some embodiments, the anti-hPG monoclonal antibody competes with a reference antibody that binds to an epitope having an amino acid sequence corresponding to the C-terminal region of hPG. In specific embodiments, the anti-hPG monoclonal antibodies compete with a reference antibody that binds to an epitope that includes hPG residues 71-74 (SEQ ID NO: 33), hPG residues 69-73 (SEQ ID NO: 34), residues 76-80 hPG (SEQ ID NO: 35) or residues 67-74 hPG (SEQ ID NO: 36).

Anti-PG monoclonal antibodies may be neutralizing. Without intending to be bound by operational theory, by binding to PG, neutralizing anti-hPG monoclonal antibodies are expected to block or inhibit its ability to interact with its signaling partner(s). This, in turn, inhibits the signal transduction pathway in colorectal tumor cells, which would otherwise lead to proliferation, reduced differentiation, and cell death. In some embodiments, anti-PG neutralizing monoclonal antibodies bind the N-terminal region of hPG. In specific embodiments, anti-PG neutralizing monoclonal antibodies compete for PG binding with anti-hPG MAb 1, MAb 2, MAb 3, MAb 4, MAb 15, MAb 16, MAb 17, MAb 18, MAb 19, or MAb 20. In other embodiments, neutralizing anti-PG monoclonal antibodies bind the C-terminal region of hPG. In specific embodiments, anti-PG neutralizing monoclonal antibodies compete for PG binding with anti-hPG MAb 5, MAb 6, MAb 7, MAb 8, MAb 9, MAb 10, MAb 11, MAb 12, MAb 13, MAb 21, MAb 22, or MAb 23.

A specific assay to determine if an anti-PG monoclonal antibody is neutralizing can be performed as described below. CRC LS174T cells are seeded in a 6-well plate as described in Example 7 below at a concentration of approximately 50,000 cells per well. The cells are then treated at 12-hour intervals for 48 hours with the anti-PG monoclonal antibody of interest or control monoclonal antibody as described in Example 7 at antibody concentrations that are approximately 5 μg/ml. The test antibody is defined as neutralizing in this assay if the number of CRCs of cancer cells treated with the test antibody shows a statistically significant decrease of at least 10% in the number of surviving cells compared to the number of cells treated with a control, non-specific antibody , using a two-tailed Mann-Whitney test (with differences considered significant when p<0.05). The total number of cells is corrected for the number of cells at the beginning of the treatment period, which is referred to as T0.

As used herein, an antibody (Ab) refers to an immunoglobulin molecule that specifically binds to or is immunologically reactive with a particular antigen and includes polyclonal, monoclonal, genetically engineered, and otherwise modified forms of antibodies, including, but not limited to, chimeric antibodies, humanized antibodies; and antigen-binding antibody fragments, including, for example, Fab', F(ab') ₂ , Fab, Fv, rIgG, and scFv fragments. In various embodiments, the anti-hPG monoclonal antibodies comprise all or part of the constant region of the antibody. In some embodiments, the constant region is isothy selected from IgA (eg IgA1 or IgA2), IgD, IgE, IgG (eg IgG1, IgG2, IgG3 or IgG4) and IgM.

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The term monoclonal antibody, as used in this application, is not limited to antibodies that are obtained using hybridoma technology. A monoclonal antibody is one that is derived from a single clone, including any eukaryotic, prokaryotic or phage clone, by any means available or known in the art. Monoclonal antibodies useful for the present invention can be obtained using a wide variety of techniques known in the art, including the use of hybridoma, recombinant and phage display techniques, or combinations thereof. In many applications of the present invention, including in vivo use of anti-hPG monoclonal antibodies in humans and in vitro detection assays, chimeric, primatized, humanized or human antibodies can be suitably used.

The term scFv refers to a single Fv chain of an antibody in which the heavy chain and light chain variable domains from a conventional antibody have been combined to form a single chain.

Reference to VH refers to the variable region of an immunoglobulin heavy chain of an antibody, including the heavy chain of an Fv, scFv, or Fab. Reference to VL refers to the variable region of an immunoglobulin light chain, including Fv, scFv, dsFv or Fab light chain. Antibodies (Ab) and immunoglobulins (Ig) are glycoproteins with the same structural characteristics. While antibodies exhibit binding specificity for a specific target, immunoglobulins include both antibodies and other antibody-like molecules that lack target specificity. Native antibodies and immunoglobulins are usually heterotetrameric glycoproteins weighing approximately 150,000 daltons, which consist of two identical light (L) chains and two identical heavy (H) chains. Each heavy chain has at one end a variable domain (VH) followed by a series of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at its other end.

The anti-hPG monoclonal antibodies of this application include complementarity determining regions (CDRs). CDRs are well known as hypervariable regions in both light and heavy chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). As is known in the art, the amino acid position/definite boundary of the hypervariable region of an antibody may vary depending on the context and various definitions known in the art. Some positions within a variable domain may be considered as hybrid hypervariable positions in the sense that these positions may be assumed to be in the hypervariable region according to a set of criteria and at the same time may be expected to be outside the hypervariable region. according to a different set of criteria. One or more of these positions may also be found in extended hypervariable regions. This application provides antibodies that include modifications at these hybrid hypervariable positions. The native heavy and light chain variable domains each comprise four FR regions, primarily by adopting a β-sheet configuration linked by CDRs that form loops that connect and in some cases form part of the β-sheet structure. The CDRs in each strand are held together in close proximity by FR regions and, together with CDRs from the other strand, contribute to the formation of the antibody's target binding site (See Kabat et al., Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda). , Md., 1987).

Several anti-hPG monoclonal antibodies having high specificity and affinity for hPG and good antitumor activity have been identified, and their CDRs and heavy and light chain variable regions have also been sequenced. The mouse heavy and light chain variable domains are referred to herein as mVH and mVL followed by the number of the corresponding monoclonal antibody, for example mV _H 3 and mV _L 3 for anti-hPG Mab 3. Anti-hPG monoclonal antibodies have three variable light chain CDRs and three variable heavy chain CDRs referred to as V _H CDR 1, 2, or 3 and V _L CDR 1, 2, or 3, respectively, followed by the number of a typical anti-hPG monoclonal antibody. For example, V _L CDR 1 MAb 3 is referred to as V _L CDR 1.3 and V _H CDR 1, and MAb 3 is referred to as V _H CDR 1.3. Similarly, the variable domains of the human heavy and light chains are referred to herein as hV _H and hV _L followed by the number of the corresponding monoclonal antibody.

In some embodiments, anti-hPG monoclonal antibodies raised against the N-terminal portion of hPG have three variable light chain CDRs and three variable heavy chain CDRs, where

V _L CDR 1 is selected from QSIVHSNGNTY (V _L CDR 1.3; SEQ ID NO: 4), QSLVHSSGVTY (V _L CDR 1.4; SEQ ID NO: 10), QSLLDSDGKTY (V _L CDR 1.16; SEQ ID NO: 50), and SQHRTYT (V _L CDR 1.19; SEQ ID NO: 51);

VL CDR 2 is selected from KVS (VL CDR 2.3 and (VL CDR 2.4; SEQ ID NO: 5), LVS (V _L CDR 2.16; SEQ ID NO: 53) and VKKDGSH (V _L CDR 2.19; SEQ ID NO: 54 );

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V _L CDR 3 is selected from FQGSHVPFT (V _L CDR 3.3; SEQ ID NO: 6), SQSTHVPPT (V _L CDR 3.4; SEQ ID NO: 11), WQGTHSPYT (V _L CDR 3.16; SEQ ID NO: 57), and GVGDAIKGQSVFV (V _L CDR 3.19; SEQ ID NO: 58);

V _H CDR 1 is selected from GYIFTSYW (V _H CDR 1.3; SEQ ID NO: 1), GYTFSSSW (V _H CDR 1.4; SEQ ID NO: 7), GYTFTSYY (”V _H CDR 1.16; SEQ ID NO: 39), and GYSITSDYA (V _H CDR 1.19; SEQ ID NO: 40);

V _H CDR 2 is selected from FYPGNSDS (V _H CDR 2.3; SEQ ID NO: 2), FLPGSGST (V _H CDR 2.4; SEQ ID NO: 8), INPSNGGT (V _H CDR 2.16; SEQ ID NO: 43), and ISFSGYT (V _H CDR 2.19; SEQ ID NO: 44); And

V _H CDR 3 is selected from TRRDSPQY (V _H CDR 3.3; SEQ ID NO: 3), ATDGNYDWFAY (V _H CDR 3.4; SEQ ID NO: 9), TRGGYYPFDY (V _H CDR 3.16; SEQ ID NO: 47) and AREVNYGDSYHFDY (V _H CDR 3.19; SEQ ID NO: 48).

See table. 1A.

In some embodiments, anti-hPG monoclonal antibodies raised against the N-terminal portion of hPG have three variable light chain CDRs and three variable heavy chain CDRs, where

V _L CDR 1 is selected from KSLRHTKGITF (V _L CDR 1.8; SEQ ID NO: 49) and QSLLDSDGKTY (V _L CDR 1.13; SEQ ID NO: 50);

V _L CDR 2 is selected from QMS (V _L CDR 2.8; SEQ ID NO: 52) and LVS (V _L CDR 2.13; SEQ ID NO: 53);

V _L CDR 3 is selected from AQNLELPLT (V _L CDR 3.8; SEQ ID NO: 55) and WQGTHFPQT (V _L CDR 3.13; SEQ ID NO: 56);

V _H CDR 1 is selected from GFTFTTYA (V _H CDR 1.8; SEQ ID NO: 37) and GFIFSSYG (V _H CDR 1.13; SEQ ID NO: 38); V _H CDR2 is selected from ISSGGTYT (”V _H CDR 2.8; SEQ ID NO: 41) and INTFGDRT (V _H CDR 2.13; SEQ ID NO: 42); And

V _H CDR 3 is selected from ATQGNYSLDF (V _H CDR 3.8; SEQ ID NO: 45) and ARGTGTY (V _H CDR 3.13; SEQ ID NO: 46).

See table. IB.

Table 1A

N-terminal anti-hPG monoclonal antibodies

Immunogen Hybridoma (deposit no.) MAb Mouse CDR sequences Mouse V _H and V _L sequences Humanized V _H and V _L sequences (designed) N1 43B9G11 MAbl N1 WE5H2G7 MAb2 N2 6B5B11C10 MASS V _H GYIFTSYW (SEQ ID CDR 1.3 NO:1) V _H FYPGNSDS (SEQ ID CDR 2.3 NO:2) V _H TRRDSPQY (SEQ ID CDR 3.3 NO:3) V _L QSIVHSNGNTY (SEQ ID CDR 1.3 NO:4) V _L KVS (SEQ ID CDR 2.3 NO:5) V _L FQGSHVPFT (SEQ ID CDR 3.3 NO:6) mV _H 3 (SEQ ID NO. 12) mV _L 3 (SEQIDNO:13) hV _H 3 (SEQ ID NO:21) hV _L 3 (SEQ ID NO:22) N2 20D2C3G2 MAb4 V _H GYTFSSSW (SEQ ID CDR 1.4 NO:7) V _H FLPGSGST (SEQ ID CDR 2.4 NO:8) V _H ATDGNYDWFAY (SEQ ID CDR 3.4 NO:9) V _L QSLVHSSGVTY (SEQ ID CDR 1.4 NO:10) V _L KVS (SEQ ID CDR 2.4 NO:5) V _L SQSTHVPPT (SEQ ID CDR 3.4 NO:11) mV _H 4 (SEQ ID NO: 14) mV _L 4 (SEQ ID NO: 15) hV _H 4 (SEQ ID NO:23) hV _L 4 (SEQ ID NO:24) N2 1E9A4A4 (1-4376) MAb 15

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N2 1E9D9B6 MAb 16 V _H GYTFTSYY (SEQ ID CDR 1.16 NO:39) V _H INPSNGGT (SEQ ID CDR 2.16 NO:43) V _H TRGGYYPFDY (SEQ ID CDR 3.16 NO:47) V _L QSLLDSDGKTY (SEQ ID CDR 1.16 NO:50) V _L LVS (SEQ ID CDR 2.16 NO:53) V _L WQGTHSPYT (SEQ ID CDR 3.16 NO:57) mV _H 16 (SEQIDNO:61) mV _L 16 (SEQ ID NO:65) hV _H 16a (SEQ ID NO:84) hV _H 16b (SEQ ID NO:86) hV _H 16c (SEQ ID NO:88) hV _L 16a (SEQ ID NO:85) hV _L 16b (SEQ ID NO:87) hV _L 16c (SEQ ID NO:89) N2 1C8D10F5 MAb 17 N2 1A7C3F11 MAb 18 N2 1B3B4F11 MAb 19 V _H GYSITSDYA (SEQ ID CDR 1.19 NO:40) V _H ISFSGYT (SEQ ID CDR 2.19 NO:44) V _H AREVNYGDSYHFDY (SEQ ID CDR 3.19 NO:48) V _L SQHRTYT (SEQ ID CDR 1.19 NO:51) V _L VKKDGSH (SEQ ID CDR 2.19 NO:54) V _L GVGDAIKGQSVFV (SEQ ID CDR 3.19 NO:58) mV _H 19 (SEQ ID NO:62) mV _L 19 (SEQ ID NO:66) hV _H 19a (SEQ ID NO:90) hV _H 19b (SEQ ID NO:92) hV _H 19c (SEQ ID NO:94) hV _L 19a (SEQ ID NO:91) hV _L 19b (SEQ ID NO:93) hV _L 19c (SEQ ID NO:95) N2 1C11F5E8 MAb20 Immunogen N1 = SWKPRSQQPDAPLG Ahx Cys BSA, also represented as (SEQ ID NO:25) Ahx Cys BSA Immunogen N2 = SWKPRSQQPDAPLG Ahx Cys KLH, also represented as (SEQ ID NO:25) Ahx Cys KLH

In table. 1A, all amino acid sequences are shown using the traditional N^C orientation. For each immunogen, progastrin peptide was synthesized with a single aminohexanoic acid (Ahx) linker followed by cysteine, which was then conjugated with either bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) carrier.

Table 1B

C-terminal anti-hPG monoclonal antibodies

Immunogen Hybridoma (deposit no.) MAb Mouse CDR sequences Mouse V _H and V _L sequences Humanized V _H and V _L sequences (designed) Cl 1B4A11D11 (1-4371) MAb 5 Cl 1B6A11F2 (1-4372) MAb6 Cl 1B11E4B11 (1-4373) MAb7 Cl 1C10D3B9 MAb 8 V _H GFTFTTYA (SEQ ID CDR 1.8 NO:37) V _H ISSGGTYT (SEQ ID CDR 2.8 NO:41) V _H ATQGNYSLDF (SEQ ID CDR 3.8 NO:45) V _L KSLRHTKGITF (SEQ ID CDR 1.8 NO:49) V _L QMS (SEQ ID CDR 2.8 NO:52) V _L AQNLELPLT (SEQ ID CDR 3.8 NO:55) mV _H .8 (SEQ ID NO:59) mV _L .8 (SEQ ID NO:63) hV _H 8a (SEQ ID NO:75) hV _H 8b (SEQ ID NO:77) hV _H 8c (SEQ ID NO:79) hV _L 8a (SEQ ID NO:76) hV _L 8b (SEQ ID NO:78) hV _L 8c (SEQ ID NO:76) Cl 1D8F5B3 MAb 9 Cl 1E1C7B4 MAb 10 Cl 2B4C8C8 (1-4374) MAb 11 Cl 2B11E6G4 (1-4375) MAb 12 Cl 2C6C3C7 MAb 13 V _H GFIFSSYG (SEQ ID CDR 1.13 NO:38) V _H INTFGDRT (SEQ ID CDR 2.13 NO:42) V _H ARGTGTY (SEQ ID CDR 3.13 NO:46) V _L QSLLDSDGKTY (SEQ ID CDR 1.13 NO:50) V _L LVS (SEQ ID CDR 2.13 NO:53) V _L WQGTHFPQT (SEQ ID CDR 3.13 NO:56) mV _H .13 (SEQ ID NO:60) mV _L .13 (SEQ ID NO:64) hV _H .13a (SEQ ID NO:80) hV _H .13b (SEQ ID NO:82) hV _L 13a (SEQ ID NO:81) hV _L 13b (SEQ ID NO:83) Cl 2H9F4B7 MAb 14 C2 1F11F5E10 MAb21 C2 1F11F5G9 MAb22 C2 1A11F2C9 MAb23

Immunogen Cl = KLH Cys Ahx Ahx QGPWLEEEEEAYGWMDFGRRSAEDEN also listed as KLH Cys Ahx Ahx (SEQ ID NO:27) Immunogen C2 = DT Cys Ahx Ahx QGPWLEEEEEAYGWMDFGRRSAEDEN also listed as DT Cys Ahx Ahx (SEQ ID NO:27)

In table. 1B, all amino acid sequences are shown using the traditional N^C orientation. For each immunogen, a progastrin peptide was synthesized with a single aminohexanoic acid (Ahx) linker followed by cysteine, which was then conjugated either with a carrier based on bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).

In some embodiments, the V _H chain CDRs of the anti-hPG monoclonal antibody are V _H CDR 1.3, V _H CDR 2.3, and V _H CDR 3.3. In a specific embodiment, the V _H chain of the anti-hPG monoclonal antibody has an amino acid sequence corresponding to mV _H 3 (SEQ ID NO: 12). See fig. 2A.

In some embodiments, the V _L chain anti-hPG monoclonal antibody CDRs are V _L CDR 1.3, V _L CDR 2.3, and V _L CDR 3.3. In a specific embodiment, the V _L chain of the anti-hPG monoclonal antibody has an amino acid sequence corresponding to mV _L 3 (SEQ ID NO: 13). See fig. 2B.

In some embodiments, the CDR V _H chains of the anti-hPG monoclonal antibody are V _H CDR 1.4, V _H CDR 2.4, and V _H CDR 3.4. In a specific embodiment, the V _H chain of the anti-hPG monoclonal antibody has an amino acid sequence corresponding to mVH 4 (SEQ ID NO: 14). See fig. 2C.

In some embodiments, the anti-hPG monoclonal antibody V _L chain CDRs are V _L CDR 1.4, V _L CDR 2.4, and V _L CDR 3.4 In a specific embodiment, the anti-hPG monoclonal antibody V _L chain has an amino acid sequence corresponding to mV _L 4 (SEQ ID NO: 15). See fig. 2D.

In some embodiments, the V _H chain CDRs of the anti-hPG monoclonal antibody are V _H CDR 1.8, V _H CDR 2.8, and V _H CDR 3.8. In a specific embodiment, the V _H chain of the anti-hPG monoclonal antibody has an amino acid sequence corresponding to mV _H 8 (SEQ ID NO: 59). See fig. 2E.

In some embodiments, the V _L CDR chains of the anti-hPG monoclonal antibody are V _L CDR 1.8, V _L CDR 2.8, and V _L CDR 3.8. In a specific embodiment, the V _L chain of the anti-hPG monoclonal antibody has an amino acid sequence corresponding to mV _L 8 (SEQ ID NO: 63). See fig. 2F.

In some embodiments, the CDR V _H chains of the anti-hPG monoclonal antibody are V _H CDR 1.13, V _H CDR 2.13, and V _H CDR 3.13. In a specific embodiment, the V _H chain of the anti-hPG monoclonal antibody has an amino acid sequence corresponding to mV _H 13 (SEQ ID NO: 60). See fig. 2g.

In some embodiments, the V _L chain anti-hPG monoclonal antibody CDRs are V _L CDR 1.13, V _L CDR 2.13, and V _L CDR 3.13. In a specific embodiment, the V _L chain of the anti-hPG monoclonal antibody has an amino acid sequence corresponding to mVL 13 (SEQ ID NO: 64). See fig. 2H.

In some embodiments, the V _H chain CDRs of the anti-hPG monoclonal antibody are V _H CDR 1.16, V _H CDR 2.16, and V _H CDR 3.16. In a specific embodiment, the V _H chain of the anti-hPG monoclonal antibody has an amino acid sequence corresponding to mV _H 16 (SEQ ID NO: 61). See fig. 21.

In some embodiments, the V _L chain anti-hPG monoclonal antibody CDRs are V _L CDR 1.16, V _L CDR 2.16, and V _L CDR 3.16. In a specific embodiment, the V _L chain of the anti-hPG monoclonal antibody has an amino acid sequence corresponding to mV _L 16 (SEQ ID NO: 65). See fig. 2J.

In some embodiments, the CDR V _H chains of the anti-hPG monoclonal antibody are V _H CDR 1.19, V _H CDR 2.19, and V _H CDR 3.19. In a specific embodiment, the V _H chain of the anti-hPG monoclonal antibody has an amino acid sequence corresponding to mVH 19 (SEQ ID NO: 62). See fig. 2K.

In some embodiments, the V _L CDR chains of the anti-hPG monoclonal antibody are V _L CDR 1.19, V _L CDR 2.19, and V _L CDR 3.19. In a specific embodiment, the V _L chain of the anti-hPG monoclonal antibody has an amino acid sequence corresponding to mV _L 19 (SEQ ID NO: 66). See fig. 2L.

In some embodiments, the CDR V _H chains of the anti-hPG monoclonal antibody are V _H CDR 1.3, V _H CDR 2.3, and V _H CDR 3.3, and the CDR V _L chains are V _L CDR 1.3, V _L CDR 2.3, and V _L CDR 3.3 . In a specific embodiment, the V _H chain of the anti-PG monoclonal antibody has an amino acid sequence corresponding to mV _H 3 (SEQ ID NO: 12) and the V _L chain has a sequence corresponding to mV _L 3 (SEQ ID NO: 13).

In some embodiments, the CDR V _H chains of the anti-hPG monoclonal antibody are V _H CDR 1.4, V _H CDR 2.4, and V _H CDR 3.4, and the CDR V _L chains are V _L CDR 1.4, V _L CDR 2.4, and V _L CDR 3.4 . In a specific embodiment, the V _H chain of the anti-hPG monoclonal antibody has the amino acid sequence corresponding to mVH 4 (SEQ ID NO: 14) and the V L chain has the sequence corresponding to mVL 4 (SEQ ID NO: 15).

In some embodiments, the CDR V _H chains of the anti-hPG monoclonal antibody are V _H CDR 1.8, V _H CDR 2.8 and V _H CDR 3.8, the CDR V _L chains are V _L CDR 1.8, V _L CDR 2.8 and

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V _L CDR 3.8. In a specific embodiment, the anti-hPG monoclonal antibody is the anti-hPG MAb 8 described herein and comprises the amino acid sequence corresponding to mVH 8 (SEQ ID NO: 59) and the amino acid sequence corresponding to mV _L 8 (SEQ

ID NO: 63).

In some embodiments, the VH chain CDRs of the anti-hPG monoclonal antibody are VH CDR 1.3, VH CDR 2.13, and VH CDR 3.13, the V _L chain CDRs are V _L CDR 1.13, V _L CDR 2.13, and V _L CDR 3.13. In a specific embodiment, the anti-hPG monoclonal antibody is the anti-hPG MAb 13 described herein and includes the amino acid sequence corresponding to mVH 13 (SEQ ID NO: 60) and the amino acid sequence corresponding to mV _L 13 (SEQ ID NO: 64).

In some embodiments, the CDR VH chains of the anti-hPG monoclonal antibody are VH CDR 1.16, VH CDR 2.16, and VH CDR 3.16, and the CDR V _L chains are V _L CDR 1.16, V _L CDR 2.16, and V _L CDR 3.16. In a specific embodiment, the anti-hPG monoclonal antibody is the anti-hPG MAb 16 described herein and includes the amino acid sequence corresponding to mV _H 16 (SEQ ID NO: 61) and the amino acid sequence corresponding to mV _L 16 (SEQ ID NO : 65).

In some embodiments, the CDR VH chains of the anti-hPG monoclonal antibody are VH CDR 1.19, VH CDR 2.19, and VH CDR 3.19, and the CDR V _L chains are V _L CDR 1.19, V _L CDR 2.19, and V _L CDR 3.19. In a specific embodiment, the anti-hPG monoclonal antibody is an anti-hPG MAb 19 as described herein and includes an amino acid sequence corresponding to mVH 19 (SEQ ID NO: 62) and an amino acid sequence corresponding to mVL 19 (SEQ ID NO: 66 ).

Anti-hPG monoclonal antibodies of this application include both intact molecules and antibody fragments (such as, for example, Fab and F(ab') ₂ fragments) that are capable of specific binding to hPG. Fab and F(ab') ₂ fragments do not contain the Fc fragment of the intact antibody, are more rapidly cleared from the animal or plant circulation, and have less non-specific tissue binding than the intact antibody (Wahl et al., 1983, J. Nucl. Med. , 24:316). Antibody fragments are thus useful in therapeutic applications, among other applications.

The term antibody fragment refers to a full length portion of an antibody, a binding site, or a variable region. Examples of antibody fragments include Fab, Fab', F(ab') ₂ and Fv fragments. The Fv fragment is the minimum fragment of an antibody that contains a complete target recognition and binding site. This region consists of a dimer of one heavy and one light chain of the variable domain in a direct non-covalent relationship (VH-VL dimer). This is a configuration in which the three CDRs of each variable domain interact to determine the target binding site on the surface of the VH-VL dimer. Often, six CDRs confer target binding specificity on an antibody. However, in some cases, even a single variable domain (or half of an Fv comprising only three target-specific CDRs) may have the ability to recognize and bind a target, albeit at a lower affinity than using a single binding site. Single chain Fv or scFv antibody fragments include the VH and V _L domains of the antibody, where these domains are present in the same polypeptide chain. In general, the Fv polypeptide further includes a polypeptide linker between the VH and V _L domains that allows the scFv to form the desired structure for target binding. Single domain antibodies consist of single VH or VL domains that show sufficient affinity for hPG. In a specific embodiment, the antibody, which consists of a single domain, is a camelized (overblue) antibody (see, for example, Riechmann, 1999, Journal of Immunological Methods, 231:2538).

The Fab fragment contains a light chain constant domain and a heavy chain first constant domain (CH1). Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy-terminal end of the heavy chain of the CH1 domain, including one or more cysteine residues from the hinge region of the antibody. F(ab') fragments are obtained by cleavage of the disulfide bond between the cysteines of the hinge region of the pepsin digest F(ab') ₂ . Additional chemical fusions of antibody fragments are known to those skilled in the art.

Anti-hPG monoclonal antibodies in accordance with this application may be chimeric antibodies. The term chimeric antibody, as used herein, refers to an antibody containing variable sequences that are derived from non-human immunoglobulins, such as a rat or mouse antibody, and human immunoglobulin constant regions, typically selected from a human immunoglobulin matrix. Methods for making chimeric antibodies are known in the art. See, for example, Morrison, 1985, Science, 229(4719):1202-7; Oi et al., 1986, BioTechniques, 4:214-221; Gillies et al., 1985, J. Immunol. Methods, 125:191-202; US patents No. 5807715; 4816567; and 4816397, which are introduced in this

- 14 041823 application as a reference in its entirety.

Anti-hPG monoclonal antibodies in accordance with this application can be humanized. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antibody target-binding subsequences) that contain minimal sequences which are derived from non-human immunoglobulins. In general, a humanized antibody will include substantially all of at least one, and typically two, variable domains in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of the consensus sequence. human immunoglobulin, and may be referred to as CDR-grafted. The humanized antibody may also include at least a portion of an immunoglobulin constant region (Fc), typically such as a human immunoglobulin consensus sequence. Methods for humanizing an antibody, including methods for constructing humanized antibodies, are known in the art. See, for example, Lefranc et al., 2003, Dev. Comp. Immunol., 27:55-77; Lefranc et al., 2009, Nucl. Acids Res., 37:D1006-1012; Lefranc, 2008, Mol. Biotechnol., 40:101-111; Riechmann et al., 1988, Nature, 332:323-7; US patents No. 5530101; 5585089; 5693761; 5693762; and 6180370, which refer to Queen et al., EP, 239400; PCT publication WO 91/09967; US patent No. 5225539; EP 592106; EP 519596; Padlan, 1991, Mol. Immunol. 28:489-498; Studnicka et al., 1994, Prot. Eng. 7:805-814; Roguska et al., 1994, Proc. Natl. Acad. Sci. 91:969-973; and US Pat. No. 5,565,332, all of which are incorporated herein by reference in their entirety.

Sequences of humanized anti-hPG monoclonal antibodies were designed for mouse anti-hPG monoclonal antibodies in accordance with the present invention, as described in the examples section below. Specific embodiments of humanized antibodies include antibodies comprising (1) any three V _L CDRs and any three VH CDRs disclosed in this application;

(2) a variable region heavy chain comprising the amino acid sequence of SEQ ID NO: 21 and a variable region light chain comprising the amino acid sequence of SEQ ID NO: 22;

(3) a variable region heavy chain comprising the amino acid sequence of SEQ ID NO: 23 and a variable region light chain comprising the amino acid sequence of SEQ ID NO: 24;

(4) a variable region heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 75, 77 and 79, and a variable region light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 76 and 78;

(5) a variable region heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 80 and 82, and a variable region light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 81, and 83;

(6) a variable region heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 84, 86 and 88, and a variable region light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 85, 87 and 89;

(7) a variable region heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 90, 92 and 94, and a variable region light chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 91, 93 and 95.

Anti-hPG monoclonal antibodies according to this application can be primatized. The term primatized antibody refers to an antibody comprising simian variable regions and human constant regions. Methods for obtaining a primatized antibody are known in the art. See, for example, US Pat. Nos. 5,658,570; 5681722; and 5,693,780, which are incorporated herein by reference in their entirety.

Included in anti-hPG monoclonal antibodies are antibodies that compete with a reference antibody, such as, for example, a polyclonal anti-hPG antibody, or any of the anti-hPG monoclonal antibodies disclosed in this application. Antibodies that compete with the anti-hPG monoclonal antibodies of this application are useful in a variety of diagnostic and therapeutic applications. Specific embodiments of suitable reference anti-hPG monoclonal antibodies include the following antibodies described in this application, for example, but not limited to:

antibodies comprising any three VL CDRs and any three VH CDRs disclosed in this application;

antibodies in which the VH chain has an amino acid sequence corresponding to

- 15 041823

SEQ ID NO: 12 (mVH 3) and the VL chain has an amino acid sequence corresponding to

SEQ ID NO: 13 (mV _L 3); and antibodies in which the VH chain has the amino acid sequence corresponding to SEQ ID NO: 14 (mVH 4) and the VL chain has the sequence corresponding to SEQ ID NO: 15 (mV _L 4), antibodies in which the V _H chain has the amino acid the sequence corresponding to SEQ ID NO: 59 (mVH 8), and the V _L chain has an amino acid sequence corresponding to SEQ ID NO: 63 (mVL 8);

antibodies in which the V _H chain has the amino acid sequence of SEQ ID NO: 60 (mVH 13) and the V _L chain has the amino acid sequence of SEQ ID NO: 64 (mVL 13);

antibodies in which the V _H chain has the amino acid sequence of SEQ ID NO: 61 (mV _H 16) and the V _L chain has the amino acid sequence of SEQ ID NO: 65 (mVL 16);

antibodies in which the V _H chain has the amino acid sequence of SEQ ID NO: 62 (mV _H 19) and the V _L chain has the amino acid sequence of SEQ ID NO: 66 (mVL 19), or any combination of VH and V _L chains disclosed in this application.

Приемлемые эталонные антитела также включают антитела, полученные с помощью гибридомы, выбранной из группы, которая состоит из 43B9G11, WE5H2G7, 6В5В11С10, 20D2C3G2, 1B4A11D11, 1B6A11F2, 1В11Е4В11, 1C10D3B9, 1D8F5B3, 1Е1С7В4, 2В4С8С8, 2B11E6G4, 2С6С3С7, 2H9F4B7, 1Е9А4А4, 1E9D9B6 , 1C8D10F5, 1A7C3F11, 1B3B4F11, 1C11F5E8, 1F11F5E10, 1F11F5G9 and 1A11F2C9;

antibodies that bind to an epitope that includes hPG residues 10-14 (SEQ ID NO: 28), hPG residues 9-14 (SEQ ID NO: 29), hPG residues 4-10 (SEQ ID NO: 30), residues 2 -10 hPG (SEQ ID NO: 31) or 2-14 hPG residues (SEQ ID NO: 32); and antibodies that bind to an epitope that includes hPG residues 71-74 (SEQ ID NO: 33), hPG residues 69-73 (SEQ ID NO: 34), hPG residues 76-80 (SEQ ID NO: 35), or residues 67-74 hPG (SEQ ID NO: 36).

The ability to compete with a monoclonal antibody of the present invention for binding to a PG, eg hPG, can be assayed, for example, using a competitive assay as described below. 96-well plates were coated using an immobilized antibody (a polyclonal or monoclonal antibody that recognizes the N- or C-terminal portion of progastrin that differs from the epitope that is recognized by the reference monoclonal antibody), at a concentration that is selected in the range of 1-10 μg/ ml overnight at 4°C (0.1-1 µg/well). After blocking with 0.1% Tween-20/0.1% BSA in PBS (blocking buffer) for 2 h at 22°C, recombinant human progastrin was added at a concentration of 10 pM-1 nM (10-1000 pg/well) and incubated for 2 h at 22°C. Thereafter, the biotinylated reference anti-hPG monoclonal antibody or mixture containing the reference monoclonal antibody was added along with increasing concentrations of unlabeled test antibody and incubated for 1 hour at 22°C. After washing, the determination was carried out by incubation for 1 hour at 22° C. with a fluorogenic substrate for horseradish peroxidase, after which the relative light units (RLU) were quantified in a luminometer. Analyzes were carried out in duplicate. Antibodies that compete with the reference anti-hPG monoclonal antibody of the present invention inhibit the binding of the reference antibody to hPG. An antibody that binds the same epitope as the control antibodies will be able to compete for binding with the reference antibody and thus will significantly reduce (eg, at least 50%) the binding of the reference antibody, as evidenced by the reduction in the amount of bound label. The reactivity of a (labeled) reference antibody in the absence of a completely mismatched antibody will be a high control value. A low control value will be obtained by incubating unlabeled test antibody with cells expressing progastrin, followed by incubation of the cell/antibody mixture with labeled control antibody of exactly the same isotype when competition occurs and binding of the labeled antibody is reduced. When performing the assay, a significant decrease in the reactivity of the labeled antibody in the presence of the test antibody is indicative of the test antibody being recognized by substantially the same epitope.

Inhibition binding can be expressed as an inhibition constant or Ki, which is calculated according to the following formula:

Ki = 1C50/ (1 + [Reference Ab Concentration]/Kd) where IC50 of test antibody is the concentration of test antibody that provides a 50% reduction in reference antibody binding, a

Kd is the dissociation constant of a reference antibody, a measure of its affinity for progastrin.

Antibodies that compete with the anti-hPG monoclonal antibodies disclosed in this

- 16 041823 application may have Ki from 10 pM to 10 nM under the assay conditions described in this application.

In various embodiments, the unlabeled anti-hPG monoclonal antibody of this application reduces the binding of the labeled reference antibody by at least 40%, at least 50%, at least 60%, at least 70%, at least by 80%, at least 90%, 100%, or a percentage that fluctuates within any of the above ranges (for example, an anti-hPG monoclonal antibody in accordance with this application reduces the binding of a labeled reference antibody by 50-70% ) when the anti-hPG monoclonal antibody is used at a concentration of 0.01, 0.08, 0.4, 2, 10, 50, 100 µg/ml, or at a concentration that fluctuates within the range of the above values (for example, at a concentration , which ranges from 2 to 10 µg/ml).

When performing an antibody competition study between a reference antibody and any antibody of interest (of mismatched species or isotype), the reference antibody may first be labeled with a detectable label, such as biotin or an enzymatic (or even radioactive) label, in order to allow subsequent identification. . In this case, the labeled reference antibody (at a fixed or increasing concentration) is incubated with a known amount of progastin. The unlabeled test antibody is then added to the pre-bound complex of progastrin and labeled antibody. The intensity of the bound label is measured. If the test antibody competes with the labeled antibody by binding an overlapping epitope, then the intensity will decrease relative to the binding of the control labeled antibody in the absence of the test antibody.

Competition analyzes are known and can be adapted to the analysis above to obtain comparable results. This assay can be any of a wide variety of immunoglobulin assays that rely on antibody competition and the reference antibody will be determined by determining their label, for example using streptavidin in the case of a biotinylated antibody, or using a chromogenic substrate in the case of an enzymatic label (such , as a substrate based on 3,3'5,5'-tetramethylbenzidine (TMB) using a peroxidase enzyme) or by simply detecting a radioactive label or a fluorescent label.

Also included in this application are anti-hPG monoclonal antibodies that are derivatized, covalently modified, or conjugated with other molecules for use in diagnostic and therapeutic methods. 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 using known protecting/blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein, and etc. Any of the numerous chemical modifications can be carried out using known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. Additionally, the derivative may contain one or more non-classical amino acids.

In another example, the antibodies of the present invention can be attached to poly(ethylene glycol) (PEG) moieties. In a specific embodiment, the antibody is an antibody fragment and the PEG residues 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 naturally exist in the antibody fragment or may be introduced into the fragment using recombinant DNA techniques. See, for example, US patent No. 5219996. Multiple sites can be used to attach two or more PEG molecules. The PEG residues can be covalently linked via the thiol group of at least one cysteine residue located in the antibody fragment. When a thiol group is used as the point of attachment, suitably activated effector moieties may be used, for example selective thiol derivatives such as maleimides and cysteine derivatives.

In a specific example, the anti-hPG monoclonal antibody conjugates are 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 EP 0948544. 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); and 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. PEG can attach to cysteine at the hinge site. In one example, a PEG-modified Fab' fragment contains a maleimide group covalently linked to one thiol group at the modified hinge. The lysine residue may be covalently linked to a maleimide group, and a methoxypoly(ethylene glycol) polymer which has a molecular weight of approximately 20,000 Da can be attached to each amino group of the lysine residue. Total molecular weight

- 17 041823

The PEG attached to the Fab' fragment may thus be approximately 40,000 Da.

Anti-hPG monoclonal antibodies include labeled antibodies useful in diagnostic applications. Antibodies can be used for diagnostic purposes, for example, to determine the expression of a target of interest in a specific cell, tissue, or serum, or to monitor the development or progression of an immunological response as part of a clinical testing procedure, for example, to determine the effectiveness of a given treatment regimen. Detection can be improved by fusing the antibody with a detectable substance or label. The label may be conjugated directly or indirectly to an anti-hPG monoclonal antibody of the present application. The label may itself be detectable (eg, radioisotope labels, isotope labels, or fluorescent labels) or, in the case of an enzymatic label, may catalyze the chemical conversion of a substrate compound or composition that is detectable. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals used in various positron emission tomography techniques, and non-radioactive paramagnetic metal ions. The analyte may be bound or conjugated to the antibody (or fragment thereof) directly or indirectly via an intermediate (such as, for example, a linker known in the art) using techniques known in the art. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferase; US Pat. No. 4,737,456), luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, β-galactosidase, acetylcholinesterase , glucoamylase, lysozyme, saccharide oxidase (eg, glucose oxidase, galactose oxidase, and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (such as uricase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of acceptable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, dimethylamine-1-naphthalenesulfonyl chloride or phycoerythrin and the like; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin and aequorin; examples of acceptable isotopic materials include ¹³ C, ¹⁵ N and deuterium; and examples of radioactive materials include ¹²⁵ I, ¹³¹ I, ⁿ¹ In, or ⁹⁹ Tc.

Anti-hPG monoclonal antibodies of all origins are included in this invention. Non-limiting exemplary natural antibodies include those derived from humans, monkeys, chickens, goats, rabbits, and rodents (eg, rats, mice, and hamsters) (see, for example, Lonberg et al., WO 93/12227; US Patent No. 5,545,806; and Kucherlapati, et al., WO 91/10741; US Pat. No. 6,150,584, which are all incorporated herein by reference in their entirety). Natural antibodies are antibodies that are produced by a host animal after immunization with an antigen, such as a polypeptide.

Nucleic acids and expression systems.

The present description encompasses nucleic acid molecules encoding immunoglobulin light and heavy chain genes for anti-hPG monoclonal antibodies, vectors comprising such nucleic acids, and host cells capable of producing anti-hPG monoclonal antibodies in accordance with the invention.

The anti-hPG monoclonal antibody of this application can be produced by recombinant expression of immunoglobulin light and heavy chain genes in a host cell. For recombinant expression of an antibody, a host cell is transfected using one or more recombinant expression vectors that carry DNA fragments encoding the immunoglobulin light and heavy chain of the antibody, such that the light and heavy chains are expressed in the host cell and optionally secreted into the culture medium. host cells, antibodies can be recovered from this medium. Standard recombinant DNA techniques are used to generate antibody heavy and light chain genes, introduce these genes into recombinant expression vectors, and insert these vectors into host cells such as those described in Molecular Cloning; A Laboratory Manual, 2nd ed. (Sambrook, Fritsch and Maniatis (eds.), Cold Spring Harbor, N.Y., 1989), Current Protocols in Molecular Biology (Ausubel, F.M. et al., eds., Greene Publishing Associates, 1989) and US Pat. No. 4,816,397.

To obtain nucleic acids encoding such anti-hPG monoclonal antibodies, DNA fragments encoding the light and heavy chain variable regions are first obtained. Such DNA can be obtained by amplifying and modifying embryonic DNA or cDNA encoding variable light and heavy chain sequences, for example, using the polymerase chain reaction (PCR). Embryonic DNA sequences for the human heavy and light chain variable region genes are known in the art (see, e.g., Lefranc et al., 2003, Dev. Comp. Immunol., 27:55-77; Lefranc et al., 2009, Nucl. Acids Res., 37:D1006-1012; Lefranc, 2008, Mol. Biotechnol., 40:101-111; VBASE human germline sequence database; see also Kabat, E. A. et al., 1991,

- 18 041823

Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH publication number 91-3242; Tomlinson et al., 1992, J. Mol. Biol., 22T:116-198; and Soh et al., 1994, Eur. J.

Immunol., 24:827-836; the content of each of which is incorporated into this application by reference).

When DNA fragments encoding the V _H and V _L segments associated with an anti-hPG monoclonal antibody are obtained, these DNA fragments can be further manipulated using standard recombinant DNA techniques, for example, to convert the variable region genes into a full-length antibody gene chain, into Fab fragment genes or scFv gene. In these manipulations, a DNA fragment encoding a V _L - or VH is operably linked to another DNA fragment encoding a different protein, such as an antibody constant region or a flexible linker. The term operably linked, as used herein, is intended to mean that two DNA fragments are joined such that the amino acid sequences encoded by the two DNA fragments remain in-frame.

An isolated DNA encoding a V _H region can be converted into a full length heavy chain gene by operatively linking the DNA encoding V _H to another DNA molecule encoding heavy chain constant regions (CH1, CH ₂ , CH ₃ and optionally CH ₄ ). Human heavy chain constant region gene sequences are known in the art (see, e.g., Kabat, EA, et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH publication number 91-3242), and DNA fragments spanning these regions can be obtained using standard PCR amplification. The heavy chain constant region may be an IgG1, IgG2, IgG ₃ , IgG ₄ , IgA, IgE, IgM or IgD constant region, but in certain specific embodiments it is an IgG1 or IgG ₄ constant region. To create a heavy chain Fab gene, the VH-encoding DNA can be operatively linked to another DNA molecule encoding only one heavy chain CH1 constant region.

An isolated DNA encoding a VH region can be converted into a full length light chain gene (as well as a light chain Fab gene) by operably linking the DNA encoding V _L to another DNA molecule encoding a light chain constant region, CL. Human light chain constant region gene sequences are known in the art (see, for example, Kabat, EA et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH no. publications 91-3242), and DNA fragments spanning these regions can be obtained using standard PCR amplification. The light chain constant region may be a kappa or lambda constant region, but in some embodiments it is a kappa constant region. To create the scFv gene, DNA fragments encoding VH and VL are operatively linked to another fragment encoding a flexible linker, such as one encoding the amino acid sequence (Gly ₄ ~Ser) ₃ (SEQ ID NO: 99), such that V _H and V _L sequences can be expressed as a contiguous single strand protein containing VL and VH regions connected by a flexible linker (see, e.g., Bird et al., 1988, Science, 242:423-426; Huston et al., 1988, Proc. Natl. Acad. Sci., USA, 85:5879-5883; McCafferty et al., 1990, Nature, 348:552-554).

For the expression of anti-hPG monoclonal antibodies in accordance with the present invention, DNAs encoding partial or complete heavy or light chains obtained as described above are inserted into expression vectors such that the genes are operably linked to transcription and translation control sequences. In this context, the term operably linked is intended to mean that the antibody gene is ligated into the vector such that transcription and translation control sequences within the vector serve their function of regulating transcription and translation of the antibody gene. The expression vector and expression control sequences are chosen to be compatible with expression in the selected host cell. The antibody light chain gene and the antibody heavy chain gene can be inserted into separate vectors or, more typically, both genes are inserted into the same expression vector.

The antibody genes are inserted into the expression vector using standard techniques (eg, ligation of complementary restriction sites into the antibody gene fragment and vector, or blunt-end ligation if no restriction sites are present). Before inserting the light and heavy chain sequences derived from the anti-hPG monoclonal antibody, the expression vector may already carry antibody constant region sequences. For example, one approach for converting V _H and V _L sequences derived from an anti-hPG monoclonal antibody into full length antibody genes is to insert them into expression vectors that already encode the heavy chain constant region and the light chain constant region, respectively, as that the VH segment is operatively associated with the CH segment(s) within the vector, and that the VL segment is operatively associated with the CL segment within the vector. Additionally or alternatively, the recombinant expression vector may encode a signal peptide that promotes secretion of the antibody chain from the host cell. The antibody chain gene can be cloned into a vector such that the signal peptide is linked in frame to the amino-terminal end of the antibody chain gene. The signal peptide can be an immunoglobulin signal peptide or a heterologous signal peptide (ie, a signal peptide from a protein other than immunoglobulin).

In addition to the antibody chain genes, the recombinant expression vectors of this application carry regulatory sequences that control the expression of the antibody chain genes in the host cell. The term regulatory sequence is intended to include promoters, enhancers, and other expression control elements (eg, polyadenylation signals) that control transcription and translation of antibody chain genes. Such regulatory sequences are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology, 185 (Academic Press, San Diego, CA, 1990). One skilled in the art will appreciate that the construction of an expression vector, including the choice of regulatory sequences, may depend on such factors as the choice of the host cell to be transformed, the level of expression of the desired protein, and so on. Suitable regulatory sequences for expression in a mammalian host cell include viral elements that direct high levels of protein expression in mammalian cells, such as promoters and/or enhancers that are derived from cytomegalovirus (CMV) (such as the CMV promoter/enhancer) vacuolating simian virus 40 (SV40) (such as the SV40 promoter/enhancer), adenovirus (eg, from the adenovirus major late promoter (AdMLP)) and polyoma. For further descriptions of viral regulatory elements and their sequences, see, for example, US Pat. No. 5,168,062 which refers to Stinski, US Pat. No. 4,510,245 which refers to Bell et al., and US Pat. No. 4,968,615 which refers to Schaffner et al. .

In addition to the antibody chain genes and regulatory sequences, the recombinant expression vectors of this application may carry additional sequences, such as sequences that regulate replication of the vector in host cells (eg origins of replication) and selectable marker genes. The selectable marker gene assists in the selection of host cells into which the vector has been introduced (see, for example, US Pat. For example, typically a selectable marker gene confers resistance to drugs such as G418, hygromycin, or methotrexate in the host cell into which the vector has been introduced. Suitable selectable marker genes include the dihydrofolate reductase (DHFR) gene (for use in DHFR host cells with methoctrexate selection/amplification) and the peo gene (for G418 selection). For expression of the light and heavy chains, the expression vector(s) encoding the heavy and light chains are transfected into a host cell using standard techniques. The various forms of the term transfection are intended to cover a wide variety of techniques that are traditionally used to introduce exogenous DNA into a prokaryotic or eukaryotic host cell, e.g., electroporation, lipofection, calcium phosphate precipitation, DEAE-based transfection, dextran, and the like.

It is possible to express antibodies according to this application either in prokaryotic or eukaryotic host cells. In some embodiments, antibody expression occurs in eukaryotic cells, eg, mammalian host cells, with optimal secretion of properly assembled and immunologically active antibody. Exemplary mammalian host cells for expressing the recombinant antibody of this application include Chinese Hamster Ovary (CHO) cells (including the DHFR CHO cells described in Urlaub and Chasin, 1980, Proc. Natl. Acad. Sci., USA, 77:4216 -4220, which are used with a DHFR selectable marker, eg as described in Kaufman and Sharp, 1982, Mol. Biol., 159:601-621), NSO myeloma cells, COS cells, and SP2 cells. When recombinant expression vectors encoding antibody genes are introduced into mammalian host cells, antibodies are obtained by culturing the host cells for a period of time sufficient to effect expression of the antibody in the host cells or secretion of the antibody into the culture medium in which the host cells are grown. Antibodies can be recovered from the culture medium using standard protein purification methods. Host cells can also be used to generate parts of an intact antibody, such as Fab fragments or scFv molecules. It is understood, however, that variations on the procedure described above are within the scope of this disclosure. For example, it may be desirable to transfect a host cell with DNA encoding either the light chain or the heavy chain (but not both) of an anti-hPG monoclonal antibody of the invention.

The recombinant DNA technique can also be used to remove some or all of the DNA encoding either the light chain or the heavy chain that is not necessary for binding to hPG. Molecules expressed from such a truncated DNA molecule may also be covered by antibodies in accordance with this disclosure.

For recombinant expression of an anti-hPG monoclonal antibody of this disclosure, a host cell can be co-transfected with two expression vectors of this application, a first vector encoding a polypeptide derived from a heavy chain and a second vector encoding a polypeptide derived from the light chain. The two vectors may contain selectable markers, or they may each contain

- 20 041823 separate selectable marker. Alternatively, a single vector can be used that encodes both heavy and light chain polypeptides.

In one embodiment, additional changes or mutations may be introduced into the coding sequence of a nucleic acid encoding one or more portions of an anti-hPG monoclonal antibody, for example, to produce a nucleic acid encoding antibodies with different CDR sequences, an antibody with reduced affinity for the Fc receptor, or antibodies of various subclasses.

Anti-hPG monoclonal antibodies in accordance with this application can also be obtained using chemical synthesis (for example, using the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984, The Pierce Chemical Co., Rockford, III) . An antibody variant can also be made using cell-free methods (see, for example, Chu et al., Biochemia, No. 2, 2001 (Roche Molecular Biologicals).

In one embodiment, the anti-hPG monoclonal antibody according to this application is obtained by recombinant expression, it can be purified using any method known in the art to purify the immunoglobulin molecule, for example, by chromatography (for example, ion exchange, affinity and size exclusion chromatography), centrifugation, differential solubility, or any other standard technique for protein purification. Additionally, anti-hPG monoclonal antibodies of the present invention, or fragments thereof, can be fused to heterologous polypeptide sequences as described herein or otherwise known in the art to improve purification.

In one embodiment, the isolated anti-hPG monoclonal antibody can be, if desired, further purified, for example, by high performance liquid chromatography (see, for example, Fisher, Laboratory Techniques In Biochemistry and Molecular Biology (Work and Burdon, eds., Elsevier, 1980) or by size exclusion chromatography on a Superdex™ 75 column (Pharmacia Biotech AB, Uppsala, Sweden).

The present disclosure provides host cells capable of producing anti-hPG monoclonal antibodies. Host cells can be cells constructed using recombinant DNA techniques to express genes encoding the heavy and light chain, or hybridomas that are derived from a suitable organism and selected for their ability to produce the desired antibodies.

Host cells capable of producing anti-PG monoclonal antibodies may be hybridomas. Methods for producing hybridomas are known in the art (see, for example, Kohler and Milstein, 1975, Nature, 256:495), one such example is provided below. In general, a host animal, such as a mouse, is immunized with an immunogen, such as a peptide of interest, in order to induce the development of lymphocytes, such as spleen cells, that produce antibodies capable of specifically binding the immunogen. Alternatively, isolated lymphocytes, including spleen cells, lymph node cells, or peripheral blood lymphocytes, can be immunized in vitro. The lymphocytes are then fused with an immortalized cell line, such as a myeloma cell line, using a suitable agent (eg, polyethylene glycol) to form a hybridoma cell line. Suitable immortalized cell lines may be of mammalian origin such as mouse, bovine or human. The hybridoma cells are then cultured in any suitable medium that contains one or more substances that inhibit the growth or survival of the non-hybrid, immortalized cells. For example, when parental cells lacking the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT) are used, hybrid cells can be grown in hypoxanthine, aminopterine and thymidine containing media (HAT media), which inhibits the growth of parental non-hybrid cells.

In some embodiments, the N-terminal anti-hPG monoclonal antibodies have variable light chain (VL) CDRs that correspond to the V _L of the monoclonal antibody that is derived from hybridomas 43B9G11 (anti-hPG producing MAb 1), WE5H2G7 (anti-hPG producing MAb 2) , 6B5B11C10 (producing anti-hPG MAb 3), 20D2C3G2 (producing anti-hPG MAb 4), 1E9A4A4 (producing anti-hPG MAb 15), 1E9D9B6 (producing anti-hPG MAb 16), 1C8D10F5 (producing anti-hPG MAb 17) , 1A7C3F11 (producing anti-hPG MAb 18), 1B3B4F11 (producing anti-hPG MAb 19), and 1C11F5E8 (producing anti-hPG MAb 20).

In some embodiments, the N-terminal anti-hPG monoclonal antibodies have VL and VH CDRs that match the VL and _VH CDRs of the monoclonal antibody derived from the above hybridomas.

In some embodiments, the C-terminal anti-hPG monoclonal antibodies have VL CDRs that correspond to the VLs of a monoclonal antibody that is derived from hybridomas 1B4A11D11 (producing anti-hPG MAb 5), 1B6A11F2 (producing anti-hPG MAb 6), 1B11E4B11 (producing anti- hPG MAb 7), 1C10D3B9 (producing anti-hPG MAb 8), 1D8F5B3 (producing anti-hPG MAb 9), 1E1C7B4 (producing anti-hPG MAb 10), 2B4C8C8 (producing anti-hPG MAb 11), 2B11E6G4 (producing anti-hPG MAb 12), 2C6C3C7 (producing anti-hPG

MAb 13), 2H9F4B7 (producing anti-hPG MAb 14), 1F11F5E10 (producing anti-hPG MAb 21),

1F11F5G9 (producing anti-hPG MAb 22) and 1A11F2C9 (producing anti-hPG MAb 23).

In some embodiments, the C-terminal anti-hPG monoclonal antibodies have CDR V _L and V H that correspond to the CDR V _L and V _H of the monoclonal antibody derived from the above hybridomas.

In one embodiment, a host cell is provided that is capable of producing an anti-hPG antibody comprising a heavy chain variable region comprising SEQ ID NO: 12 and a light chain variable region comprising SEQ ID NO: 13. In one embodiment, a host cell is provided that is capable of producing anti -hPG antibody comprising a heavy chain variable region comprising SEQ ID NO: 14 and a light chain variable region comprising SEQ ID NO: 15. In one embodiment, a host cell capable of producing an anti-hPG antibody comprising a heavy chain variable region is provided, comprising SEQ ID NO: 59, and a light chain variable region comprising SEQ ID NO: 63. In one embodiment, a host cell capable of producing an anti-hPG antibody comprising a heavy chain variable region comprising SEQ ID NO: 60 and a variable region is provided light chain comprising SEQ ID NO: 64. In one embodiment, a host cell capable of producing anti-hPG anti a body comprising a heavy chain variable region comprising SEQ ID NO: 61 and a light chain variable region comprising SEQ ID NO: 65. In one embodiment, a host cell capable of producing an anti-hPG antibody comprising a heavy chain variable region comprising SEQ is provided. ID NO: 62, and a light chain variable region comprising SEQ ID NO: 66.

In some embodiments, the host cell according to this application comprises a nucleic acid selected from the nucleotide sequence encoding the heavy chain variable region polypeptide of SEQ ID NOs: 12, 14, 59, 60, 61, and 62; and a nucleic acid selected from a nucleotide sequence encoding a light chain variable region polypeptide of SEQ ID NOs: 13, 15, 63, 64, 65, and 66.

In some embodiments, the heavy chain variable region is encoded by a nucleic acid sequence selected from SEQ ID NOs: 16, 18, 67, 68, 69, and 70. In some embodiments, the light chain variable region is encoded by a nucleic acid sequence selected from SEQ ID NO: 17, 19, 71, 72, 73 and 74.

In some embodiments, polynucleotide sequences are provided that encode the heavy chain variable region of a humanized anti-hPG monoclonal antibody. Specific embodiments include polynucleotides encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 21, 23, 75, 77, 79, 80, 82, 84, 86, 88, 90, 92, and 94. B In some embodiments, polynucleotide sequences encoding the heavy chain variable region of a humanized anti-hPG monoclonal antibody are provided. Specific embodiments include polynucleotides encoding a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 24, 76, 78, 81, 83, 85, 87, 89, 91, 93 and 95.

Biological activities of anti-hPG monoclonal antibodies PG is implicated in the survival and/or proliferation of CRC tumor cells. Without intending to be bound by any theory of mechanism, it should be noted that through PG binding, neutralizing anti-PG monoclonal antibodies are expected to block or inhibit the ability of PG to interact with its signaling partner(s). in turn blocks and inhibits cellular responses to progastrin such as cell proliferation, reduced cell differentiation and/or reduced cell death, and/or tumor growth. As a consequence of such activities, the anti-hPG neutralizing monoclonal antibodies of this application can be used in a variety of in vitro, in vivo and ex vivo contexts to bind PG and block PG dependent signaling.

Accordingly, the application provides methods for inhibiting PG dependent responses in CRC cells. In general, the methods include contacting CRC cells with or exposing a cell population to a neutralizing anti-PG monoclonal antibody in an amount effective to inhibit one or more PG-induced CRC cell responses, such as proliferation and/or survival of CRC cells. Their proliferation or inhibition can be determined in vitro and in vivo according to assays to measure the increase in cell number, tumor number or tumor size over a period of time. Assays for inhibiting cell and tumor proliferation are well known in the art.

Blocking PG-dependent signaling can inhibit the survival of CRC cells by increasing cell death. Inhibition of CRC cell survival in vitro or in vivo can be determined by measuring the reduction in the number of viable cancer cells over a period of time (eg, within 24 or 48 hours). Assays for determining cell death are well known in the art. Additionally, an example of a cell survival assay is provided in this application.

- 22 041823

The study also suggests that inhibition of PG-dependent signaling in CRC tumor cells may inhibit CRC cell survival by triggering programmed cell death or apoptosis. Apoptosis induction can be determined using any of the methods known in the art, including, but not limited to, measuring changes in the expression of genes with pro- or anti-apoptosis activity. For example, an increase in expression over a period of time (eg, within 48 hours) of a pro-apoptotic gene, such as, for example, Bax, is indicative of an increase in apoptosis. Likewise, a decrease in expression over a period of time (eg, within 72 or 96 hours) of an anti-apoptotic gene, such as, for example, but not limited to, Bcl-2, is indicative of an increase in apoptosis. Techniques for determining changes in gene expression, such as quantitative real-time PCR, are well known in the art. See, for example, Hollande et al. WO 2007/135542.

Inhibition of progastrin-dependent signaling also stimulates cell differentiation. Accordingly, methods for inhibiting the proliferation and/or survival of CRC cells comprise administering an amount of a neutralizing anti-PG monoclonal antibody effective to induce proliferation of CRC cells in vitro or in vivo. Differentiation of CRC cells can be determined by measuring the increase over a period of time (for example, within 24 or 48 hours) in the expression of genetic markers for cell proliferation, such as, for example, but not limited to, Muc-2 or other markers for differentiated intestinal cells (for example, goblet cells). Changes in gene expression can be measured using any of the means known in the art. See, for example, Hollande et al. WO 2007/135542. Other genes whose expression or repression is PG dependent, such as ICAT, may also be assessed using standard methods. See ibid.

pharmaceutical compositions.

Anti-PG monoclonal antibodies can be formulated as a composition. Compositions may optionally be provided herein, which may include one or more additional therapeutic agents, such as the second therapeutic agents described below. The compositions will typically be provided as part of a sterile pharmaceutical composition, which will normally include a pharmaceutically acceptable carrier. This composition may be presented in an acceptable form (depending on the desired method of administration to the patient).

The anti-hPG monoclonal antibodies of this application may be administered to a patient using a variety of routes such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intraocular, topical, intrathecal, and intracerebroventricular. The most appropriate route for administration in each case will depend on the particular antibody, the subject, the nature and severity of the disease, and the physical condition of the subject. The antibody may be formulated as an aqueous solution and may be administered by subcutaneous injection.

The pharmaceutical composition can be conventionally presented in unit dose forms containing a predetermined amount of the anti-hPG monoclonal antibody of this dosage application. Such a unit form 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 the present invention may take various forms depending on, for example, the condition being treated or the route of administration.

Pharmaceutical compositions in accordance with this application can be prepared for storage in the form of a lyophilized preparation or aqueous solutions by mixing an antibody having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers, which are typically used in the field of technology (which are all referred to in this application by carriers), i.e. buffering agents, stabilizing agents, preservatives, isotonic agents, non-ionic surfactants, antioxidants, and various other additives. See Remington's Pharmaceutic Sciences, 16th ed. (Osol, ed. 1980). Such additives 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 can be presented at a concentration that ranges from about 2 mM to about 50 mM. Suitable buffering agents for use in accordance with the present invention include both organic and inorganic acids and their salts, such as citrate buffers (for example, a mixture of monosodium citrate-disodium citrate, a mixture of citric acid-trisodium citrate, a mixture of citric acid-monosodium citrate and etc.), 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 potassium acid-tartrate, tartaric acid-sodium hydroxide mixture, etc.), fumarate buffers (e.g. fumaric acid-monosodium fumarate, fumaric acid-disodium fumarate, monosodium fumarate-disodium fumarate, etc.), gluco

- 23 041823 Nat buffers (e.g. gluconic acid-sodium gluconate mixture, gluconic acid-sodium hydroxide mixture gluconic acid-potassium gluconate, etc.), oxalate buffers (e.g. oxalic acid-sodium oxalate mixture, oxalic acid-sodium hydroxide mixture , oxalic acid-potassium oxalate, etc.), lactate buffers (e.g. lactic acid-sodium lactate, lactic acid-sodium hydroxide, lactic acid-potassium lactate, etc.) and acetate buffers (e.g. , acetic acid-sodium acetate mixture, acetic acid-sodium hydroxide mixture, etc.). In addition, histidine buffers or buffers based on trimethylamine salts such as Tris can be used.

Preservatives can be added to slow down the growth of bacteria, and they can be added in amounts that vary from 0.2 to 1% (w/v). Suitable preservatives suitable for use in accordance with the present invention include phenol, benzyl alcohol, metacresol, methyl paraben, propyl paraben, octadecyldimethylbenzyl ammonium chloride, benzalkonium halides (e.g., chloride, bromide, and iodide), hexamethonium chloride, and alkyl parabens such as methyl or ethyl paraben, catechol, resorcinol, cyclohexanol and 3-pentanol. Isotonizing agents, sometimes known as stabilizers, may be added to provide isotonicity to the liquid composition of the present invention and include polyhydric sugar alcohols, for example trihydric or higher sugar alcohols such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol. Stabilizers are a broad category of excipients that can have a wide range of functions from a bulking agent to an additive that increases the solubility of a therapeutic agent or helps prevent denaturation or sticking to the container wall. Exemplary stabilizers may 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 and sugar alcohols such as lactose, trehalose, stachyose, mannitol, sorbitol, xylitol, myoinositol, galactitol, glycerin and the like, including cyclitols such as inositol, polyethylene glycol;

amino acid polymers;

sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, a-monothioglycerol and sodium thiosulfate;

low molecular weight polypeptides (eg, peptides containing 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, and 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 weight of the active protein.

Non-ionic surfactants and detergents (also known as wetting agents) may be added to help dissolve the therapeutic agent as well as to protect the therapeutic protein from agitation-induced aggregation, which also allows the composition to be subjected to surface shear without denaturation. squirrel. Suitable neoin surfactants include polysorbates (20, 80, etc.), polyoxamers (184, 188, etc.), Pluronic polyols, polyoxyethylene sorbitan monoesters (TWEEN®-20, TWEEN®-80, etc.). e.) 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 excipients include bulking agents (eg starch), chelating agents (eg EDTA), antioxidants (eg ascorbic acid, methionine, vitamin E), and co-solvents.

The anti-PG monoclonal antibodies may be administered alone as mixtures of one or more anti-PG monoclonal antibodies, in admixture or combination with other agents useful in the treatment of CRC, or in conjunction with other CRC therapies. Examples of acceptable combination and additional therapeutic measures are provided below.

The present invention encompasses pharmaceutical kits containing neutralizing anti-hPG monoclonal antibodies (including antibody conjugates) in accordance with the application. A pharmaceutical kit is a package that includes a neutralizing anti-hPG monoclonal antibody (for example, either in lyophilized form or as an aqueous solution) and one or more of the following:

a second therapeutic agent, for example as described below;

a device for administering an anti-hPG monoclonal antibody, such as a pen, needle, and/or

- 24 041823 syringe; and pharmaceutical grade water or antibody resuspension buffer if the antibody is in lyophilized form.

Each anti-hPG monoclonal antibody unit dose may be packaged separately 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, ten unit doses, or more ). In a specific embodiment, one or more unit doses are each placed in a syringe or pen.

effective dosages.

Neutralizing anti-PG monoclonal antibodies or compositions thereof will generally be used in an amount effective to achieve the intended result, for example in an amount effective to treat CRC in a subject in need thereof. Pharmaceutical compositions comprising anti-PG neutralizing monoclonal antibodies may be administered to patients (eg, human patients) at therapeutically effective dosages. As used herein, a therapeutically effective dose is an amount that provides a therapeutic benefit. In the context of CRC therapy, therapeutic benefit means attenuation of CRC, including any one or combination of stopping or slowing the progression of CRC (e.g., from the first stage of colorectal cancer to the next), stopping or delaying the worsening or worsening of symptoms or signs of CRC, reducing the severity of CRC, including CRC remission, inhibition of CRC tumor cell proliferation, tumor CRC size or number of tumor CRCs, or reduction of serum PG levels.

The amount of anti-PG neutralizing monoclonal antibodies that are 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 (eg, whether a second therapeutic agent is being used), the age and condition of the particular the subject being treated, the sensitivity of the patient being treated to anti-PG monoclonal antibodies. An acceptable dosage can be readily determined by one of skill in the art. Ultimately, the attending physician will determine the acceptable dosage used. This dosage will be repeated as often as necessary. If side effects develop, then the amount and/or frequency of dosing will be changed or reduced in accordance with normal clinical practice. The exact dosage and treatment regimen will be determined by monitoring 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, an initial dose for use in animals may be formulated to achieve a circulating blood or serum concentration of the anti-PG monoclonal antibody that is at or above the binding affinity level of the antibody for progastrin as measured in vitro. Calculating dosages to achieve such circulating blood or serum concentrations taking into account the bioavailability of a particular antibody is within the ability of the skilled artisan. For guidance, the reader may be referred to Fingl & Woodbury, General Principles in Goodman and Gilman's The Pharmaceutic Basis of Therapeutics, Chapter 1, latest edition, Pagamonon Press, and the references therein.

Initial dosages may be estimated from in vivo data such as animal models. Animal models suitable for testing the efficacy of compounds in the treatment of CRC are well known in the art. In addition, animal models of CRC are described in the examples section below. One of ordinary skill in the art can conventionally adapt this information to determine acceptable dosages for administration to humans.

An effective dose of an anti-hPG neutralizing monoclonal antibody of this application may range from about 0.001 mg/kg to about 75 mg/kg per single (eg, bolus) administration, multiple administration, or continuous administration, or to achieve a serum concentration of 0. 01-5000 μg/ml serum per single (e.g. bolus) administration, multiple administration, or continuous administration, or any effective range or value that falls within this range, depending on the condition being treated, route of administration, and age, weight and state of the subject. In some embodiments, each dose may range from about 0.5 micrograms to about 50 micrograms per kilogram of body weight, such as from about 3 micrograms to about 30 micrograms 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 condition of the patient. The therapeutic regimen for administration may last from 2 weeks to infinity, for 2 weeks to 6 months, 3 months to 5 years, 6 months to 1 year or 2 years, 8 to 18 months, or the like. Optionally, the therapeutic regimen provides repeated administrations, for example, once a day, daily, twice a day, every other day, two days later, five days later, a week later, two weeks later, or one month later. Repeated administrations may be at the same dose or at a different dose. The administration may 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 anti-hPG monoclonal antibody may be administered as a single dose or over a course of a therapeutic regimen, e.g., over a course that is one week, two weeks, three weeks, one month, three months, six months, one year or more .

Therapeutic ways.

The ability of the anti-hPG neutralizing monoclonal antibodies of the present invention to block PG-dependent responses, including cell proliferation, makes them useful in the treatment of colorectal cancer. Accordingly, in another aspect, the present disclosure provides methods for treating CRC in patients in need thereof. In general, the methods comprise administering to a patient a therapeutically effective amount of an anti-hPG neutralizing monoclonal antibody of the invention.

The subject or patient to whom the anti-hPG monoclonal antibody of this application is administered is preferably a mammal, such as a non-primate animal (eg, cow, pig, horse, dog, cat, rat, etc.) or primate (e.g. monkey or human). The subject or patient may be a human, such as an adult patient or a pediatric patient.

Patients who are eligible for anti-hPG monoclonal antibody therapy are those diagnosed as having CRC. CRC may be of any type or at any stage of clinical development or manifestation. Eligible subjects include patients with CRC tumors (operable or inoperable), patients whose tumors have been surgically removed or resected, patients with a CRC tumor comprising cells that carry a mutation in an oncogene such as, for example, RAS or APC, patients who have received or are receiving other therapy for CRC in combination with or in addition to anti-hPG monoclonal antibody therapy. Other therapies for CRC include, but are not limited to, chemotherapy treatment, radiation therapy, surgical removal, and treatment with one or more other therapeutic antibodies, as detailed below.

Anti-hPG antibody therapy may be combined with or used in addition to one or more other treatments. Other treatments include, but are not limited to, chemotherapy treatment, radiation therapy, surgical removal, and antibody-based therapy as described herein.

Anti-hPG monoclonal antibody therapy may be complementary to other therapies, including surgical removal.

Combination therapy, as described herein, involves administering at least two agents to a patient, the first of which is the anti-hPG neutralizing monoclonal antibody of this application and the second of which is a second therapeutic agent. The anti-hPG neutralizing monoclonal antibody and the second therapeutic agent may be administered simultaneously, sequentially, or separately.

As used herein, the anti-hPG neutralizing monoclonal antibody and the second therapeutic agent are administered sequentially if they are administered to the patient on the same day, for example, during the same patient visit. Sequential administration may be 1, 2, 3, 4, 5, 6, 7, or 8 hours apart. In contrast, the anti-hPG monoclonal antibody of this application and the second therapeutic agent are reported to be administered separately if they are administered to a patient on different days, for example, the anti-hPG monoclonal antibody of this application and the second therapeutic agent may be administered at intervals of 1, 2 or 3 days, 1, 2 weeks or a month. In the methods of this application, administration of the anti-hPG monoclonal antibody of this application may precede or follow administration of the second therapeutic agent.

As a non-limiting example, the anti-hPG neutralizing monoclonal antibody and the second therapeutic agent may be administered simultaneously for a period of time followed by a second period of time during which administration of the anti-hPG monoclonal antibody of this application and the second therapeutic agent alternate.

The combination therapies of the present invention can result in more than an additive or synergistic effect, providing therapeutic benefits where neither the anti-hPG neutralizing monoclonal antibody nor the second therapeutic agent is administered in an amount that is therapeutically effective on its own. Thus, such agents can be administered in lower amounts, reducing the likelihood and/or severity of side effects.

The second therapeutic agent may be a chemotherapeutic agent. Chemotherapeutic agents include, but are not limited to, radioactive molecules, toxins, also referred to as cytotoxins or cytotoxic agents, which include any agent that is destructive to cell viability, agents, liposomes, or other carriers containing chemotherapeutic compounds. Examples of acceptable chemotherapeutic agents

- 26 041823 agents include, but are not limited to, 1-dehydrotestosterone, 5-fluorouracil decarbazine, 6-mercaptopurine, 6-thioguanine, actinomycin D, adriamycin, aldesleukin, alkylating agents, sodium allopurinol, altretamine, amifostine, anastrozole, anthramycin (AMC )), antimitotic agents, cis-dichlorodiamine platinum(II) (DDP) cisplatin), diaminodichloroplatinum, anthracyclines, antibiotics, antimetabolites, asparaginase, BCG live (intravesicular), betamethasone sodium phosphate and betamethasone acetate, bicalutamide, bleomycin sulfate, busulfan, leucovorin calcium, calicheamicin, capecitabine, carboplatin, lomustine (CCNU), carmustine (BSNU), chlorambucil, cisplatin, cladribine, colchicine, conjugated estrogens, cyclophosphamide, cyclotosfamide, cytarabine, cytochalasin B, cytoxan, dacarbazine, dactinomycin, dactinomycin (formerly actinomycin) , daunorubicin HCL, daunorubicin citrate, denileukin diftitox, decrazoxane, dibromomannitol, dihydroxy anthracine dione, docetaxel, dolasetron mesylate, doxorub icin HCL, dronabinol, E. coli L-asparaginase, emetine, epoetin-α, Erwinia L-asparaginase, esterified estrogens, estradiol, estramustine sodium phosphate, ethidium bromide, ethinyl estradiol, etidronate, etoposide citrovorum factor, etoposide phosphate, filgrastim, floxuridine, fluconazole, fludarabine phosphate, fluorouracil, flutamide, folinic acid, gemcitabine HCL, glucocorticoids, goserelin acetate, gramicidin D, granisetron HCL, hydroxyurea, idarubicin HCL, ifosfamide, interferon a-2b, irinotecan HCL, letrozole, leucovorin calcium, leuprolide acetate, levamisole HCL, lidocaine, lomustine, maytansinoid, mechloramine HCL, medroxyprogesterone acetate, megestrol acetate, melphalan HCL, mercaptopurine, mesna, methotrexate, methyltestosterone, mithramycin, mitamycin C, mitotane, mitoxantrone, nilutamide, octreotide acetate, ondasetron HCL, oxaliplatin, paclitaxel, paclitaxel disodium, pentostatin, pilocarpine HCL, plimycin, polyfeprosan 20 with carmustine implant, sodium porfimer, procaine, procarbazine HCL, propr anolol, rituximab, sargramostin, streptozotocin, tamoxifen, taxol, tegafur, teniposide, tenoposide, testolactone, tetracaine, thiotepa chlorambucil, thioguanine, thiotepa, topotecan HCL, toremifene citrate, trastuzumab, tretinoin, valrubicin, vinblastine sulfate, vincristine sulfate, and vinorelbine tartrate.

The anti-hPG neutralizing monoclonal antibodies disclosed herein may be administered to a patient in need of colorectal cancer treatment who is 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 chemotherapeutic agents disclosed in this application.

As is known in the art, chemotherapeutic regimens for colorectal cancer using combinations of different chemotherapeutic agents have been standardized in clinical studies. Such regimens are often known by acronyms 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.

Neutralizing anti-hPG monoclonal antibodies may also be combined with other therapeutic antibodies. Accordingly, anti-hPG monoclonal antibody therapies may be combined with a different monoclonal antibody, such as, for example, but not limited to, an anti-EGFR (EGF receptor) monoclonal antibody or an anti-VEGF monoclonal antibody, or may be administered in addition to it. Specific examples of anti-EGFR antibodies include cetuximab and panitumumab. A specific example of an anti-VEGF antibody is bevacizumab.

Determination of progastrin using anti-hPG antibodies.

Anti-PG monoclonal antibodies, either neutralizing or non-neutralizing, are often useful for applications that depend on the definition of PG, such as diagnosing CRC or minitoring the effects of treatment in a subject with CRC. Accordingly, in one aspect, the present invention provides a method for diagnosing colorectal cancer in a patient, comprising determining the amount of progastrin in a sample from the patient using an anti-hPG monoclonal antibody in accordance with the present invention. In general, methods for diagnosing colorectal cancer in a patient include measuring progastrin in a sample obtained from the patient using an antihPG monoclonal antibody according to this application, where measuring the amount of progastrin in the sample in the range of 20 to 400 pM is indicative of colorectal cancer. Progastrin can be measured, for example, in blood, serum, plasma, tissue and/or cell samples. Determination of hPG can be carried out using assays known in the art and/or described in this application, such as ELISA, ELISA sandwich, Western blot, immunoprecipitation, BIACORE technique, and the like.

As noted in this application, progastrin is the penultimate of a number of different polypeptides that are formed as a result of post-translational processing of the gastrin gene

- 27 041823 product. Diagnostic, monitoring and other methods described in this application specifically determine hPG in contrast to other products of the gastrin gene, including degradation products. Accordingly, in specific embodiments, hPG is determined using an ELISA as disclosed herein, where two anti-hPG antibodies are used that target the N- and C-terminal end of hPG, respectively. In some embodiments, one of the two antibodies used to determine is an anti-hPG monoclonal antibody, as described in this application. hPG levels that range from 20 to 400 pM are indicative of colorectal cancer.

In general, the procedure for determining hPG levels using anti-hPG monoclonal antibodies is as follows. Prepare a surface, such as wells in a 96 well plate, to which a known amount of the first, immobilized anti-hPG antibody binds. The immobilized antibody may be, for example, an anti-hPG antibody that binds to the C- or N-terminal hPG. After blocking, the sample for analysis is applied to the surface, followed by an incubation period. The surface is then washed to remove unbound antigen and a solution containing a second anti-hPG antibody is used for detection. The detection antibody can be any of the anti-hPG monoclonal antibodies described herein, provided that the detection antibody binds a different epitope than the capture antibody. For example, if the immobilized antibody binds the C-terminal peptide region of hPG, then an acceptable detection antibody would be one that binds the N-terminal peptide region of hPG. Progastrin levels can then be determined directly (if, for example, the detection antibody is conjugated to a detectable label) or indirectly (using a labeled secondary antibody that binds the anti-hPG detection antibody).

In a specific embodiment, hPG levels are measured as described below for the test sample. 96 well microtiter plates are coated with 0.5 to 10 μg/ml rabbit C-terminal anti-hPG polyclonal antibody and incubated overnight. The plates are then washed three times with PBS-Tween (0.05%) and blocked with 2% (w/v) skimmed dried milk PBS-Tween (0.05%). Separately prepare test samples, controls (blank or PG-negative plasma or serum samples), and approximately 5 pM (0.5x10'1 M) to approximately 0.1 nM (1x10'1 M) hPG reference ( lyophilized hPG diluted in PG-negative plasma or serum) in a suitable diluent (eg PBS-Tween 0.05%). Samples are incubated on coated plates for 2-4 hours at 37°C or alternatively 12 to 16 hours at 21°C. After incubation, the plates are washed three times with PBS-Tween (0.05%) and incubated with 0.001-0.1 μg/ml N-terminal anti-hPG monoclonal antibody as described herein fused with horseradish peroxidase (HRP (Nakane et. 15 min at 21° C. The reaction is stopped by adding 100 µl of 0.5 M sulfuric acid and the optical density is measured at 405 nm The hPG levels of the test sample are determined by comparison with a standard curve constructed from measurements obtained on the hPG reference standard.

Typically, patients are diagnosed based on invasive procedures, such as histological evaluation of biopsy tissue, as well as other invasive procedures, such as colonoscopy. CRC is classified into 5 stages, which range from stage 0 (tumor confined to the inner lining of the colon or rectum), to stage 1 (tumor in the inner wall of the colon or rectum), to stage 2 (cancer that extends through the intestinal wall but not found in nearby lymph nodes), to stage 3 (cancer that is found in the lymph nodes and tissue that surrounds the colon or rectum) to stage 4 (cancer that has spread to other parts of the body). From a histological perspective, colorectal tumors are present in a wide range of neoplasms that range from benign growths to invasive cancers and are primarily epithelial-derived tumors (i.e., adenomas and adenocarcinomas). Lesions can be classified into three groups: non-neoplastic polyps, neoplastic polyps (adenomatous polyps, adenomas) and various cancers. Adenomatous polyps are benign tumors that can undergo malignant transformation and are classified into three histological types, in order of increasing malignant potential: tubular, tubular-villous, and villous. Adenocarcinomas have also been categorized according to their histology into mucinous (colloidal) adenocarcinomas; ring cell adenocarcinomas; fibrous tumors; and neuroendocrine tumors.

In contrast to currently available means for diagnosing CRC, the present invention provides methods for diagnosing subjects with CRC in the absence of any histological analysis or staging of the disease and is based on measuring hPG levels that can be determined from a blood sample. In addition, the methods of the present invention are useful in selecting CRC patients eligible for anti-hPG.

- 28 041823 monoclonal therapy, regardless of how the patient was diagnosed.

Serum PG levels are also useful in assessing the effectiveness of CRC treatments. Accordingly, the present disclosure provides a method for monitoring the effectiveness of colorectal cancer therapy, comprising determining PG levels in a patient undergoing CRC treatment. Methods for monitoring the effectiveness of therapy against colorectal cancer include repeating the determination of hPG levels using an anti-PG monoclonal antibody in accordance with the present invention in a patient with colorectal cancer undergoing treatment for colorectal cancer, where the decrease in circulating hPG levels in the patient for a certain interval treatment is indicative of the effectiveness of the treatment. For example, after a first measurement of circulating hPG levels in a patient, a second measurement may be performed concurrently with or after receiving treatment for colorectal cancer. The two measurements are then compared and the reduction in hPG levels is indicative of a therapeutic benefit.

In one aspect, the invention provides diagnostic kits containing anti-hPG monoclonal antibodies (including antibody conjugates). A diagnostic kit is a package that includes at least one anti-hPG monoclonal antibody according to this application (for example, either in lyophilized form or as an aqueous solution) and one or more reagents useful for performing a diagnostic assay (for example, diluents , a labeled antibody that binds to an anti-hPG monoclonal antibody, an acceptable substrate for the labeled antibody, hPG in a form suitable for use as a positive control and reference standard, negative control). In specific embodiments, the kit includes two anti-hPG antibodies, wherein at least one of the antibodies is an anti-hPG monoclonal antibody. Optionally, the second antibody is a polyclonal anti-hPG antibody. In some embodiments, the kit in accordance with the present invention includes an N-terminal anti-hPG monoclonal antibody, as described in this application.

Anti-hPG antibodies can be labeled as described above. Alternatively, the kit may include a labeled antibody that binds to an anti-hPG monoclonal antibody and is enzyme-conjugated. If the anti-hPG monoclonal antibody or other antibody is conjugated to an enzyme for detection, then the kit may include the substrates and cofactors required for the enzyme (eg, a substrate precursor that provides a detectable chromophore or fluorophore). In addition, other additives may include agents such as stabilizers, buffers (eg blocking buffer or lytic buffer, etc.). The anti-hPG monoclonal antibodies included in the diagnostic kit may be immobilized on a solid surface, or alternatively a solid surface (eg, glass slide) on which the antibody can be immobilized may be included in such a kit. The relative amounts of the various reagents can vary over a wide range to provide concentrations of reagents in solution that greatly optimize the sensitivity of the assay. Antibodies and other reagents may be provided as (alone or in combination) dry powders, usually lyophilized, including excipients which, when dissolved, will provide a reagent solution having an acceptable concentration.

Kits may include instructional materials containing instructions (eg, prescriptions) for performing diagnostic methods. While guidance materials typically include written or printed materials, they are not limited to such. A means capable of storing such instructions and transmitting them to the final user is contemplated by the present invention. Such media include, but are not limited to, electronic media (eg, magnetic disks, tapes, cartridges, chips), optical media (eg, CD ROM), and the like. Such means may include links to Internet sites that provide such instructional materials.

10. Examples

The following examples are illustrative and are not intended to be limiting.

Example 1. Obtaining monoclonal antibodies against progastrin.

Immunogens for human progastrin.

Several immunogens have been developed to develop hybridomas that produce monoclonal antibodies against human progastrin. Previously used antigens to generate antibodies, such as full length human progastrin, and immunogens that are based on 70-80 hPG residues have failed to generate a monoclonal immune response or have produced a PG-specific monoclonal antibody. As described in more detail below, antigens of 14 amino acids and longer that contain sequences unique to hPG at either the N-terminal or C-terminal end of the protein were capable of inducing an adequate immune response in animals and were used to generate hybridomas that produce over 20 different monoclonal antibodies to hPG. Surprisingly, it has been found that immunogens that include 55-80 hPG residues, some of which are also found in other peptides,

- 29 041823 derived from the progastrin gene have been successfully used to generate monoclonal antibodies that were specific for hPG. The table below summarizes the immunogens tested.

table 2

Experiment Immunogen Numbers of positive clones 1 Human progastrin (SEQ ID NO:20) 0* 1 (SEQ ID NO:26)-Ahx-Cys-BSA 2 2 (SEQ ID NO:26)-Ahx-Cys-KLH 2 3 (SEQ ID NO:26)-Ahx-Cys-KLH 8 1 BSA-Cys-Ahx-Ahx-(SEQ ID NO:97) 0 2 KLH-Cys-Ahx-Ahx-(SEQ ID NO:97) 0 3 KLH-Cys-Ahx-Ahx-(SEQ ID NO:96) 10 DT-Cys-Ahx-Ahx-(SEQ ID NO:96) 3

* Immunized mice did not show an immune response.

The immunogens given in table. 2 were prepared according to techniques known in the art using chemical synthesis of a peptide sequence and a linker followed by cross-linking of a carrier based on bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or diphtheria toxin (DT) with a suitable crosslinking agent (e.g. MBS (m-maleimidobenzoyl-N-hydrosuccinimide ester), glutaraldehyde, or sulfo-SMCC (sulfosuccinimidyl 4-[N-maleimidomethyl]cyclohexane-1-carboxylate) (Coligan J.E. et al., Current protocols in Immunology, Volume 2, New York: John Wiley and Sons, 1996, pp. 9.0.1-9.0.15 Harlow D.L., Antibodies: A Laboratory Manual New York: Cold Spring Harbor Laboratory, 1998, p.72 -87) The linkers used were one or two aminohexanoic acid (Ahx) residues fused to a cysteine residue.

In each of the three Balb/c experiments, mice were injected 4-5 times with N-terminal immunogens and 2-4 times with C-terminal immunogens. With each injection, 10 μg of the immunogen was administered with adjuvant Ribi, Alun, or Freund's adjuvant.

Cell fusion and hybridoma screening.

Serum obtained from each mouse was analyzed by ELISA against immunogen and collected splenocytes from mice with the strongest immune response. Splenocytes were fused with Sp2 myeloma cells using polyethylene glycol and plated on 96 well plates at a density of 15,000-35,000 cells per well. The fused cells were selected using a medium containing hypoxanthine, aminopterin and thymidine (HAT medium).

Hybridoma supernatants were screened using ELISA for the ability to bind the immunogen and full length progastrin. Three rounds of screening were performed to ensure that only those hybridomas were selected that stably produce antibodies recognizing the full length hPG and the immunogen.

Screening of hybridomas and monoclonal antibodies to determine binding to various PG peptides was performed using the ELISA technique as described below. This procedure was used to screen for PG binding of fused splenocytes, first and second cycle subclones, and to test the specificity of the antibody for PG compared to other peptides that are derived from the gastrin gene.

Briefly, 96-well plates were incubated overnight at 4°C with acceptable concentration(s) of test peptide in phosphate buffered saline (PBS), after which the wells were washed three times with wash solution (PBS and 0.1% Tween-20) and incubated for 2 hours at 22°C with 100 μl of blocking solution (PBS, 0.1% Tween-20, 0.1% bovine serum albumin or casein hydrolyzate) per well. After blocking, wells were washed three times and the primary antibody, the antibody to be analyzed, was added. For initial screening for fused splenocytes, 50 μl of culture supernatant from each culture being evaluated was added to each well of the plate. In assays that were performed on monoclonal antibodies, 100 μl of test antibody in PBS and 0.1% Tween-20 was added to each well. The plates were then incubated for 2 hours at 22°C, after which the primary antibody solution was discarded and after a washing step (3x100 µl of wash solution as above) was replaced with a blocking solution containing a secondary antibody that binds the primary antibody and is fused with the enzyme. The secondary antibody was a goat anti-mouse IgG (Fc) antibody fused to horseradish peroxidase. After a 1 hour incubation with the secondary antibody, 100 µl of a substrate solution (e.g., Fast OPD or O-phenylenediamine dihydrochloride, which is available from Sigma-Aldrich Co., prepared according to the manufacturer's instructions) was added to each well and incubated in the dark for 20 min at 22°C. Then the reaction was stopped

- 30 041823 by adding 50 µl of 4N sulfuric acid and the amount of catalyzed substrate was determined by measuring the optical density (O.D.) at 492 nm. Substrate conversion is proportional to the amount of primary (test) antibody bound to the antigen. Experiments were performed in duplicate and OD measurements were plotted as a function of antigen or antibody concentration depending on the purpose of the experiment. Samples were considered positive for antiPG antibodies if the measured O.D. was in the range of 0.2-1.5. This interval O.D. used to identify antibodies that bind the immunogenic peptide used to inoculate animals.

Typical materials and reagents used in the assay are shown below.

Component Source Reference number Greiner MicroIon 96 cell plates Dutscher #655092 YUH DPBS Dutscher # P04-53500 Twin 20 Sigma #63158 BSA (for blocking) Euromedex # 04-100-810-C Hydrolyzed casein (when used instead of BSA) Sigma 22090 Hybridoma supernatants or N-terminal or C-terminal monoclonal antibodies BioRealites As described in this application Goat anti-mouse IgG (Fc) fused with peroxidase Thermo #31439 Fast OPD Sigma # P9187 95-97% sulfuric acid Sigma #30743

Typical peptides used in hybridoma and monoclonal antibody screening are as follows.

Screening Peptides Source Reference number BSA Euromedex # 04-100-810-C Recombinant human progastrin BioRealites McQueen et al., 2002, J. Protein Chem., 21(7):465471. Human gastrin I (G-17) Sigma #53673 Glycine-extended gastrin 17 (G-Gly) Auspep # S10082 KLH Pierce (Perbio) #77653 C-terminal flanking peptide (CTFP) Auspep #R41345

Recombinant human progastrin was prepared as described by McQueen et al., 2002, J. Protein Chem., 21:465-471) with minor modifications. Briefly, BL21 DE3 Star bacterial cells (InVitrogen) were transformed with a vector containing a full length human progastrin sequence in a PGEX-GST-TEV backbone (GE Healthcare).

Bacteria were grown in LB medium containing 0.5 mM IPTG for 3 h at 37°C. The bacterial pellets were broken up using a French press and soluble as well as insoluble fractions were separated by centrifugation. Thereafter, the GST labeled hPG was isolated using a glutathione affinity column and PG was cleaved from the GST using the tobacco etch virus NIa (TEV) protease. Finally, PG was dialyzed against a final solution (10 mM Hepes, 0.5% BSA, pH 7.4).

First, hybridomas were cloned, then subcloned and propagated. Positive hybridomas were selected based on the following criteria:

(1) specificity for PG and immunogen;

(2) relative affinity of the antibody;

(3) growth of hybridoma cells;

(4) antibody secretion; and (5) monoclonal hybridomas.

The analyzes and selection criteria were as follows.

For specificity, the supernatants of the hybridomas under study were evaluated by ELISA as described above (50 μl of 1 in 2 dilutions of the supernatant in PBS assay medium). Hybridomas were considered positive if the measured O.D. ranged from 0.2 to 1.5 in the assay using hPG or the immunogen used to inoculate mice. As an additional matter of specificity, clones were selected based on the lack of binding to other peptides that are derived from the gastrin gene. Lack of binding was measured as a non-statistically significant difference between the signal from test cells and the mean signal from control cells containing only PBS.

To characterize affinity, serial dilutions of the antibody were evaluated by ELISA for binding to hPG as described above. Standard dilutions used in N-terminal antibody assays were 0, 0.1, 0.3, 1, 3, 10, 30, 100 ng/mL. Standard dilutions, is

- 31 041823 used in C-terminal antibody assays were 0, 0.03, 0.1, 0.3, 1, 3, 10 ng/mL.

The growth of hybridoma cells during repeated serial culture cycles was assessed by microscopic observation 2 days after seeding. The cells were expected to proliferate and fill the well 48 hours after seeding. Serial dilutions were made (typically the first dilution at 1:5, followed by at least 2 additional dilutions at 1:10), followed by microscopic observations 48 hours later to confirm adequate growth. Hybridoma cells that met these criteria were diluted and seeded again and then subjected to microscopic examination 48 hours later. These dilution-seeding-observation cycles were repeated three times until a hybridoma was considered to meet the growth criteria.

Antibody secretion was analyzed by performing hPG ELISA as described above on serial dilutions (1/2, 1/20, 1/200, 1/500, 1/1000, 1/2000) of cell-free supernatants.

Monoclonality was determined by seeding the clone or hybridoma in a 96-well plate at a density of 0.6 cells/well and incubated for 2 weeks. After 2 weeks, supernatants were assessed for hPG binding by ELISA and the clonal nature of the population was determined based on the presence of a uniform OD value for at least 90% of wells containing live cells.

Monoclonal antibodies against progastrin full length.

Each of the three mice was inoculated with recombinant human progastrin (described above). The immunogen did not elicit any detectable immune response in mice: PG binding could not be determined using ELISA as described above. No linking took place.

N-terminal monoclonal antibodies against progastrin.

As indicated above, N-terminal monoclonal antibodies were generated against an antigen containing hPG residues 1-14 linked to bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) using an Ahx-Cys linker at the C-terminal end of residue 14 of the antigen ( SWKPRSQQPDAPLG-Ahx-Cys (SEQ ID NO: 26)).

In the first experiment, three mice were inoculated with BSA-linked N-terminal peptide. Of the three fusions that were performed using splenocytes from two of the three mice, one fusion did not produce clones that showed PG or immunogen binding. Of the other two fusions seeded on 96-well plates, one provided 4 PG binding and PG-specific hybridomas, of which one stable IgG-producing subclone was reconstituted. The second fusion also resulted in one stable hybridoma subclone that produces IgG1, binds PG, and is PG-specific. In total, out of three mice, 17 first hybridoma cycles (or 0.74% of hybridomas screened) were positive for PG and immunogen binding, of which 9 positive cell lines were subcloned, of which two were IgG positive cell lines. Thus, the first experiment produced two clones after a couple of cycles of subcloning that retained a strong positive signal against the immunogen and hPG (positive for PG binding) and did not bind other peptides that are derived from the gastrin gene (positive for PG specific): hybridomas 43B9G11 and WE5H2G7 producing anti-hPG Mab 1 and anti-hPG Mab 2, respectively.

In a second experiment, mice were inoculated with KLH-linked N-terminal peptide. Two fusions were performed using Sp2 myeloma cells. Of these, only one fusion generated PG- and immunogen-positive clones that were also PG-specific. Of these, 1920 hybridomas were sown. Many hybridomas analyzed were positive with immunizing peptides but not with progastrin, or were not specific for PG. Specifically, 297 hybridomas showed a strong positive signal for the immunizing peptide (approximately 15.5% of 1920), of which 124 were also positive for progastrin binding (6.5%). There were 36 hybridomas, or 1.8%, that were positive for progastrin but not for the immunogen used. Only 12 hybridomas out of 1920 seeded produced antibodies that specifically bind to progastrin but not to other gastrin gene products (0.6% of total hybridomas, 3.6% of clones that were positive for the peptide and/or progastrin in the first screening). Of the 12 selected clones, only 2 were stable enough to develop into permanent clones and were frozen for long term storage. Thus, in this second experiment, almost 2000 hybridomas were seeded, only 2 clones were recovered, 6B5B11C10 (producing anti-hPG MAb 3) and 20D2C3G2 (producing anti-hPG MAb 4), producing anti-hPG Mab 3 and Mab 4, respectively, which express monoclonal antibodies capable of binding hPG and the immunizing peptide, as well as having specificity for progastrin compared to other gastrin gene products and high affinity for hPG. Both exemplary antibodies were of the IgG1 isotype.

In the third experiment, mice were inoculated with the same immunogen as in the second experiment. Fusions with Sp2 myeloma cells were performed using splenocytes obtained from the two mice with the strongest immune response. Hybridomas sown from these mergers

- 32 041823 (3840 single fusion hybridomas each, per mouse) and supernatants were analyzed for PG and immunogen binding as well as PG specificity. From each fusion, 6 hybridomas showed PG-specificity, of which 3 subclones were selected as meeting the requirements for further selection for growth, monoclonality, antibody secretion, relative affinity. Thus, a total of 2.9% of the hybridomas examined after seeding (220/7680) were positive for PG and immunogen, of which 0.15% were positive clones (12/7680), seeded with recovered final subclones constituting 0.15% of original hybridomas (6/7680). This experiment provided hybridomas 1E9A4A4 (producing anti-hPG MAb 15), 1E9D9B6 (producing anti-hPG MAb 16), 1C8D10F5 (producing anti-hPG MAb 17), 1A7C3F11 (producing anti-hPG MAb 18), 1B3B4F11 (producing anti-hPG MAb 19) and 1C11F5E8 (producing anti-hPG MAb 20).

The table below shows the number of clones seeded for each experiment, the immunogen used, and the number and percentage of hybridomas producing monoclonal antibodies that recognize both the immunogen and full length progastrin.

Table 3

Experiment Immunogen seeded clones PG ⁺ hybridoma supernatants PG-specific clones PG-specific, IgG-producing subclones 1 (SEQ ID NO:26)-AhxCys-BSA 2304 17 (0.74%) 9 (0.39%) 2 (0.087%) 2 (SEQ ID NO:26)-AhxCys-KLH 1920 124 (6.5%) 12 (0.6%) 2 (0.1%) 3 (SEQ ID NO:26)-AhxCys-KLH 7680 220 (2.9%) 12 (0.15%) 6 (0.1%)

C-terminal monoclonal antibodies against progastrin.

C-terminal monoclonal antibodies were generated against an antigen containing residues 55-80 hPG associated with either KLH or DT using a Cys-Ahx-Ahx linker at the N-terminal end of the antigen containing 26 residues (Cys-Ahx-Ahx- QGPWLEEEEEAYGWMDFGRRSAEDEN (SEQ ID NO: 96)). Attempts to generate hybridomas with a smaller antigen containing only 70-80 hPG residues, Cys-AhxAhx-FGRRSAEDEN (SEQ ID NO: 97), conjugated to either BSA or KLH, did not produce any clone.

Carried out three experiments. In the first two experiments, which used a shorter peptide, 0 subclones were recovered. Specifically, in the first experiment, 4 mice were injected with a C-terminal peptide corresponding to SEQ ID NO: 97 associated with BSA. None of the resulting fusions produced IgG-producing hybridomas. In the second experiment, 6 mice were injected, 3 for each of the indicated options, with a peptide corresponding to SEQ ID NO: 97 associated with KLH at its N-terminal end, or a peptide corresponding to SEQ ID NO: 97 associated with KLH at its N-terminal end. its C-terminal end. For the first immunogen, fusions were performed and hybridomas were recovered, but no subclones positive for PG binding and PG specificity were isolated. For the second immunogen, none of the mice developed an immune response and no fusions were performed.

In the third experiment, a 26 amino acid peptide was used, including C-terminal sequences that are not unique to hPG. Mice were injected with an immunogen that includes a peptide corresponding to SEQ ID NO: 96 and associated with either KLH or DT. Fusions with Sp2 myeloma cells were performed using splenocytes obtained from the two mice with the strongest immune response. At the same time, 3840 hybridomas were seeded from one fusion per mouse. A total of 7680 screened hybridomas, of which 382 (5%) were PG positive and specific for PG, 13 (0.17%) were stable, recovered positive subclones: 1B4A11D11 (producing anti-hPG MAb 5), 1B6A11F2 ( producing anti-hPG MAb 6), 1B11E4B11 (producing anti-hPG MAb 7), 1C10D3B9 (producing anti-hPG MAb 8), D8F5B3 (producing anti-hPG MAb 9), 1E1C7B4 (producing anti-hPG MAb 10), 2B4C8C8 ( producing anti-hPG MAb 11), 2B11E6G4 (producing anti-hPG MAb 12), 2C6C3C7 (producing anti-hPG MAb 13), 2H9F4B7 (producing anti-hPG MAb 14), 1F11F5E10 (producing anti-hPG MAb 21), 1F11F5G9 ( producing anti-hPG MAb 22) and 1A11F2C9 (producing anti-hPG MAb 23).

The table below shows the number of clones seeded for each experiment, the immunogen used, and the number and percentage of hybridomas producing monoclonal antibodies that bind PG and the immunogen that are PG-specific and meet the selection criteria (growth, monoclonality, relative affinity). etc.) after three rounds of subcloning.

- 33 041823

Table 4

Experiment Immunogen seeded clones PG ⁺ hybridoma supernatants PG specific clones PG-specific, IgG-producing subclones 1 BSA-nnHKep-(SEQ ID NO:97) 3072 10 (0.32%) 9 (0.29%) 0 2 KLH-nnHKep-(SEQ ID NO:97) 1920 27 (0.47%) 4 (0.07%) 0 (SEQ ID NO:97) linker-KLH 0 0 0 3 KLH-nuHKep-(SEQ ID NO:96) 3840 192 (5%) 17 (0.44%) 10 (0.26%) DT-nHHKep-(SEQ ID NO:96) 3840 190 (4.95%) 13 (0.34%) 3 (0.08%)

Monoclonal antibodies and the stable hybridomas from which they are derived are detailed in the table below.

Table 5

monoclonal antibody hybridoma Mouse 1g MAb 1 43B9G11 IgGi MAY 2 WE5H2G7 IgGi MAY 3 6V5V11S10 IgGi MAY 4 20D2C3G2 IgGi MAY 5 1B4A11D11 IgGi MAY 6 1B6A11F2 IgGi MAY 7 1B11E4B11 IgGi MAY 8 1C10D3B9 IgGi MAY 9 1D8F5B3 IgGi MAY 10 1Е1С7В4 IgGi MAY 11 2B4S8S8 IgGi MAY 12 2B11E6G4 IgGi MAY 13 2S6SZS7 IgGi MAY 14 2H9F4B7 IgGi MAY 15 1E9A4A4 IgGi MAY 16 1E9D9B6 IgGi MAY 17 1C8D10F5 H/O MAY 18 1A7C3F11 IgG2 MAY 19 1B3B4F11 IgG2 MAY 20 1C11F5E8 IgG2 MAY 21 1F11F5E10 IgG2 MAY 22 1F11F5G9 IgG2 MAY 23 1A11F2C9 IgG2

Hybridoma cell lines 1B4A11D11 (MAb 5, CNCM registration number I-4371), 1B6A11F2 (MAb 6, CNCM registration number I-4372), 1B11E4B11 (MAb 7, CNCM registration number I-4373), 2B4C8C8 (MAb 11, CNCM registration number I-4374), 2B11E6G4 (MAb 12, CNCM registration number I-4375) and 1E9A4A4 (MAb 15, registration number and CNCM I-4376) were deposited under the Budapest Treaty and received on October 6, 2010 by the National Collection of Microorganism Cultures ( CNCM), Pasteur Institute, Paris, France.

Cloning of anti-hPG monoclonal antibodies.

Monoclonal antibodies produced by hybridomas can be cloned and sequenced using techniques known to those skilled in the art. Monoclonal antibodies from hybridomas are given in table. 5 above were sequenced as described below.

Monoclonal antibody coding sequences obtained using hybridomas 6B5B11C10 and 20D2C3G2 were cloned and sequenced using standard techniques. Briefly, total RNA was isolated from frozen cell pellets using the RNABee reagent, AMSBio catalog number CS-104B, which was used according to the manufacturer's instructions. cDNA for the V regions was prepared from mRNA using reverse transcriptase polymerase chain reaction (RT-PCR) followed by rapid 5' cDNA end amplification (RACE). cDNA synthesis was performed using constant site specific primers, after which the first strand product was purified and a terminal deoxynucleotide transferase was used to attach the homopolymer ends to the 3' ends of the cDNA. The extended cDNA sequences were then amplified by PCR using primer pairs (one primer for each homopolymer tail) and either the VH or VL region, respectively. The heavy and light chain variable region of the PCR products was then cloned into a TA cloning vector (pGEM-T easy, Promega cat. no. A 1360) and sequenced using standard techniques. Cm.

fig. 2A, 2B (MAb 3), FIG. 2C, 2D (MAb 4).

The sequences encoding monoclonal antibodies that are produced by hybridomas 1C10D3B9, 2C6C3C7, 1B3B4F1 and 1E9D9B61 were determined as described below. Total RNA was isolated from frozen cell pellets using the RNAqueous®-4PCR kit (Ambion cat. no. AM1914) using the manufacturer's instructions. Heavy chain V mRNA was amplified using a set of six degenerate primer pools (HA to HF) and light chain V RNA was amplified using a set of eight degenerate primer pools, seven for the cluster (KA to KG) and one for λ cluster (LA). cDNA for variable regions was obtained from mRNA using RT-PCR. cDNA synthesis was performed using constant region specific primers followed by PCR using pools of degenerate primers for the mouse 5' signal sequence and primers for the 3' constant region for each of IgGVH, IgKVL and IgλVL. (Jones et al., 1991, Rapid PCR cloning of fulllength mouse immunoglobulin variable regions, Bio/Technology, 9:88-89). The heavy and light chain variable region PCR products were then cloned into a TA cloning vector (p-GEM-T easy, Promega cat. no. A 1360) and sequenced using standard techniques. See fig. 2E, 2F (MAb 8), 2G, 2H (MAb 13), 2I, 2J (Mab 16) and 2K, 2L (Mab 19).

Example 2 Binding affinity for an anti-hPG antibody.

A. Relative affinity.

Relative affinity of representative monoclonal antibodies was measured using the ELISA method described above, in which wells were coated with a peptide solution containing 50 ng of progastrin and then incubated in the presence of increasing concentrations of each monoclonal antibody as shown below.

monoclonal antibody Investigated concentration range N-terminal anti-hPG monoclonal antibodies MAbs 1-4 0.001-1 µg/ml N-terminal anti-hPG monoclonal antibodies MAbs 3, 15-20 0.1-100 ng/ml C-terminal anti-hPG monoclonal antibodies MAbs 5-14, 21-23 0.03-30 ng/ml

Fig. 3A shows the relative affinity of four anti-hPG monoclonal antibodies, MAbs 1-4, tested at antibody concentrations from 1 ng/mL to 1 μg/mL. Fig. 3B shows the relative affinity of the N-terminal anti-hPG monoclonal antibodies MAbs 3 and 15-20 tested at antibody concentrations of 0.1-100 ng/mL. Fig. 3C shows the relative affinity of the C-terminal anti-hPG monoclonal antibodies MAbs 5-14 and 21-23 tested at antibody concentrations of 0.03-30 ng/mL.

B. Measurement of the affinity constant.

To provide a more absolute affinity quantification for monoclonal antibodies, affinity constants were measured using the Proteon method (BioRad) according to Nahshol et al. its integrity. Briefly, anti-mouse IgG antibody (50 μg/ml) was first applied to the sensor chip, making sure that the signal detected by the chip decreased to 10,000-11,500 RU (response units) after injection. Then, a mouse monoclonal antibody, which is being analyzed, was injected (typical concentration: 30 μg/ml). Sufficient binding of the antibody to be assayed was determined based on an additional signal that is at least 500 RU on the sensor chip. The time-dependent interaction of the anti-progastrin monoclonal antibody and progastrin was then measured by injecting varying concentrations of progastrin, eg 200, 100, 50, 25 and 12.5 nM, and the level of dissociation was determined. Typically, multiple channels were available to test multiple antibodies in parallel in a single experiment, making it possible to evaluate the binding of an antibody of interest at varying concentrations of PG in parallel. Typically, one channel was reserved for a mouse monoclonal antibody that is not specific for PG as a control for non-specific binding, and the other channel was injected using dilution buffer along with baseline for background signal. In general, no binding was detected in the channels injected with the non-specific mouse antibody. Antibodies showing a high level of association in this kit, which can result in saturation of the captured mAb with progastrin, can be assayed against lower concentrations of progastrin (50, 25, 12.5, 6.25, and 3.125 nM), allowing more accurate measurement.

Affinity constants (KD) were calculated as the ratio between the dissociation constant (kd) and

- 35 041823 association constant (ka). Experimental values were confirmed by analysis of statistically relevant similarity between experimental curves based on binding measurements and theoretical profiles. The mathematical model used in the Proteon experiments to choose to fit any of the experimental curves to the theoretical model was a Langmuir model based on a 1:1 interaction between progastrin molecules and anti-progastrin monoclonal antibodies.

Table 6

monoclonal antibody The measured value of the affinity constant Kd (M) Anti-hPG MAb 1 2.5 μM (2.5 x 10' ⁶ M) Anti-hPG MAb 2 185 nM (1.85 x 10'M) Anti-hPG MAb 3 6.4 nM (6.4 x 10' ⁹ M) Anti-hPG MAb 4 3.5 nM (3.5 x 10' ⁹ M) Anti-hPG MAb 5 13 pM (1.30 x Yu ^-11 M) Anti-hPG MAb 6 0.6 nM (6.38 x 10' ^1u M) Anti-hPG MAb 7 58 pM (5.84 x Yu ^-11 M) Anti-hPG MAb 8 0.1 nM (1.08 x 10' ^1u M) Anti-hPG MAY 10 3.6 nM (3.62 x 10' ⁹ M) Anti-hPG MAY 11 0.3 nM (3.12 x 10' ^1u M) Anti-hPG MAY 12 0.4 nM (4.43 x 10' ^1u M) Anti-hPG MAY 13 0.6 nM (6.12 x 10' ^1u M) Anti-hPG MAY 14 6.8 pM (6.86 x10' ¹² M) Anti-hPG MAY 15 0.2 nM (2.11 x 10' ^1u M) Anti-hPG MAY 16 0.2 nM (2.78 x 10' ^1u M) Anti-hPG MAY 17 8.3 nM (8.29 x 10' ⁹ M) Anti-hPG MAY 18 1.2 nM (1.24 x 10' ⁹ M) Anti-hPG MAY 19 0.7 nM (7.79 x 10' ^1u M) Anti-hPG MAY 20 0.2 nM (2.47 x 10' ^1u M) Anti-hPG MAY 21 3.9 nM (3.90 x 10' ⁹ M) Anti-hPG MAY 22 5 nM (4.94 x 10' ⁹ M) Anti-hPG MAY 23 0.4 μM (3.99 XU ^-/ M)

Example 3 Aggregation of anti-hPG antibodies.

Antibody aggregation can reduce therapeutic efficacy by reducing the amount of antibodies available to bind to the target protein. Antibodies with very low levels of aggregation are preferred for therapeutic applications. Each batch of antibody will vary from other batches of the same monoclonal antibody. In general, it is desirable to use batches with low aggregation. To quantify antibody aggregation, antibody solutions were placed in a spectrofluorimeter (Photon Technology International) and irradiated at 280 nm. Scattering was measured at 280 nm, while emission due to aromatic acids (mainly tryptophan) was measured at 330 nm.

The level of aggregation was then quantified by calculating the ratio between OD values measured at 280 nm versus 330 nm as described in Nomine et al., 2003, Biochemistry, 42:4909-4917. A higher ratio of 280/330 nm indicates higher aggregation. The concentration of antibody used was 15 μg/ml and was 5-15 times higher than that used for in vitro treatments.

The results are presented in table. 7 below and in FIG. 4.

- 36 041823

Table 7

Sample Ratio 280/330 nm Bovine serum albumin (u=1) 6.4 Anti-hPG MAb 1 (n=1) 15.42 Anti-hPG MAb 2 (n=2) 9.81 Anti-hPG MAb 3 (n=2) 3.08 Anti-hPG MAb 4 (n=2) 1.94

Example 4 Binding specificity of anti-hPG monoclonal antibodies.

PG is only one of the peptide products of the gastrin gene. Other gastrin gene products play a role in normal homeostasis, but the role of PG in CRC makes it a useful target for therapeutic and diagnostic purposes. The monoclonal antibodies described herein are specific for full length progastrin compared to other peptides resulting from gastrin gene expression and processing, such as glycine-extended gastrin (G17-gly), amidated (gastrin 17) gastrins, and C -terminal flanking peptide (CTFP). These peptides are present in the circulating blood. The binding specificity of anti-hPG monoclonal antibodies is determined by assaying antibodies against human progastrin and other peptides derived from the gastrin gene using the ELISA assay described in Example 1 above.

Specifically, wells were coated with a solution containing one of the following peptides at the indicated amounts: progastrin, keyhole limpet hemocyanin (KLH), amidated gastrin-17 (G17), glycine-extended gastrin 17 (Ggly), or C-terminal flanking peptide (CTFP ). Screening peptides Test quantity Recombinant progastrin 50 ng (exp. 1) 25 ng (exp. 2) Human amidated gastrin I (G-17) 50 and 250 ng Glycine-extended gastrin 17 (G17-Gly) 50 and 250 ng KLH 50 and 250 ng C-terminal flanking peptide (CTFP) 50 and 250 ng

In the first experiment, 3 ng/ml (0.3 ng) of anti-hPG MAbs 3 and 1 μg/ml (0.1 μg) of each of the anti-hPG MAbs 1, 2, and 4 were used. See FIG. 5A. In the second experiment, the binding specificity of anti-hPG MAbs 5-14 and 21-23 was determined at a concentration of 0.3 ng/ml (see Fig. 5B), and the binding specificity of anti-hPG MAbs 3, 15-20 was analyzed at a concentration of 1 ng /ml (see Fig. 5C).

Antibodies that show little response to high amounts of KLH, which is fused to an antigenic peptide, were used in some immunogens to immunize mice in Example 1 above. There was no detectable effect of BSA on PG binding by any antibody, including those raised against immunogens that included BSA as a carrier.

All antibodies showed high specificity for binding to full length hPG (assayed at 50 and then 25 ng) compared to other peptides that are derived from the gastrin gene, such as amidated gastrin-17, glycine-extended gastrin 17 (Ggly) and a C-terminal flanking peptide, a 5 amino acid peptide that is cleaved from progastrin during normal polypeptide processing to form gastrin. Exemplary antibodies showed no detectable binding (signal above PBS background only) to any of the peptides derived from the gastrin gene other than hPG.

Example 5: Competitive Analysis.

The ability of an anti-hPG monoclonal antibody to compete with an anti-hPG polyclonal antibody for binding to progastrin was determined using an ELISA with an immobilized anti-hPG antibody and an anti-hPG antibody for determination. Anti-hPG MAb 3 were analyzed as follows: 96-well plates were precoated with a C-terminal anti-progastrin polyclonal antibody raised against a peptide containing residues 71-80 hPG. The plates were then incubated with 100 pM progastrin followed by the addition of 10 μg/ml of a biotinylated N-terminal anti-hPG polyclonal antibody formed against a peptide consisting of the amino acid sequence of SEQ ID NO: 25 and increasing concentrations of an anti-hPG MAB 3 monoclonal antibody. Binding of a biotinylated N-terminal anti-hPG polyclonal antibody was determined by incubating the plates with streptavidin-HRP followed by OPD treatment according to standard protocols. Binding was measured by quantitative luminescence.

The results show that increasing concentrations (μg/ml) of anti-hPG MAb 3 reduced the ability of the anti-hPG antibody to bind to progastrin, demonstrating that the monoclonal antibody competes with the polyclonal antibody (see FIG. 6).

Example 6 Mapping of epitopes.

Specific epitopes bound by exemplary monoclonal antibodies were mapped using the SPOT technique and alanine scanning as described in Laune, D. et al., 2002, J. Immunol. Methods, 267:53-70; and Laune, D., 1997, J. Biol. Chem., 272:30937-30944 respectively. In the SPOT technique, 15 amino acid peptide sequences containing a putative epitope were generated and applied to nitrocellulose membranes, which were then probed using the test antibody to determine the minimum epitope sequence that was recognized by the antibody. Alanine scanning was used to identify residues within an epitope that are critical for antibody binding: each residue within a putative epitope is mutated one by one to an alanine residue and the alanine-containing peptides are then probed with the antibody of interest.

Families of epitopes identified for a typical antibody in accordance with this application. For the N-terminal anti-hPG monoclonal antibody MAb 1-4 and 15-20, epitopes include at least the following sequences: DAPLG (SEQ ID NO: 28), PDAPLG (SEQ ID NO: 29), PRSQQPD (SEQ ID NO: 30), WKPRSQQPD (SEQ ID NO: 31), or WKPRSqQpDAPLG (SEQ ID NO: 32), as shown in Table 1. 8 below.

Table 8

MAY PG peptide antigen: SWKPRSQQPDAPLG SEQID NO MAY 2 WKPRSQQPDAPLG 32 MAY 4 WKPRSQQPDAPLG 32 MAb 1 PDAPLG 29 MAY 3 DAPLG 28 MAY 17 WKPRSQQPD 31 MAY 18 WKPRSQQPD 31 MAY 19 WKPRSQQPD 31 MAY 20 WKPRSQQPD 31 MAY 15 PRSQQPD thirty MAY 16 PRSQQPD thirty

For C-terminal anti-hPG monoclonal antibodies MAb 5-7, 9-12, 14 and 21-23, epitopes include at least the following sequences: FGRR (SEQ ID NO: 33), MDFGR (SEQ ID NO: 34), AEDEN (SEQ ID NO: 35) and GWMDFGRR (SEQ ID NO: 36), as shown in table. 9 below.

Table 9

MAb PG peptide antigen: QGPWLEEEEEAYGWMDFGRRSAEDEN SEQID NO MAb 14 GWMDFGRR 36 MAb 11 MDFGR 34 MAb 5 FGRR 33 MAb 6 FGRR 33 MAb 7 FGRR 33 MAb 9 FGRR 33 MAb 10 FGRR..E 33 MAb 12 FGRR 33 MAb 23 AEDEN 35

Example 7 Neutralizing activity of an anti-hPG antibody against cancer cell lines.

(A) Neutralizing activity of anti-hPG monoclonal antibodies.

Anti-hPG monoclonal antibodies reduce cell survival in reference colorectal cancer cell lines. Acceptable colorectal cancer cell lines are known in the art. For example, HCT116, LS174T, SW480, and SW620 are cell lines that are commonly used to study colon cancer, which also produce and secrete progastrin. Monoclonal antibodies to PG were analyzed for their ability to inhibit proliferation in these various cell lines. Cell survival of each of HCT116, LST174T, SW480 and SW620 was analyzed using different anti-hPG monoclonal antibodies.

For each experiment, 50,000 cells were seeded in 6 well plates in medium containing fetal calf serum and incubated for 8 h. h for 48 h in the absence of fetal calf serum with 1 µg/ml monoclonal control antibody (mouse anti-human IgG1, Calbiochem, cat. no. #411451) (CT mAb) or with 1 µg/ml anti-hPG MAb 1 -4 as stated. In further quantification of monoclonal antibodies, antibodies were determined to be those used at a concentration of approximately 3-5 μg/ml. The number of cells at T0 was counted in the control cell for each experiment. For HCT116 cells, experiments were carried out in the presence of 0.5% fetal calf serum. 48 hours after the start of treatment, the number of surviving cells in each well was counted three times in a blind experiment. The reduction in cell proliferation or CRC survival was determined by counting surviving cells treated with anti-hPG MAB as a percentage of cells treated with control MAb. The number of cells at the start of treatment (T0) was subtracted from the number of test and control cells measured after 48 h. the difference between each number of cells and the number of cells determined in T0 was calculated. The resulting number of anti-hPG MAB treated cells was then expressed as a percentage of the number of cells treated with the control MAb.

Fig. 7A-7C show the effect of anti-hPG MAb 3 and anti-hPG MAb 4 on the survival of SW480 cells, LS174T cells and HCT116 cells from reference experiments. Results are the mean +/- standard error of the mean of four cells obtained from two independent experiments. Treatment with anti-hPG monoclonal antibodies significantly reduced the number of cells compared to treatment with a control antibody. Statistical significance was determined using Student's t-test: *=p<0.05, **=p<0.01 and ***=p<0.001. In each cell line, anti-hPG antibodies reduced cell survival. In one cell line, LST174T, the number of cells at the end of 48 hours of treatment with anti-hPG antibodies was lower than the number of cells at the beginning of the experiment, suggesting that the antibodies cause cell death in addition to inhibition of cell proliferation.

Tab. 10 shows the survival percentage of SW480 colorectal cancer cells treated with each of the four anti-hPG monoclonal antibodies compared to a control monoclonal antibody (mouse anti-human IgG1, Calbiochem, Cat #411451) (CT mAb).

Table 10

SW480 (TO = 26,667) Number of cells - TO % of control P (treated vs. control) two-sample Mann-Whitney test Control mAb 36050 +/- 3228 Anti-hPG MAb 1 30425 +/- 3098 84.4 0.3556 Anti-hPG MAb 2 28925 +/- 2757 80.2 0.0476 Anti-hPG MAb 3 6050 +/- 1788 16.8 < 0.0001 Anti-hPG MAb 4 17560 +/- 3439 48.7 0.0002

Compared with controls, treatment with anti-hPG monoclonal antibodies reduced cancer cell survival by 83.2% (anti-hPG MAb 3), 51.3% (anti-hPG MAb 4), 19.8% (anti-hPG MAb 2) and 15.6% (anti-hPG MAb 1).

The tables below show the percent survival of LS174T and HCT-116 cells treated with anti-hPG MAb 3 and 4 compared to a control monoclonal antibody. The data is presented graphically in the respective boxes of FIG. 7.

Table 11

NST-116 (TO = 42,750) Number of cells - TO % of control P (treated vs. control) two-sample Mann-Whitney test Control MAb 151 250 +/- 9071 MASS 62 750 +/- 9194 41.5% < 0.0001 MAb4 82 250 +/-7435 54.4% <0.0001

Table 12

LS 174T (TO = 51,666) Number of cells -THAT % of control P (treated vs. control) two-sample Mann-Whitney test Control MAb 85 334 +/- 7520 MASS -6666 +/- 5000 - 8 % 0.0084 MAb4 +8334 +/- 2500 7% 0.0085

Under the conditions of the in vitro assay, complete inhibition of cell growth is not expected. In cell culture, progastrin is continuously secreted by cancer cells and accumulates in the cell culture medium. It is expected that progastrin levels will rise over time more than would occur in the circulating blood of the body, increasing the ratio of target protein to antibody and diluting the neutralizing effect of the antibody. Thus, the neutralizing effect observed with the use of antibodies in vitro is expected to be stronger than in vivo, where progastrin secreted by tumor cells is carried out by the blood stream, reducing its accumulation in situ.

Inhibition of cell proliferation by anti-hPG MAb 5-23 was determined in one or

- 39 041823 more CRC cell lines SW620, HCT116 and LS47T. Assays were performed in 6-well plates as described above using 5 μg/ml test control (anti-hPG) monoclonal antibodies. 50,000 cells were seeded per well for HCT116 and LS174T and 100,000 for SW620 cells. The tables below provide the percentage of surviving treated cells relative to control treatments from representative experiments. The averaged results are graphically presented in FIG. 7G-7I for SW620, LS174T, and HCT-116 cell lines, respectively.

Table 13

Number of cells - TO % of control P (treated vs. control) two-sample Mann-Whitney test Experiment 1 SW 620 (TO = 103,067) Control MAb 82 100 +/- 15489 - MAY 5 54 511 +/-8292 66% < 0.0001 MAY 6 44367 +/- 9321 54% < 0.0001 MAY 7 49279 +/- 8009 60% < 0.0001 MAY 8 32673 +/- 4680 40% < 0.0001 MAY 9 73283 +/- 3835 89% 0.1305 MAY 10 70178 +/- 4173 85% 0.0618 Experiment 2 SW 620 (TO = 118,553) Control MAb 81 347 +/-6062 MAY 11 46 974 +/-7422 58% 0.0003 MAY 12 52 980 +/- 10529 65% 0.0002 MAY 13 38 933 +/- 5284 48% 0.0003 MAY 14 83 767 +/- 9484 103% 0.21 MAY 21 59 497 +/- 2828 73% 0.0002 MAY 22 64 227 +/- 7123 79% 0.0013 MAY 23 83 914 +/- 5629 103% 0.82 Experiment 3 SW 620 (TO = 116,283) Control MAb 101 333 +/- 17 626 - MAY 15 66 052 +/-7739 65% < 0.0001 MAY 16 58 883 +/- 9950 58% < 0.0001 MAY 17 76 688 +/- 5578 75.5% 0.0014 MAY 18 75 874 +/- 10129 75% 0.0005 MAY 19 70 242 +/- 10 851 69% < 0.0001 MAY 20 66 470 +/- 7557 66% < 0.0001

Table 14

Number of cells - TO % of control P (treated vs. control) two-sample Mann-Whitney test Experiment 1 LS 174T (TO = 60,944) Control MAb 107 956 +/- 5859 MAY 13 62,341 +/- 10,683 58% 0.0003 MAY 16 65 389 +/- 8185 61% 0.0002 Experiment 2 LS 174T (TO = 86,389) Control Mab 241 711 +/- 11 620 MAY 14 246 444 +/- 19563 102% insignificant MAY 19 204 433 +/- 8946 84.5% 0.0005 Experiment 3 LS 174T (TO = 79,667) Control MAb 135,800 +/- 18,338 MAY 8 57 333 +/- 12657 42% < 0.001

-40 041823

Table 15

Number of cells - TO % of control P (treated vs. control) two-sample Mann-Whitney test Experiment 1 NST-116 (TO = 49,286) Control MAb 78 214 +/- 6230 MAY 13 28 805 +/- 3477 36% < 0.0001 MAY 16 56 484 +/- 8333 72% < 0.0001 MAY 19 68 945 +/- 8795 88% 0.0302 Experiment 2 NST-116 (TO = 60,944) Control MAb 122 456 +/- 1697 MAY 8 75 867 +/- 15627 62% < 0.0001 MAY 16 87011 +/- 5091 71% < 0.0001

(B) Neutralizing activity of an anti-hPG polyclonal antibody.

Analyzes were performed as described above with the following modifications. An N-terminal anti-hPG polyclonal antibody was used as described in Example 5. As a control, 3 μg/ml polyclonal antibody (polyclonal rabbit anti-human IgG, Affinity BioReagents, ref #SA1-600) (CT pAB) was used. For anti-PG treatments, 3 μg/ml anti-hPG polyclonal antibody was used for all cell lines. SW480 and LS174T were treated with control or N-terminal anti-hPG polyclonal antibody for 24-48 h in DMEM without FCS, while HCT116 was treated with anti-hPG polyclonal antibody for 48 h in DMEM with 0.5 %FCS. Surviving cells were then trypsinized and counted compared to cells treated using an equivalent concentration of control (anti-human IgG) polyclonal antibody.

The results of representative experiments are shown in the tables below and in FIG. 7D-7F. Treatment with the anti-hPG polyclonal antibody significantly reduced the number of cells compared to treatment with the control antibody. Statistical significance was determined using Student's t-test: *=p<0.05, **=p<0.01 and ***=p<0.001. The number of cells was expressed in relation to the number of cells in culture at the beginning of the experiment (T0). For each experiment, cells in each of the 4 wells were counted three times. As with anti-hPG monoclonal antibodies, colorectal cancer cell proliferation was inhibited by the anti-hPG polyclonal antibody, demonstrating that the anti-tumor effects seen with anti-progastrin polyclonal antibodies are sufficiently predictive of the activity of the monoclonal antibody to block the effects of progastrin on tumor cells. .

Table16

Number of cells -TO % of control P (treated vs. control) two-sample Mann-Whitney test Experiment 1 SW 480 (TO = 26,667) Control PAL 37 580 +/- 4233 PGPAb 7833 +/- 3660 21% 0.0001 Experiment 2 NST-116 (TO = 58 750) Control PAL 105 350 +/- 8660 PGPAb 7833 +/- 3660 21% < 0.05 Experiment 3 LS174T (TO = 112,500) Control PAL 207,500 +/- 10,000 PGPAb 102 500 +/- 5000 49.5% < 0.01

Example 8 The neutralizing effect of anti-hPG monoclonal antibodies is eliminated when the antibodies are pre-incubated with purified hPG.

To demonstrate that the neutralizing effect of anti-hPG monoclonal antibodies is mediated by binding to hPG, LS174T cells were cultured in the presence of a typical antibody, anti-hPG MAb 8, which was pre-incubated with and without hPG. As positive and negative controls, cells were cultured with hPG alone, control antibody alone, and control antibody pre-incubated with hPG.

In particular, 33.3 nM (5 μg/ml) of anti-hPG MAb 8 was pre-incubated for 1 hour at room temperature with a 20-fold molar excess of recombinant hPG, or 667 nM (6.67 μg/ml). Recombinant human progastrin prepared as described in Example 1 was used. In parallel, 33.3 nM (5 μg/ml) mouse anti-human IgG1 (General BioScience, ref. AB23420) was similarly pre-incubated with and without hPG.

5000 LS174T cells were seeded in each well of 96 well plates in medium containing 10% fetal calf serum and incubated for 8 h, after which the cells were transferred to medium containing no serum and grown for a further 12 h. without serum for 12 h, cells were treated with one of the following agents every 12 h: control antibody, control antibody pre-incubated with hPG, anti-hPG MAb 8 only, anti-hPG MAb 8 pre-incubated with hPG , and only hPG. 48 hours after the first treatment, the remaining viable cells were quantified by incubating the plates with Promega CellTiter 96 Aqueous One Solution and recording the absorbance at 490 nm. The absorbance measured for cells treated with the control monoclonal antibody (control MAb) was taken as 100% and all other experimental conditions were measured in comparison to the absorbance of cells treated with the control MAb. The results are shown in the table below and also in FIG. 8.

Table 17

Absorption % of control P (treated vs. control) two-sample Mann-Whitney test Processing only PG 0.244 +/- 0.088 132.5% 0.099 (not significant) Control MAb 0.184 +/- 0.084 100% not applicable Anti-hPG MAb 8 0.057 +/- 0.06 31% 0.001 Control MAb +hPG 0.321 +/- 0.079 174.5% 0.002 Anti-hPG MAb8 + hPG 0.271 +/- 0.076 147.6% 0.0229

The addition or incubation of the antibody with hPG increases the number of viable cells in culture. In contrast, treatment of cells with anti-hPG MAb 8 taken alone causes a significant decrease in the number of viable cells. Thus, the ability of anti-hPG monoclonal antibodies to neutralize PG activity is eliminated by the addition of hPG, which is expected to bind to the antibody and saturate the antibody. This result confirms the specificity of the neutralizing activity of anti-hPG monoclonal antibodies.

Example 9 In vivo anti-tumor activity of an anti-hPG antibody.

A number of experimental in vivo models have been developed for the study of colorectal cancer. Mouse xenograft assays have been developed in which tumor tissue or cells from human cell lines are transplanted into immunodeficient (so-called athymic nude) mice. Pocard M. et al., In vivo (1996), 10(5):463-469. Several transgenic mouse models have also been developed. Mouse models include heterozygous mutations in the adenomatous colon polyposis (APC) gene such as Apc ^Min , Apc1638N, Apc716, or ApcΔ14. The tumor APC suppressor gene encodes a cytosolic protein, APC, which upon interaction binds to β-catenin and blocks it in the cytosol within a multiprotein complex that targets β-catenin to the proteasome for degradation, thus preventing β-catenin from activating the transcription factor Tcf-4 in core. Heyer et al., Oncogene, 18:5325-5333 (1999). APCΔ14 mice carry a heterozygous exon 14 deletion within the adenomatous colon polyposis (APC) gene. Similar to what occurs in over 70% of patients with sporadic colorectal cancer, somatic loss of heterozygosity (LOH) at the second Apc allele occurs in intestinal cells, leading to constitutive activation of the β-catenin/Tcf-4 transcription complex and spontaneous development of intestinal tumors in these animals. The molecular origin of these adenomas and carcinomas, as well as the tumor morphology (including vascularization, inflammatory response, and presence of immune cells) with much greater similarity to those of human tumors compared to mouse xenograft models, makes APCΔ14 a highly relevant model for human colorectal cancer therapy research. Colnot et al., 2004, Lab. Invest., 84:1619-1630. Other transgenic mouse models are based on mutations in genes such as MSH2, MSH6, CDX2, K-RAS, as well as lines that combine mutations in APC with mutations in other oncogenes. Heyer et al., 1999, Oncogene, 18:5325-5333, Janssen KP et al., 2002, Gastroenterology, 123:492-504. These models are widely used to study colorectal cancer and explore hypotheses regarding the treatment of colorectal cancer.

Transgenic mice that carry a heterozygous 14 deletion of the adenomatous colon polyposis gene (LPCA14) serve as models for colorectal cancer by developing tumors similar to those found in human colon cancer. In the first experiment, LRCA14 mice were treated with a preparation containing equal amounts of anti-hPG polyclonal antibodies raised against (1) the peptide corresponding to SEQ ID NO: 25 and (2) the C-terminal peptide as described by Hollande et al., WO 07/135542. Mice aged 3.5 months were treated for 5 weeks with either a control polyclonal antibody or an anti-hPG polyclonal antibody (two mice per treatment). The bodies were administered by intraperitoneal injection twice a week at a dose of 10 mg/kg (injection volume 150 μl). At the end of the treatment, mice were sacrificed and gut washed with PBS, intestine dissected for digital photography, and fixed in 4% para-formaldehyde for immunohistochemical analysis. The number of tumors and images of colorectal tissue were recorded.

Morphological evaluation of intestinal tissue demonstrated that anti-hPG antibodies do not affect the restoration of healthy mouse intestinal epithelium. Tumors were counted in the treatment and control groups. The total number of tumors for mice treated with control antibody was 27 compared to 4 for mice treated with antihPG antibody. Thus, anti-hPG antibodies reduce the number of tumors by more than 6.5 times compared with control antibodies, but do not affect the restoration of healthy mouse intestinal epithelium.

In a second experiment, the effect of an anti-hPG antibody was examined in LPCA14 mice and normal (control) mice (C57BL6J). Mice aged 4 months were treated twice a week for 6 weeks with either a control polyclonal antibody, rabbit anti-human IgG antiserum (Jackson ImmunoResearch (reference number 309-005-0089), or with an anti-hPG polyclonal antibody by intraperitoneal injections twice a week at a dose of 9 mg/kg (injection volume 150 μl) After 6 weeks of treatment, two hours before sacrifice, mice were injected with bromodeoxyuridine (BrdU) (2 mg per mouse, intraperitoneal injection) 6 LRSA14 mice were treated with anti -hPG polyclonal antibody and 4 with control polyclonal antibody Control antibody and anti-hPG antibody were administered to 6 normal mice (C57BL6J) each. -hPG antibodies to normal intestinal tissue.

Investigated the number of tumors and size (height and length) in the treated group against the control group LPCA14 mice. Tumor size was determined by measuring images taken from the intestines of each animal prepared as described below. The intestines were washed as described above, the intestines were dissected lengthwise, embedded in paraffin and prepared for immunohistochemistry using the Swiss roll technique. Tumor length and height were measured using Image J software.

The results are presented in table. 18 and in FIG. 8L and 8B. The results for tumor size show a statistically significant difference between control and antihPG treated groups. Statistical significance was determined using the Mann-Whitney test: *=p<0.05, **=p<0.01 and ***=p<0.001. Mice treated with the control antibody showed a total of 125 tumors with an average of 31.25 tumors per mouse. Anti-hPG treated mice exhibited 46 tumors or an average of 7.6 tumors per mouse. This difference is statistically significant (Mann-Whitney test, P=0.0095), demonstrating that anti-hPG antibodies significantly reduce the number of colorectal cancer tumors in vivo.

Table 18

Processing (number of mice) Number of tumors per mouse Control RAI (4) 23 48 28 26 Anti-hPG PAI (6) 2 16 15 9 2 2

Lnti-hPG antibodies significantly reduced colorectal cancer tumors in vivo, as measured by reduction in the number and size of tumors in a mouse model of colorectal cancer, without any apparent side effects on normal colorectal epithelium. Thus, the treatment of colorectal cancer tumors using anti-hPG provides a therapeutic advantage in vivo.

Example 10 Construction of humanized anti-hPG antibodies.

Humanized antibodies were constructed in silico (by computer simulation) from mouse anti-hPG monoclonal antibodies MAb 3, 4, 8, 13, 16, and 19. Construction is humanized

- 43 041823 antibodies were carried out according to the procedure described in Lefranc et al., 2003, Dev. Comp. Immunol., 27:55-77; Lefranc et al., 2009, Nucl. Acids Res., 37:D1006-1012; Lefranc, 2008, Mol. Biotechnol., 40:101-111. For each of the mouse monoclonal antibodies, the corresponding humanized VH and V _L peptide sequences were determined by identifying the CDR and framework regions in the mouse sequences using the IMGT-ONTOLOGY database (Duroux et al., 2008, Biochimie, 90:570-583; Giudicelli and Lefranc, 1999, Bioinformatics, 15:1047-1054) and the IMGT® database and related tools (Ehrenmann et al., 2010, Nucl. Acids. Res., 38:D301-307; Brochet et al., 2008, Nucl. Acids. Res., 36:W503-508), followed by identification of the amino acid sequence of the closest human framework sequence in IMGT/GENE-DB (Giudicelli et al., 2005, Nucl. Acids. Res., 33:D256-261 ) and grafting the CDR sequences onto human framework regions. Specifically, the nucleotide and amino acid sequences of the mouse VH and VL domains were analyzed using IMGT/V-QUEST and IMGT/DomainGapAlign to determine CDR boundaries and framework regions, determine CDR length, and identify anchor amino acids. Anchor amino acids are residues at positions 26, 39, 55, 66, 104 and 118 of IMGT Collier de Perles that support CDR 1-IMGT, CDR 2-IMGT, CDR 3-IMGT (Kaas and Lefranc, 2007, Current Bioinformatics, 2 :21-30; Kaas et al., Brief Funct Genomic Proteomic, 2007, 6:253264). The nearest V (variable) and J (connection) genes for mouse sequences were identified and the most suitable genes were selected. Individual amino acids in murine framework regions were maintained if they were considered to possibly contribute to antibody specificity by comparison with known 3D structures (Kaas et al., 2004, Nucl. Acids. Res., 32:208- 210) when using IMGT Collier de Perles on two coats.

VH CDRs for MAb 3 were defined as those that are 8, 8, and 8 amino acids long for CDRs 1, 2, and 3, respectively. The closest embryonic mouse gene to the sequence of the mouse VH MAb 3 region, IGHVl-5*01, differed by 10 residues, 3 of which mapped to CDR and were retained in the humanized anti-hPG monoclonal antibody construct (I29 in CDR 1, F56 and S65 in CDR 2). In addition, V39, G55 and R66 were considered potentially important for maintaining specificity and were retained in the construct. The closest human embryonic gene was IGHV1-3*01 (63.3% identity at the amino acid level based on IGMT/DGapAlign). 27 sequence differences in framework regions 1-3 were found between mouse and human embryonic genes. In framework region 4, threonine-123 and leucine-124 were replaced with leucine-123 and valine-124 in order to match the human IGHJ1*01 gene. In general, the humanized V _H region for MAb 3 is 87.8% identical to the variable region for IGHV1-46*03 and 88 of the 91 amino acids in the four framework regions are identical.

VL CDRs for MAb 3 were defined as those that are 11, 3, and 9 amino acids long for CDRs 1, 2, and 3, respectively. The closest embryonic mouse gene to the sequence of the mouse VL MAb 3 region, IGKV1-117*01, differed by one residue that was mapped to the framework region. The closest human embryonic gene was IGKV2-30*02 (81% identity at the amino acid level based on IGMT/DomainGapAlign). 14 sequence differences in framework regions 1-3 were found between mouse and human embryonic genes. Residues E40 and F68 were maintained in the engineered humanized sequence. In framework region 4, leucine-124 was replaced with valine-124 to match the human IGKJ4*01 gene. In general, the humanized Vk sequence for MAb 3 is 93% identical to IGKV2D-29*02 and 87 of the 89 amino acids in the four framework regions are identical.

The constructed VH and Vk sequences are provided in the table below.

Table 19

SEQID NO: Amino acid sequence V chain 21 QVQLVQSGAEVKKPGASVKVSCKASGYIFTSYWVHWVRQAP GQRLEWMGGFYPGNSDSRYSQKFQGRVTITRDTSASTAYMEL SSLRSEDTAVYYCTRRDSPQYWGQGTLVTVSS hv _H 3 22 DVVMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLEWFQ QRPGQSPRRLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCFQGSHVPFTFGGGTKVEIK LUKZ

VH CDRs for MAb 4 were defined as those that are 8, 8, and 11 amino acids long for CDRs 1, 2, and 3, respectively. The closest embryonic mouse gene to the sequence of the mouse MAb 4 V _H region, IGHV1-9*01, differed in 11 residues, 4 of which were mapped to the CDR framework region and maintained in the humanized anti-hPG monoclonal antibody construct (S35, S36 and S37 in CDR 1, F56 in CDR 2). In addition, D66 was considered potentially important for maintaining specificity and was retained in the construct. The closest human embryonic gene was IGHV1-46*03 (64.9% identity at the amino acid level based on IGMT/DomainGapAlign). 27 sequence differences in framework regions 1-3 were found

- 44 041823 between mouse and human embryonic genes. In framework region 4, alanine-128 was replaced with serine-128 to match the human IGHJ5*01 gene. In general, the humanized VH sequence for MAb 4 is 91.8% identical to the variable region for IGHV1-46*03 and 90 of the 91 amino acids in the four framework regions are identical.

VL CDRs for MAb 4 were defined as those that are 11, 3, and 9 amino acids long for CDRs 1, 2, and 3, respectively. The closest embryonic mouse gene to the mouse VL MAb 4 sequence, IGKV1-110*01, differed in 3 residues, 2 of which mapped to CDR 1 (serine34 and valine-36) and were retained in the humanized anti-hPG monoclonal antibody construct. The closest human embryonic gene was IGKV2D-29*02 (81% amino acid level identity based on IGMT/DomainGapAlign). Eleven sequence differences in framework regions 1-3 were found between mouse and human embryonic genes. In framework region 4, serine-120 was replaced with glutamine-120 to match the human IGKJ2*01 gene. In general, the humanized Vk sequence for MAb 4 is 92% identical to IGKV2D-29*02 and 100% identical to the four framework regions.

The constructed VH and Vk sequences are provided in the table below.

Table 20

SEQID NO: Amino acid sequence V chain 23 QVQLVQSGAEVKKPGASVKVSCKASGYTFSSSWMHWVRQAPG QGLEWMGIFLPGSGSTDYAQKFQGRVTMTRDTSTSTVYMELS SLRSEDTAVYYCATDGNYDWFAYWGQGTLVTVSS hV _H 4 24 DIVMTQTPLSLSVTPGQPASISCKSSQSLVHSSGVTYLYWYL QKPGQSPQLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCSQSTHVPPTFGQGTKLEIK hVK4

The VH CDRs for MAb 8 were defined as those that are 8, 8, and 10 amino acids long for CDRs 1, 2, and 3, respectively. The closest embryonic mouse gene to the sequence of the mouse MAb 8 VH region, IGHV5-9-3*01, differed in 5 residues, 4 of which were mapped to the CDR framework region and maintained in the humanized anti-hPG monoclonal antibody construct. The closest human embryonic lineage genes were IGHV3-21*01 and *02 (80.4% amino acid level identity based on IGMT/DomainGapAlign). 12 sequence differences in framework regions 1-3 were found between mouse and human embryonic genes. In framework region 4, serine-123 and leucine-124 were replaced with threonine-123 and valine-124, respectively, in order to match the human IGHJ6*01 gene. In general, the humanized VH region for MAb 8 is 92.8% identical to the variable region for IGHV3-21*01 and *02 and 100% identical to the four framework regions. There is a potential N-glycosylation site in the mouse VH CDR 3 for MAb 8.

CDR V _L for MAb 8 were defined as those that are 11, 3 and 9 amino acids long for CDR 1, 2 and 3, respectively. The closest embryonic mouse gene to the sequence of the mouse VL MAb 8 region, IGKV2-109*01, differed in 6 residues, 4 of which were mapped to CDR 1 and protected in the humanized anti-hPG monoclonal antibody construct. The closest human embryonic lineage genes were IGKV2-28*01 and IGKV2D-28*01 (75% identity at the amino acid level based on IGMT/DomainGapAlign). 12 sequence differences in framework regions 1-3 were found between mouse and human embryonic genes. In framework region 4, alanine-120, leucine-124, and leucine-126 were replaced by glycine-120, valine-124, and isoleucine-126, respectively, in order to match the human IGKJ4*01 gene. In general, the humanized Vk sequence for MAb 8 is 87% identical to IGKV2-28*01 and IGKV2D-28*01 and 100% identical to the four framework regions.

The constructed VH and Vk sequences are provided in the table below.

- 45 041823

Table 21

SEQID NO: Amino acid sequence V chain 75 EVQLVESGGGLVKPGGSLRLSCAASGFT FTTYAMNWVRQAPGKGLEWVSSISSGGT YTYYADSVKGRFTISRDNAKNSLYLQMN SLRAEDTAVYYCATQGNYSLDFWGQGTT VTVSS hV _H 8a 76 DIVMTQSPLSLPVTPGEPA SISCRSSKSLRHTKGITFLD WYLQKPGQSPQLLIYQMSNRASGVPD RFSGSGSGTDFTLKISRVEAEDVG VYYCAQNLELPLTFGGGTKVEIK hVK8a 77 EVQLVESGGGLVKPGGSLRLSCAA SGFTFTTYAMSWVRQAPGKGLEWVS SISSGGTYTYYADSVKG RFTISRDNAKNSLYLQMNSLRAEDTA VYYCATQGNYSLDFWGQGTTVTVSS hV _H 8b 78 DIVMTQSPLSLPVTPGEPASISC RSSKSLRHTKGITFLYWYLQKPG QSPQLLIYQMSNRASGVPD RFSGSGSGTDFTLKISRVEAED VGVYYCAQNLELPLTFGGGTKVEIK hVK8b 79 EVQLVESGGGLVKPGGSLRLSCAASGF TFTTYAMSWVRQAPGKGLEWVSTISSG GTYTYYADSVKGRFTISRDNAKNSLYL QMNSLRAEDTAVYYCATQGNYSLDFWGQGTTVTVSS hV _H 8b

The VH CDRs for MAb 13 were defined as those that are 8, 8, and 7 amino acids long for CDRs 1, 2, and 3, respectively. The closest embryonic mouse gene to the sequence of the mouse VH MAb 13 region, IGHV5-6-3*01, differed in 10 residues, 5 of which were mapped to the CDR framework region and maintained in the humanized anti-hPG monoclonal antibody construct. The closest human embryonic lineage genes were IGHV3-7*01 and *02 (78.6% amino acid level identity based on IGMT/DomainGapAlign). 13 sequence differences in framework regions 1-3 were found between mouse and human embryonic genes. In framework region 4, threonine-123 and leucine-124 were replaced with leucine-123 and valine-124, respectively, in order to match the human IGHJ4*01 gene. In general, the humanized VH region for MAb 13 is 91.8% identical to the variable region for IGHV37*01 and *02 and 100% identical to the four framework regions.

The VL CDRs for MAb 13 were defined as those that are 11, 3, and 9 amino acids long for CDRs 1, 2, and 3, respectively. The closest embryonic mouse gene to the sequence of the mouse MAb 13 Vl region, IGKV1-135*01, differed by one residue located in the framework region. The closest human embryonic lineage genes were IGKV2-30*01 and *02 (81% amino acid level identity based on IGMT/DomainGapAlign). 13 sequence differences in framework regions 1-3 were found between mouse and human embryonic genes. In framework region 4, leucine-124 was replaced with valine-124 to match the human IGKJ4*01 gene. In general, the humanized Vk sequence for MAb 13 is 94% identical to IGKV2-30*01 and *02 and 100% identical to the four framework regions.

The constructed VH and Vk sequences are provided in the table below.

Table 22

SEQID NO: Amino acid sequence V chain 80 EVQLVESGGGLVQPGGSLRLSCAASGFIFSSYGMSWVRQAP GKGLEWVANINTFGDRTYYVDSVKGRFTISRDNAKNSLYLQ MNSLRAEDTAVYYCARGTGTYWGQGTLVTVSS hV _H .13a 81 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWFQ QRPGQSPRRLIYLVSNRDSGVPDRFSGSGSGTDFTLKISRVE AEDVGVYYCWQGTHFPQTFGGGTKVEIK hVK13a 82 EVQLVESGGGLVQPGGSLRLSCAASGFIFSSYGMSWVRQ APGKGLEWVASINTFGDRTYYVDSVKGRFTISRDNAKNS LYLQMNSLRAEDTAVYYCARGTGTYWGQGTLVTVSS hV _H 13b 83 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNW FQQRPGQSPRRLIYLVSKRDSGVPDRFSGSGSGTDFTLKIS RVEAEDVGVYYCWQGTHFPQTFGGGTKVEIK hVK13b

V _H CDRs for MAb 16 were defined as those that are 8, 8, and 10 amino acids long for CDRs 1, 2, and 3, respectively. The closest fetal mouse gene to the sequence of the mouse VH MAb 16 region, IGHV1-53*01, differed in 7 residues, 2 of which were mapped to the CDR framework region and maintained in the humanized anti-hPG monoclonal antibody construct. The closest human embryonic line genes were IGHV1-46*01 and *03 (71.4% identical

- 46 041823 amino acid level based on IGMT/DomainGapAlign). 25 sequence differences in framework regions 1-3 were found between mouse and human embryonic genes. In framework region 4, leucine-124 was replaced with valine-124 in order to match the human IGHJ6*01 gene. In general, the humanized VH sequence for MAb 16 is 96.9% identical to the variable region for IGHV1-46*01 and *03 and 100% identical to the four framework regions.

VL CDRs for MAb 16 were defined as those that are 11, 3, and 9 amino acids long for CDRs 1, 2, and 3, respectively. The closest embryonic mouse gene to the sequence of the mouse VL region of MAb 16, IGKV1-135*01, differed in 4 residues located in the framework region. The closest genes in the human embryonic lineage were IGKV2-30*01 and *02 (79% identity at the amino acid level based on IGMT/DomainGapAlign), which differ by one amino acid in CDR 1. 15 sequence differences in framework regions 1-3 were found between mouse and human embryonic genes. In framework region 4, glycine-120 was replaced by glutamine-120 in order to match the human IGKJ2*01 gene. In general, the humanized Vk sequence for MAb 16 is 94% identical to IGKV2-30*01 and *02 and 100% identical to the four framework regions.

The constructed VH and Vk sequences are provided in the table below.

Table 23

SEQID NO: Amino acid sequence V chain 84 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMHWVRQAPG QGLEWMGIINPSNGGTSYAQKFQGRVTMTRDTSTSTVYMELSS LRSEDTAVYYCTRGGYYPFDYWGQGTTVTVSS hV _H 16a 85 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLNWFQQ RPGQSPRRLIYLVSNRDSGVPDRFSGSGSGTDFTLKISRVEAE DVGVYYCWQGTHSPYTFGQGTKLEIK hVK16a 86 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQG LEWMGIINPSNGGTSYAQKFQGRVTMTRDTSTSTVYMELSSLRSE DTAVYYCTRGGYYPFDYWGQGTTVTVSS hV _H 16b 87 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLYWFQQRP GQSPRRLIYLVSNRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCWQGTHSPYTFGQGTKLEIK hVK16b 88 QVQLVQSGAEVKKPGASVKVSCKASGYTFTSYYMYWVRQAPGQGL EWMGEINPSNGGTNYAQKFQGRVTMTRDTSTSTVYMELSSLRSED TAVYYCTRGGYYPFDYWGQGTTVTVSS hV _H 16c 89 DVVMTQSPLSLPVTLGQPASISCRSSQSLLDSDGKTYLYWFQQRP GQSPRRLIYLVSERDSGVPDRFSGSGSGTDFTLKISRVEAEDVGV YYCWQGTHSPYTFGQGTKLEIK hVK16c

The VH CDRs for MAb 19 were defined as those that are 9, 7, and 14 amino acids long for CDRs 1, 2, and 3, respectively. The closest fetal mouse gene to the sequence of the mouse VH MAb 19 region, IGHV3-2*01, differed in 5 residues, 2 of which were mapped to the framework region CDR and maintained in the humanized anti-hPG monoclonal antibody construct. The closest human embryonic gene was IGHV4-30-4*01 (72.4% amino acid level identity based on IGMT/DomainGapAlign). However, since this gene is polymorphic, IGHV4-31*02 and *03 were chosen (71.4% identity at the amino acid level based on IGMT/DomainGapAlign). 21 sequence differences in framework regions 1-3 were found between mouse and human embryonic genes. In framework region 4, isoleucine-123 was replaced with leucine-123 to match the human IGHJ4*01 gene. In general, the humanized VH sequence for MAb 19 is 91.9% identical to the variable region for IGHV4-31*02 and *03 and 100% identical to the four framework regions.

The VL CDRs for MAb 19 were defined as those that are 7, 7, and 13 amino acids long for CDRs 1, 2, and 3, respectively. The closest embryonic mouse gene to the sequence of the mouse VL region of MAb 19, IGLV3*01, differed in 8 residues, 5 of which are located in the CDR. The closest human embryonic lineage genes were IGLV4-69*01 and *02 (69.9% amino acid level identity based on IGMT/DomainGapAlign). 23 sequence differences in framework regions 1-3 were found between mouse and human embryonic genes. In framework region 4, valine-124 was replaced with leucine-124 in order to match the human IGLJ3*01 gene. Alternatively, the IGJK4*01 gene can be used for 4 framework regions. In general, the humanized Vk sequence for MAb 19 is 92.4% identical to IGLV4-69*01 and *02 and 100% identical to the four framework regions.

The constructed VH and Vk sequences are provided in the table below.

-

Claims (42)

Table 24 SEQID NO: Amino acid sequence V chain 90 QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDYAWSWIRQH PGKGLEWIGYISFSGYTYYNPSLKSRVTISVDTSKNQFSLK LSSVTAADTAVYYCAREVNYGDSYHFDYWGQGTLVTVSS V _H 19a 91 QLVLTQSPSASASLGASVKLTCTLSSQHRTYTIAWHQQQPEK GPRYLMKVKKDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSE DEADYYCGVGDAIKGQSVFVFGGGTKVEIK VK19a 92 QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDYAWNWIRQHP GKGLEWIGYISFSGYTYYNPSLKSRVTISVDTSKNQFSLKLSS VTAADTAVYYCAREVNYGDSYHFDYWGQGTLVTVSS V _H 19b 93 QLVLTQSPSASASLGASVKLTCTLSSQHRTYTIEWHQQQPEKG PRYLMKVKKDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSED EADYYCGVGDAIKGQSVFVFGGGTKVEIK VK19b 94 QVQLQESGPGLVKPSQTLSLTCTVSGYSITSDYAWNWIRQHPGK GLEWIGYISFSGYTSYNPSLKSRVTISVDTSKNQFSLKLSSVTA ADTAVYYCAREVNYGDSYHFDYWGQGTLVTVSS V _H 19c 95 QLVLTQSPSASASLGASVKLTCTLSSQHRTYTIEWHQQQPEKGP RYLMEVKKDGSHSKGDGIPDRFSGSSSGAERYLTISSLQSEDE ADYYCGVGDAIKGQ SVFVFGGGTKVEIK Vk19c All publications, patents, patent applications, and other documents referred to in this application are incorporated by reference in their entirety for all purposes, to the extent that each individual publication, patent, patent application, or other document is individually cited for that purpose. to be entered as a link for all purposes. While various specific embodiments have been illustrated and described, it will be understood that various changes can be made without departing from the spirit and scope of the invention(s). CLAIM 1. A monoclonal antibody that specifically binds the N-terminal region of human progastrin (hPG), which antibody includes VL and VH CDRs having sequences selected from one of the following groups of VL and VH CDR sequences: (i) VH CDR 1.3 containing the amino acid sequence of SEQ ID NO: 1, VH CDR 2.3 containing the amino acid sequence of SEQ ID NO: 2, VH CDR 3.3 containing the amino acid sequence of SEQ ID NO: 3, VL CDR 1.3 containing the amino acid sequence of SEQ ID NO: 4, VL CDR 2.3 containing the amino acid sequence of SEQ ID NO: 5, and VL CDR 3.3 containing the amino acid sequence of SEQ ID NO: 6; (ii) VH CDR 1.4 containing the amino acid sequence of SEQ ID NO: 7, VH CDR 2.4 containing the amino acid sequence of SEQ ID NO: 8, VH CDR 3.4 containing the amino acid sequence of SEQ ID NO: 9, VL CDR 1.4 containing the amino acid sequence of SEQ ID NO: 10, VL CDR 2.4 containing the amino acid sequence of SEQ ID NO: 5, and VL CDR 3.4 containing the amino acid sequence of SEQ ID NO: 11; (iii) VH CDR 1.16 containing the amino acid sequence of SEQ ID NO: 39, VH CDR 2.16 containing the amino acid sequence of SEQ ID NO: 43, VH CDR 3.16 containing the amino acid sequence of SEQ ID NO: 47, VL CDR 1.16 containing the amino acid sequence of SEQ ID NO: 50, VL CDR 2.16 containing the amino acid sequence of SEQ ID NO: 53, and VL CDR 3.16 containing the amino acid sequence of SEQ ID NO: 57; and (iv) VH CDR 1.19 containing the amino acid sequence of SEQ ID NO: 40, VH CDR 2.19 containing the amino acid sequence of SEQ ID NO: 44, VH CDR 3.19 containing the amino acid sequence of SEQ ID NO: 48, VL CDR 1.19 containing the amino acid sequence SEQ ID NO: 51, VL CDR 2.19 containing the amino acid sequence of SEQ ID NO: 54, and VL CDR 3.19 containing the amino acid sequence of SEQ ID NO: 58. 2. A monoclonal antibody according to claim 1, which includes VH and VL chains having sequences selected from one of the following groups of V _H and V _L sequences: (i) mVH 3 (SEQ ID NO: 12) and mVL 3 (SEQ ID NO: 13); (ii) mVH 4 (SEQ ID NO: 14) and mVL 4 (SEQ ID NO: 15); (iii) mVH 16 (SEQ ID NO: 61) and mVL 16 (SEQ ID NO: 65); and (iv) mVH 19 (SEQ ID NO: 62) and mVL 119 (SEQ ID NO: 66). 3. Monoclonal antibody according to claim 1, which is humanized. 4. A monoclonal antibody according to claim 3, which includes VH and VL chains having sequences selected from one of the following groups of VH and VL sequences: (i) hVH 3 (SEQ ID NO: 21) and hVL 3 (SEQ ID NO: 22); (ii) hVH 4 (SEQ ID NO: 23) and hVL 4 (SEQ ID NO: 24); - 48 041823 (iii) hVH 16a (SEQ ID NO: 84) and hVL 16a (SEQ ID NO: 85); (iv) hVH 16b (SEQ ID NO: 86) and hVL 16b (SEQ ID NO: 87); (v) hVH 16c (SEQ ID NO: 88) and hV _L 16c (SEQ ID NO: 89); (vi) hVH 19a (SEQ ID NO: 90) and hV _L 19a (SEQ ID NO: 91); (vii) hVH 19b (SEQ ID NO: 92) and hVL 19b (SEQ ID NO: 93); and (viii) hVH 19c (SEQ ID NO: 94) and hVL 19c (SEQ ID NO: 95). 5. A monoclonal antibody according to any one of claims 1 to 4 which has an affinity in the range of from about 1 pM to about 7 nM. 6. A monoclonal antibody according to any one of claims 1 to 5 which neutralizes hPG activity in an in vitro assay which is performed using LS174T cells. 7. A monoclonal antibody according to any one of claims 1 to 6 that binds to an epitope comprising an amino acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NOs: 28, 29, 30, 31 and 32. 8. The pharmaceutical composition is intended for the treatment of cancer, where the specified composition includes a monoclonal antibody according to any one of claims 1 to 7 and an excipient, carrier and/or diluent. 9. A polynucleotide encoding the light chain variable region (V _L ) of an anti-hPG monoclonal antibody according to any one of claims 1-7. 10. A polynucleotide encoding the heavy chain variable region (VH) of an anti-hPG monoclonal antibody according to any one of claims 1-7. 11. A polynucleotide encoding the V _L of an anti-hPG monoclonal antibody according to any one of claims 1 to 7 and the V H of an anti-hPG monoclonal antibody according to any one of claims 1 to 7. 12. An expression vector comprising a polynucleotide according to claim 9 and a polynucleotide according to claim 10; or a polynucleotide according to claim 11. 13. A host cell for producing an anti-hPG monoclonal antibody transformed with the expression vector of claim 12. 14. A method for producing an anti-hPG monoclonal antibody, comprising (a) culturing the host cell according to claim 13 under suitable conditions; and (b) isolating the anti-hPG monoclonal antibody from the culture medium or hybridoma cells. 15. A monoclonal antibody that specifically binds the C-terminal region of human progastrin (hPG), which antibody comprises V _L and VH CDRs having sequences selected from one of the following groups of VL and VH CDR sequences: (i) VH CDR 1.13 containing the amino acid sequence of SEQ ID NO: 38, VH CDR 2.13 containing the amino acid sequence of SEQ ID NO: 42, VH CDR 3.13 containing the amino acid sequence of SEQ ID NO: 46, V _L CDR 1.13 containing the amino acid sequence SEQ ID NO: 50, V _L CDR 2.13 containing the amino acid sequence of SEQ ID NO: 53, and V _L CDR 3.13 containing the amino acid sequence of SEQ ID NO: 56. 16. A monoclonal antibody according to claim 15, which contains V H and V _L chains having sequences selected from one of the following groups of V _H and V _L sequences: (i) mVH 13 (SEQ ID NO: 60) and mV _L 13 (SEQ ID NO: 64). 17. The monoclonal antibody of claim 15 which is humanized. 18. A monoclonal antibody according to claim 17, which includes V _L and VH chains having sequences selected from one of the following groups of VL and VH sequences: (i) hVH 13a (SEQ ID NO: 80) and hVL 13a (SEQ ID NO: 81); and (ii) hVH 13b (SEQ ID NO: 82) and hVL 13b (SEQ ID NO: 83). 19. A monoclonal antibody according to any one of claims 15-18, which has an affinity ranging from about 1 pM to about 7 nM. 20. A monoclonal antibody according to any one of claims 15-19 which neutralizes hPG activity in an in vitro assay which is performed using LS174T cells. 21. A monoclonal antibody according to any one of claims 15-20 that binds to an epitope comprising an amino acid sequence corresponding to a sequence selected from the group consisting of SEQ ID NOs: 33, 34, 35 and 36. 22. A pharmaceutical composition for the treatment of cancer, said composition comprising a monoclonal antibody according to any one of claims 15 to 21 and an excipient, carrier and/or diluent. 23. A polynucleotide encoding the light chain variable region (V _L ) of an anti-hPG monoclonal antibody according to any one of claims 15-21. 24. A polynucleotide encoding the heavy chain variable region (VH) of an anti-hPG monoclonal antibody according to any one of claims 15-21. 25. A polynucleotide encoding the V _L of an anti-hPG monoclonal antibody according to any one of claims 15-21 and the VH of an anti-hPG monoclonal antibody according to any one of claims 15-21. - 49 041823 26. An expression vector for producing an anti-hPG monoclonal antibody, comprising the polynucleotide of claim 23 and the polynucleotide of claim 24; or a polynucleotide according to claim 25. 27. A host cell for producing an anti-hPG monoclonal antibody transformed with the expression vector of claim 26. 28. A method for producing an anti-hPG monoclonal antibody, comprising (a) culturing the host cell according to claim 27 under suitable conditions; and (b) isolating the anti-hPG monoclonal antibody from the culture medium or hybridoma cells. 29. The use of an anti-hPG monoclonal antibody according to any one of claims 1-7 or 15-21 for the preparation of a medicament for the treatment of colorectal cancer. 30. The use of an anti-hPG monoclonal antibody according to any one of claims 1 to 7 or 15 to 21 for the preparation of a medicament for inhibiting colorectal cancer cell proliferation. 31. The use of an anti-hPG monoclonal antibody according to any one of claims 1-7 or 15-21 for the preparation of a medicament for inhibiting the progastrin-dependent signal transduction cascade in a cell. 32. A method for diagnosing colorectal cancer in a subject in vitro, comprising quantifying progastrin in a serum sample of the subject using a monoclonal antibody according to any one of claims 1-7 or 15-21, where the amount of progastrin in the range from about 20 to 400 pM is indicative of colorectal cancer. 33. A method for selecting an in vitro patient suitable for anti-hPG therapy, comprising quantifying progastrin in a serum sample from a colorectal cancer patient using a monoclonal antibody according to any one of claims 1-7 or 15-21, where the amount of progastrin is in the range of approximately 20 to 400 pM indicates the suitability of anti-hPG therapy. 34. A method for monitoring the effectiveness of colorectal cancer treatment in vitro, comprising quantifying the level of progastrin in the blood of a subject who has been treated for colorectal cancer as a function of time using a monoclonal antibody according to any one of claims 1-7 or 15-21, where time-dependent a decrease in the subject's serum progastrin level indicates the effectiveness of the treatment. 35. A method for inhibiting a progastrin-dependent signal transduction cascade in a cell in vitro, comprising exposing a cell that secretes hPG with an amount of a neutralizing anti-PG monoclonal antibody effective to inhibit the progastrin-dependent signal transduction cascade according to any one of claims 1 to 7 or 15-21. 36. A kit for determining hPG, comprising (i) an N-terminal anti-hPG monoclonal antibody according to any one of claims 1 to 7 and an antibody that specifically binds the C-terminal region of hPG; or (ii) a C-terminal anti-hPG monoclonal antibody according to any one of claims 15-21 and an antibody that specifically binds the N-terminal region of hPG. 37. The kit according to claim 36, where the antibody that specifically binds the C-terminal region of hPG (i) is a polyclonal antibody; or an antibody that specifically binds the N-terminal region of hPG(ii) is a polyclonal antibody. 38. A hybridoma capable of secreting an anti-hPG monoclonal antibody according to any one of claims 15 to 21, wherein said hybridoma is CNCM I-4371 hybridoma, which was deposited pursuant to the Budapest Treaty on October 6, 2010 by the National Collection of Cultures of Microorganisms (CNCM) , Pasteur Institute, Paris, France. 39. A hybridoma capable of secreting an anti-hPG monoclonal antibody according to any one of claims 15 to 21, wherein said hybridoma is CNCM I-4372 hybridoma, which was deposited under the Budapest Treaty on October 6, 2010 by the National Collection of Cultures of Microorganisms (CNCM) , Pasteur Institute, Paris, France. 40. A hybridoma capable of secreting an anti-hPG monoclonal antibody according to any one of claims 15 to 21, wherein said hybridoma is CNCM I-4373 hybridoma, which was deposited under the Budapest Treaty on October 6, 2010 by the National Collection of Cultures of Microorganisms (CNCM) , Pasteur Institute, Paris, France. 41. A hybridoma capable of secreting an anti-hPG monoclonal antibody according to any one of claims 15 to 21, wherein said hybridoma is CNCM I-4374 hybridoma, which was deposited under the Budapest Treaty on October 6, 2010 by the National Collection of Cultures of Microorganisms (CNCM) , Pasteur Institute, Paris, France. 42. A hybridoma capable of secreting an anti-hPG monoclonal antibody according to any one of claims 15 to 21, wherein said hybridoma is a CNCM I-4375 hybridoma that was deposited under the Budapest Treaty on October 6, 2010 by the National Collection of Microorganism Cultures -