Combination therapy

Abstract

The present invention relates to methods of treating colorectal cancer in a subject in need thereof. The method comprises co-administering to the subject an effective amount of: a) a DKK1 antibody or antigen-binding fragment thereof; b) a VEGF or a VEGFR inhibitor; and c) optionally one or more chemotherapeutic agents or a pharmaceutically acceptable salt of any of the foregoing. The present invention also provides a pharmaceutical composition and a kit comprising a combination of a DKK1 antibody, and a VEGF or VEGFR inhibitor, and optionally one or more chemotherapeutic agents.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 388,553, filed on July 12, 2022. The entire teachings of the above application are incorporated herein by reference. Background Art

[0003] Colorectal cancer (CRC) is the third most prevalent cancer, with 1,931,590 cases diagnosed in 2020, and is the second leading cause of cancer-related deaths, with 935,173 deaths worldwide in 2020 (World Health Organization 2021). The global prevalence of CRC is higher in men than in women (746,298 vs. 614,304) (White et al., 2018). Despite the favorable contribution of screening programs, 25% of patients have advanced disease at diagnosis, while another 50% of patients subsequently develop metastasis, resulting in a higher mortality rate (Van Cutsem et al., 2011). Advanced colorectal cancer (CRC) remains a serious, life-threatening disease. Therefore, there is a continuing need for new and improved treatments for cancer patients. Summary of the Invention

[0004] The invention described herein is directed to a method of treating colorectal cancer in a subject in need of such treatment.

[0005] In a first embodiment, the method comprises co-administering to a subject having colorectal cancer an effective amount of a DKK1 antibody or antigen-binding fragment thereof, a VEGF or VEGFR inhibitor; and optionally one or more chemotherapeutic agents or pharmaceutically acceptable salts of any of the foregoing.

[0006] In another embodiment, the present invention relates to a pharmaceutical composition comprising: a DKK1 antibody or an antigen-binding fragment thereof; bevacizumab; and optionally one or more chemotherapeutic agents or a pharmaceutically acceptable salt of any of the foregoing.

[0007] In yet another embodiment, the present invention is a pharmaceutical composition comprising: a) a DKK1 antibody or an antigen-binding fragment thereof; b) a VEGF or VEGFR inhibitor; and c) one or more chemotherapeutic agents or a pharmaceutically acceptable salt of any of the foregoing.

[0008] In yet another embodiment, the present invention is a pharmaceutical composition for use in the treatment of colorectal cancer, the pharmaceutical composition comprising: a) a DKK1 antibody or an antigen-binding fragment thereof; b) a VEGF or VEGFR inhibitor; and c) one or more chemotherapeutic agents.

[0009] In yet another embodiment, the present invention is a pharmaceutical composition for use in the preparation of a medicament for treating colorectal cancer, the pharmaceutical composition comprising: a) a DKK1 antibody or an antigen-binding fragment thereof; b) a VEGF or VEGFR inhibitor; and c) one or more chemotherapeutic agents.

[0010] In yet another embodiment, the present invention is a kit comprising: a) a DKK1 antibody or an antigen-binding fragment thereof; b) a VEGF or VEGFR inhibitor; c) one or more chemotherapeutic agents or pharmaceutically acceptable salts thereof; and d) instructions for use.

[0011] In yet another embodiment, the present invention is a kit comprising: a) a DKK1 antibody or an antigen-binding fragment thereof; b) bevacizumab; c) one or more chemotherapeutic agents or pharmaceutically acceptable salts thereof; and d) instructions for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The foregoing will be apparent from the following more particular description of various example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters represent like parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the invention.

[0013] Figure 1 The overall design of the randomized phase 2 clinical trial is described.

[0014] Figures 2A and 2B show the results of co-administration of DKN-01 and the PI3K inhibitor apellix in a xenograft model of colorectal cancer.

[0015] Figure 3 Shown are the results of the duration of co-administration of mDKN-01 and anti-PD-1 antibody in a xenograft model of colorectal cancer.

[0016] Figure 4 Shown is a consort diagram of Part A of the DeFianCE study.

[0017] Figure 5 Table 1 is shown, which characterizes the patients studied in Part A of the DeFianCe study.

[0018] Figure 6 is a graph showing the best overall response among evaluable patients studied in Part A of the DeFianCe study.

[0019] Figure 7A is a graph showing the best overall response among evaluable patients studied in Part A of the DeFianCe study, who were categorized by the presence of a KRAS mutation.

[0020] Figure 7B is a graph showing the best overall response among evaluable patients studied in Part A of the DeFianCe study, categorized by the presence of liver metastases.

[0021] Figure 7C is a graph showing the best overall response among evaluable patients studied in Part A of the DeFianCe study, who were categorized by prior bevacizumab treatment.

[0022] Figure 7D is a graph showing the best overall response among evaluable patients studied in Part A of the DeFianCe study who were categorized by rapid disease progression.

[0023] Figure 8 is a "swim" graph showing study duration, overall survival after therapy for patients currently on the study, and follow-up for best overall response. DETAILED DESCRIPTION

[0024] A description of example embodiments of the invention follows.

[0025] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0026] While the invention has been particularly shown and described with reference to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims.

[0027] Colorectal cancer

[0028] Colorectal cancer (CRC), also known as bowel cancer, colon cancer, or rectal cancer, is any cancer that affects the colon, rectum, or both. The American Cancer Society estimates that approximately 1 in 21 men and 1 in 23 women in the United States will develop colorectal cancer in their lifetime.

[0029] Colorectal cancer can be stage 0, stage I, stage IIA, stage IIB, stage IIC, stage IIIIA, stage IIIB, stage IIIC, stage IVA, stage IVB, or stage IVC as defined by the TNM system of the American Joint Committee on Cancer (AJCC).

[0030] In some embodiments, the colorectal cancer is metastatic or advanced colorectal cancer (i.e., colorectal cancer that has spread from the primary site to other parts of the body, such as the lungs, liver, or any other organ other than the primary site). The methods described herein relate to treating subjects with colorectal cancer. In some embodiments, the colorectal cancer is colorectal adenocarcinoma. In some embodiments, the colorectal cancer is metastatic colorectal cancer (mCRC). In some embodiments, the colorectal cancer is mismatch repair function normal (pMMR) mCRC. In some embodiments, the CRC is not pMMR. In some embodiments, the CRC is mismatch repair deficiency (dMMR) colorectal cancer. In some embodiments, the colorectal cancer is microsatellite stable (MSS) CRC. In some embodiments, the colorectal cancer is microsatellite unstable (MSI) CRC. In some embodiments, the colorectal cancer is pMMR / MSSmCRC. In some embodiments, the colorectal cancer is pMMR / MSS mCRC and has not responded to treatment previously performed with a single agent checkpoint inhibitor. In some embodiments, CRC is KRAS wild type and has not responded to treatment previously performed with an EGFR inhibitor. In some embodiments, the colorectal cancer is an advanced cancer. In some embodiments, the colorectal cancer is specific to the rectum. In another embodiment, the colorectal cancer is specific to the colon. In yet another embodiment, the colorectal cancer involves both the colon and the rectum.

[0031] MSS CRC: Each of our cells contains DNA with genes that provide instructions for how to grow, perform specific activities, divide, or die. The DNA in our cells also contains segments of short, repetitive DNA sequences called microsatellites. This microsatellite DNA serves as a biomarker for the stability of our DNA. When the number of microsatellite repeats is the same in all cells of the body, the DNA is considered stable, also known as microsatellite stable, or MSS.

[0032] DNA mismatch repair (MMR) is a quality control and "spell-check" process responsible for ensuring that DNA is copied without errors. When this is correct, the number of microsatellite repeats is the same in all cells. The MMR process relies on four main proteins: MLH1, MSH2, MSH6, and PMS2, which work together to repair errors in DNA.

[0033] In some cancer patients, the MMR process works perfectly. In these people, the cancer cells have normal mismatch repair (pMMR), and their tumor cells have the same number of repeat sequences as their healthy cells. This is also known as microsatellite stability, or MSS.

[0034] In some embodiments, the colorectal cancer has a mutation, chromosomal alteration, or translocation affecting one or more of the WNT / β-catenin, MAPK / PBK, TGF-β, or TP53 pathways. In some embodiments, the colorectal cancer has a gene mutation selected from the group consisting of c-MYC, KRAS, NRAS, HRAS, BRAF, PIK3CA, PTEN, SMAD2, or SMAD4. In some embodiments, the mCRC has a BRAF or KRAS mutation. In some embodiments, the BRAF mutation is a V600E substitution mutation. In some embodiments, the KRAS mutation is a V9, G12, G13, V14, L19, Q22, D33, A59, G60, Q61, R68, K117, A146, R164, K176, or K180 substitution. In some embodiments, the KRAS mutation is G12D, G12V, G12C, G12A, G12S, or other G12 variants. In other embodiments, the KRAS mutation is G13D, G12R, Q61H, Q61P, Q61K, Q61R, Q61L, Q61H, R68S, A11-G12dup, Y71C, P34L, L19F, Q22K, A59T, A146V, K117N, A146T, G13C, Q61E, E98*, and A155D. In some embodiments, the NRAS mutation is selected from G12, G13, G60, Q61, E123, or P185. In other embodiments, the NRAS mutation is selected from G12D, G13D, G13R, G12C, Q61L, Q61K, Q61H, Q61R, G12V, G12A, E132K, R164C, and E76K. In some embodiments, the HRAS mutation is G12, G13, Q61, K117, R164, or P167. In some embodiments, the colorectal cancer is KRAS wild-type. In some embodiments, the colorectal cancer is BRAF wild-type. In another embodiment, the colorectal cancer does not have a BRAFV600E mutation.

[0035] Evaluation of DKK1 levels in tumor, serum, and plasma

[0036] In another embodiment, the subject's tumor (a subject with colorectal cancer) has detectable DKK1 expression levels as determined by one or more of various standard mRNA or protein detection methods known in the art, such as chromogenic in situ hybridization (RNAscope), immunohistochemistry, qPCR, RNA-Seq, and NanoString.

[0037] H score and % positive value

[0038] The level of expression of the target gene product (e.g., expression of DKK1) can be evaluated by immunohistochemistry or chromogenic in situ hybridization techniques. A convenient semi-quantitative measure of expression level is to calculate the % positive value (% of tumor cells stained by the DKK-1 RNA detection reagent) or to assign an H score (or "tissue" score) to the tumor sample. For the H score, the amount or intensity of staining of each cell in a fixed field of view is determined (0, 1+, 2+, or 3+). For in situ hybridization techniques, the number of dots per cell can be determined, where 0 is no dot detected per cell, 1+ is 1-3 dots / cell, 2+ is 4-9 dots / cell, and 3+ is 10+ dots / cell. The H score can then be based on the main amount of staining (or number of dots / cell), or more complexly, can include the sum of the individual percentages of each level of staining (or number of dots / cell) seen. By one approach, the percentage of cells at each level of staining intensity (or number of dots / cell) is calculated, and finally the H score is assigned using the following formula:

[0039] H score = [1 x (cells 1+%) + 2 x (cells 2+%) + 3 x (cells 3+%)]

[0040] The final H score (ranging from 0 to 300) gives greater relative weight to greater intensity or amount of staining (e.g., dots / cell) in a given tumor sample. The sample can then be considered positive or negative based on a specific discrimination threshold. See, e.g., Hirsch FR, Varella-Garcia M, Bunn PA Jr, et al: Epidermal growth factor receptor in non-small-cell lung carcinomas: Correlation between gene copy number and protein expression and impact on prognosis. J Clin Oncol 21:3798-3807, 2003; and John T, Liu G, Tsao MS: Overview of molecular testing in non-small-cell lung cancer: Mutational analysis, gene copy number, protein expression and other biomarkers of EGFR for the prediction of response to tyrosine kinase inhibitors. Oncogene 28:S14-S23, 2009.

[0041] In various embodiments, the H-score (eg, predetermined values ​​of the H-score) can be from 0 to 300, eg, 0, 1, 2, 3, etc. Example predetermined values ​​of the H-score are: 1, 2, 3, 4, and 5. In some embodiments, the predetermined values ​​of the H-score are: 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 4, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 135, 136, 137, 1 38, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196 6, 197, 198, 199, 200, 201, 202, 103, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253,254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, or 300.

[0042] In an alternative embodiment, the measurement value of DKK1 expression can be a value for the fraction of tumor cells that stain positive for DKK1 (% positive or tumor percentage score (TPS)). First, based on the number of spots in the cell, the amount of staining (0, 1+, 2+, or 3+) for each tumor cell in a fixed field of view is determined. After all tumor cells are assigned as "positive" (for example, a single staining spot is detected by RNAscope chromogenic in situ hybridization), the percentage of positive tumor cells is determined by adding up all stained tumor cells and dividing by the total number of tumor cells. The % positive can range from 0 to 100.

[0043] In various embodiments, the % Positive value (e.g., a predetermined value for % Positive) can be from 0% to 50%, such as from 1% to 5%. Example predetermined values ​​for % Positive are: 1% or greater, 2% or greater, 3% or greater, 4% or greater, or 5% or greater. In certain embodiments, the predetermined values ​​for % Positive are: 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110 %, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.

[0044] RNAscope analysis

[0045] One method for calculating the H-score of a sample is developed and commercially available from Advanced Cell Diagnostics, Inc. In situ hybridization techniques, such as those described at the URL https: / / acdbio.com / and available from Flagship Biosciences, Broomfield, CO. This technique relies on an optical signal from a hybridization probe homologous to the target mRNA. The signal can be detected by bright field or epifluorescence microscopy. The technique allows for the detection of single molecules. See, for example, RNAscope: A Novel In Situ RNA Analysis Platform for Formalin-Fixed Paraffin-Embedded Tissues. Wang F, Flanagan J, Su N, Wang LC, Bui S, Nielson A, Wu X, Vo HT, Ma XJ, Luo Y (2012). J of Mol Diagnostics, 14(1): 22-29.

[0046] Enzyme-linked immunosorbent assay

[0047] Serum and plasma DKK1 levels can be measured using a modified immunoassay with electrochemiluminescence detection. In this assay, serum or plasma samples containing DKK1 are captured by anti-hDKK1 (MAB 1096) coated on a microtiter plate. Unbound material is washed away, leaving only the captured DKK1. DKK1 is released from the MAB 1096-coated plate by acidification (leaving MAB 1096 bound to the plate). The released DKK1 is transferred to an MSD plate, where DKK1 is directly bound to the plate. Bound DKK1 is detected by adding biotinylated anti-hDKK1 (MAB 1096) and then developing with streptavidin-ruthenium and MSD read buffer. Results are reported as the concentration of DKK1 in the serum or plasma sample (ng / mL).

[0048] DKK1 and DKK1 Antibodies

[0049] Dickkopf-1 (DKK1) is a secreted regulator of the Wnt signaling pathway that affects many biological processes, such as stem cell maintenance, cell fate determination, cell proliferation, survival, migration, and polarity determination during development and adult tissue homeostasis. DKK1 has been most widely characterized as an inhibitor of canonical Wnt / β-catenin-dependent signaling, and this has been associated with promoting an immunosuppressive tumor microenvironment. DKK1 is also involved in promoting tumor growth and metastasis by activating non-canonical (β-catenin-independent Wnt signaling) and PI3K / AKT signaling pathways. DKK1 also regulates bone homeostasis during development and in adult organisms. DKK1 inhibits osteoblastogenesis, or the differentiation of mesenchymal stem cells into osteoblasts (OBs), a process promoted by Wnt signaling. As a result, the balance of OBs (bone formation) / osteoclasts (OCs) (bone resorption) shifts toward increased bone resorption, ultimately leading to osteolytic lesions. Certain tumors are associated with the development of osteolytic bone disease mediated by increased OC bone resorption and impaired OB bone formation.

[0050] In another embodiment, the colorectal cancer has increased expression levels of DKK1 as determined by one or more of various standard mRNA or protein detection methods known in the art (eg, chromogenic in situ hybridization or immunohistochemistry).

[0051] DKK1 antibodies have been previously described (see, e.g., U.S. Patent No. 8,148,498, which is incorporated herein by reference in its entirety). The DKK1 antibodies disclosed herein are therapeutically useful DKK1 antagonists that possess many desirable properties. For example, the DKK1 antibodies reduce DKK1-mediated inhibition of alkaline phosphatase (a marker of osteoblast activity) and treat various types of cancer (e.g., non-small cell lung cancer).

[0052] A full-length antibody, when present in nature, is an immunoglobulin molecule comprising two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The amino-terminal portion of each chain includes a variable region of approximately 100-110 amino acids, which is primarily responsible for antigen recognition via the complementarity determining regions (CDRs) contained therein. The carboxyl-terminal portion of each chain defines a constant region, which is primarily responsible for effector functions.

[0053] These CDRs are interspersed with more conserved regions, called framework regions ("FRs"). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) consists of three CDRs and four FRs, arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are referred to as "LCDR1, LCDR2, and LCDR3" and the three CDRs of the heavy chain are referred to as "HCDR1, HCDR2, and HCDR3." These CDRs contain most of the residues that form specific interactions with the antigen. The numbering and positioning of the CDR amino acid residues within the LCVR and HCVR regions follow the well-known Kabat numbering convention.

[0054] The light chain is divided into kappa or lambda, and is characterized by specific constant regions known in the art. The heavy chain is divided into gamma, μ, alpha, delta or epsilon, and the isotype of the antibody is defined as IgG, IgM, IgA, IgD or IgE, respectively. IgG antibodies can be further divided into subclasses, for example, IgG1, IgG2, IgG3, IgG4. Every type of heavy chain is characterized by a specific constant region with a sequence well known in the art.

[0055] As used herein, the term "monoclonal antibody" (Mab) refers to an antibody that is derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone, rather than the method by which it is produced. The Mabs of the present invention are preferably present in a homogeneous or substantially homogeneous population. A complete Mab contains two heavy chains and two light chains.

[0056] Unless otherwise indicated, the term "DKK1 antibody" encompasses both full-length antibodies as well as antigen-binding fragments of DKK1 antibodies.

[0057] Such "antigen-binding fragments" of monoclonal antibodies include, for example, Fab fragments, Fab' fragments, F(ab')2 fragments, and single-chain Fv fragments, as well as bispecific and / or multivalent antibodies that can utilize the CDRs of the DKK1 antibody. Monoclonal antibodies and their antigen-binding fragments can be produced, for example, by recombinant technology, phage display technology, synthetic technology (e.g., CDR transplantation), or a combination of such technologies, or other technologies known in the art. For example, mice can be immunized with human DKK1 or a fragment thereof, the resulting antibodies can be recovered and purified, and they can be evaluated by methods known in the art to determine whether they have similar or identical binding and functional properties to the antibody compounds disclosed herein. Antigen-binding fragments can also be prepared by conventional methods. Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art and can be found, for example, in Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, Chapters 5-8 and 15, ISBN 0-87969-314-2.

[0058] The monoclonal DKK1 antibodies disclosed herein are engineered to contain framework regions that are substantially human or fully human surrounding CDRs derived from non-human antibodies. "Antigen-binding fragments" of such human engineered antibodies include, for example, Fab fragments, Fab' fragments, F(ab')2 fragments, and single-chain Fv fragments. "Framework region" or "framework sequence" refers to any one of framework regions 1 to 4. Human engineered antibodies and antigen-binding fragments thereof encompassed by the antibodies disclosed herein include molecules in which any one or more of framework regions 1 to 4 are substantially or fully human, i.e., in which any of the possible combinations of separate substantially or fully human framework regions 1 to 4 are present. For example, this includes molecules in which framework region 1 and framework region 2, framework region 1 and framework region 3, framework regions 1, 2, and 3, etc. are substantially or fully human. Substantially human frameworks are those that have at least about 80% sequence identity with known human germline framework sequences. Preferably, substantially human frameworks have at least about 85%, about 90%, about 95%, or about 99% sequence identity with known human germline framework sequences.

[0059] Several different methods can be used to generate human engineered antibodies that show similar functional properties in addition to those disclosed herein. The specific antibody compounds disclosed herein can be used as templates or parent antibody compounds to prepare additional antibody compounds. In one method, the parent antibody compound CDR is transplanted into a human framework with high sequence identity to the parent antibody compound framework. The sequence identity of the new framework will generally be at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 99% identical to the sequence of the corresponding framework in the parent antibody compound. This transplant may result in a reduction in binding affinity compared to the parent antibody. If this is the case, the framework can be backmutated to the parent framework at certain positions based on the specific criteria disclosed by Queen et al. (1991) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 88: 2869. Additional references describing methods that can be used to humanize mouse antibodies include U.S. Pat. Nos. 4,816,397; 5,225,539 and 5,693,761; the computer programs ABMOD and ENCAD, as described in Levitt (1983) J. Mol. Biol. 168:595-620; and the method of Winter and co-workers (Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323-327; and Verhoeyen et al. (1988) Science 239:1534-1536). Methods for identifying residues to be considered for backmutation are known in the art (see, e.g., U.S. Pat. No. 8,148,498).

[0060] The DKK1 antibody administered in the methods of treatment described herein comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3.

[0061] In one embodiment, the DKK1 antibody comprises a LCDR1 having the amino sequence of SEQ ID NO: 1, a LCDR2 having the amino sequence of SEQ ID NO: 2, a LCDR3 having the amino sequence of SEQ ID NO: 3, a HCDR1 having the amino sequence of SEQ ID NO: 4, a HCDR2 having the amino sequence of SEQ ID NO: 5, and a HCDR3 having the amino sequence of SEQ ID NO: 6.

[0062] In another embodiment, the DKK1 antibody comprises a LCVR having the amino acid sequence of SEQ ID NO: 7 and a HCVR having the amino acid sequence of SEQ ID NO: 8. In a specific embodiment, the LCVR comprises the amino acid sequence of SEQ ID NO: 11 and the HCVR comprises the amino acid sequence of SEQ ID NO: 12.

[0063] In another embodiment, the DKK1 antibody comprises a heavy chain (HC) having the amino acid sequence of SEQ ID NO: 17 and a light chain (LC) having the amino acid sequence of SEQ ID NO: 18. The DKK1 antibody or antigen-binding fragment thereof comprising the HC and LC amino acid sequences of SEQ ID NO: 17 and SEQ ID NO: 18, respectively, is referred to herein as DKN-01. In particular, DKN-01 has the molecular / empirical formula C 6394 H 9810 N 1698 O 2012 S 42 , and the molecular weight is 144015 Daltons (intact).

[0064] In certain embodiments, the DKK1 antibodies disclosed herein are IgG4 antibodies that have neutralizing activity against human DKK1 comprising the sequence shown in SEQ ID NO: 22 or a fragment thereof. For example, canonical Wnt signaling is important for osteoblast differentiation and activity. Wnt-3a in combination with BMP-4 induces pluripotent mouse C2C12 cells to differentiate into osteoblasts, with a measurable alkaline phosphatase ("AP") (a marker of osteoblast activity) endpoint. DKK1 (an inhibitor of canonical Wnt signaling) inhibits the differentiation and production of AP. Neutralizing DKK1 antibodies prevents DKK1-mediated inhibition of AP. Antibodies that block DKK1 inhibitory activity prevent the loss of AP activity (see U.S. Patent No. 8,148,498). In specific embodiments, the DKK1 antibody with neutralizing activity is DKN-01, which is an IgG4 antibody.

[0065] The DKK1 antibodies disclosed herein have a high affinity (Kd) for DKK1 (e.g., human DKK1, SEQ ID NO: 22), as described in U.S. Patent No. 8,148,498. For example, the DKK1 antibodies of the present invention have a Kd of 0.5 x 10 -12 M and 3.0x10 -11 Kd between M.

[0066] sequence

[0067] The following is the sequence of the DKN-01 antibody that can be employed in practicing the various exemplary embodiments described herein.

[0068] LCDR1

[0069] His Ala Ser Asp Ser Ile Ser Asn Ser Leu His (SEQ ID NO:1)

[0070] LCDR2

[0071] Tyr Xaa Arg Gln Ser Xaa Gln (SEQ ID NO:2)

[0072] wherein Xaa at position 2 is Gly or Ala; and Xaa at position 6 is Ile or Glu

[0073] LCDR3

[0074] Gln Gln Ser Xaa Ser Trp Pro Leu His(SEQ ID NO:3)

[0075] wherein Xaa at position 4 is Glu or Ala

[0076] HCDR1

[0077] Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser (SEQ ID NO:4)

[0078] HCDR2

[0079] Thr Ile Ser Gly Gly Gly Phe Gly Thr Tyr Tyr Pro Asp Ser Val Lys(SEQID NO:5)

[0080] HCDR3

[0081] Pro Gly Tyr Xaa Asn Tyr Tyr Phe Asp Ile (SEQ ID NO:6)

[0082] wherein Xaa at position 4 is His or Asn

[0083] LCVR

[0084] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro GlyGluArg Ala Thr Leu Ser Cys His Ala Ser Asp Ser Ile Ser Asn Ser LeuHis Trp TyrGln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr TyrXaa Arg Gln Ser XaaGln Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly SerGly Thr Asp Phe Thr Leu ThrIle Ser Ser Leu Glu Pro Glu Asp Phe AlaVal Tyr Tyr Cys Gln Gln Ser Xaa SerTrp Pro Leu His Phe Gly Gly GlyThr Lys Val Glu Ile Lys (SEQ ID NO:7)

[0085] wherein Xaa at position 51 is Gly or Ala; Xaa at position 55 is Ile or Glu and Xaa at position 92 is Glu or Ala.

[0086] HCVR

[0087] Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr ThrMet Ser TrpVal Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala ThrIle Ser Gly Gly GlyPhe Gly Thr Tyr Tyr Pro Asp Ser Val Lys Gly ArgPhe Thr Ile Ser Arg Asp AsnAla Lys Asn Ser Leu Tyr Leu Gln Met AsnSer Leu Arg Ala Glu Asp Thr Ala ValTyr Tyr Cys Ala Arg Pro Gly TyrXaa Asn Tyr Tyr Phe Asp Ile Trp Gly Gln GlyThr Thr Val Thr Val SerSer(SEQ ID NO:8)

[0088] Where Xaa at position 102 is His or Asn

[0089] LCVR

[0090] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro GlyGluArg Ala Thr Leu Ser Cys His Ala Ser Asp Ser Ile Ser Asn Ser LeuHis Trp TyrGln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr TyrGly Arg Gln Ser IleGln Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser GlyThr Asp Phe Thr Leu ThrIle Ser Ser Leu Glu Pro Glu Asp Phe Ala ValTyr Tyr Cys Gln Gln Ser Glu SerTrp Pro Leu His Phe Gly Gly Gly ThrLys Val Glu Ile Lys(SEQ ID NO:9)

[0091] HCVR

[0092] Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr ThrMet Ser TrpVal Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala ThrIle Ser Gly Gly GlyPhe Gly Thr Tyr Tyr Pro Asp Ser Val Lys Gly ArgPhe Thr Ile Ser Arg Asp AsnAla Lys Asn Ser Leu Tyr Leu Gln Met AsnSer Leu Arg Ala Glu Asp Thr Ala ValTyr Tyr Cys Ala Arg Pro Gly TyrHis Asn Tyr Tyr Phe Asp Ile Trp Gly Gln GlyThr Thr Val Thr Val SerSer(SEQ ID NO:10)

[0093] LCVR

[0094] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro GlyGluArg Ala Thr Leu Ser Cys His Ala Ser Asp Ser Ile Ser Asn Ser LeuHis Trp TyrGln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr TyrAla Arg Gln Ser IleGln Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser GlyThr Asp Phe Thr Leu ThrIle Ser Ser Leu Glu Pro Glu Asp Phe Ala ValTyr Tyr Cys Gln Gln Ser Glu SerTrp Pro Leu His Phe Gly Gly Gly ThrLys Val Glu Ile Lys(SEQ ID NO:11)

[0095] HCVR

[0096] Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr ThrMet Ser TrpVal Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala ThrIle Ser Gly Gly GlyPhe Gly Thr Tyr Tyr Pro Asp Ser Val Lys Gly ArgPhe Thr Ile Ser Arg Asp AsnAla Lys Asn Ser Leu Tyr Leu Gln Met AsnSer Leu Arg Ala Glu Asp Thr Ala ValTyr Tyr Cys Ala Arg Pro Gly TyrAsn Asn Tyr Tyr Phe Asp Ile Trp Gly Gln GlyThr Thr Val Thr Val SerSer(SEQ ID NO:12)

[0097] LCVR

[0098] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro GlyGluArg Ala Thr Leu Ser Cys His Ala Ser Asp Ser Ile Ser Asn Ser LeuHis Trp TyrGln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr TyrGly Arg Gln Ser IleGln Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser GlyThr Asp Phe Thr Leu ThrIle Ser Ser Leu Glu Pro Glu Asp Phe Ala ValTyr Tyr Cys Gln Gln Ser Ala SerTrp Pro Leu His Phe Gly Gly Gly ThrLys Val Glu Ile Lys(SEQ ID NO:13)

[0099] LCVR

[0100] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro GlyGluArg Ala Thr Leu Ser Cys His Ala Ser Asp Ser Ile Ser Asn Ser LeuHis Trp TyrGln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr TyrAla Arg Gln Ser GluGln Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly SerGly Thr Asp Phe Thr Leu ThrIle Ser Ser Leu Glu Pro Glu Asp Phe AlaVal Tyr Tyr Cys Gln Gln Ser Ala SerTrp Pro Leu His Phe Gly Gly GlyThr Lys Val Glu Ile Lys(SEQ ID NO:14)

[0101] HC

[0102] Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr ThrMet Ser TrpVal Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala ThrIle Ser Gly Gly Gly P He Gly Gly Ser Asp Tyr Tyr ArgPhe Thr Ile Ser Arg Asp AsnAla Lys Asn Ser Leu Tyr Leu Gln Met AsnSer Leu Arg Ala Glu Asp Thr Ala ValTyr Tyr Cys Ala Arg Pro Gly TyrHis Asn Tyr Tyr Phe Asp Ile Trp Gly Glyn GlyThr Thr Val Thr Val Thr Val Ser Ser Pro Ala Ser Cys Ser ArgSer Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys AspTyr PhePro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly ValHisThr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser SerVal Val ThrVal Ser Thr AsnVal Asp His Lys ProSer Asn Thr Lys Val Asp Lys Arg Val Glu Ser LysTyr Gly Pro Pro Cys Pro ProCys Pro Ala Pro Glu Ala Ala Gly ProSer Val Phe Leu Phe Pro Pro Lys ProLys Asp Thr Leu Met Ile Ser ArgThr Pro GluVal Thr Cys Val Val Val Asp ValSer Gln Glu Asp Pro GluVal Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val HisAsn Ala Lys ThrLys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val SerVal LeuThr Val Leu Asp ValSer Gln Glu Lys As Glu Lys Cys Lys ValSerAsn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys GlyGln ProArg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu MetThr Lys Asn GlnVal Ser Leu Thr Cys Leu Val Gly Lys Phe Tyr Pro Gly Ser Gln Pro Glu Asn Asn Tyr LysThr Thr Pro Pro Val Leu Asp SerAsp Gly Ser Phe Phe Leu Tyr Ser ArgLeu Thr Val Asp Lys Ser Arg Trp Gln GluGly Asn Val Phe Ser Cys SerVal Met His Glu Ala Leu His Asn His Tyr Thr Gln Ser Leu Leu Ser GlyS Q NO:15)

[0103] LC

[0104] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro GlyGluArg Ala Thr Leu Ser Cys His Ala Ser Asp Ser Ile Ser Asn Ser LeuHis Trp TyrGln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr TyrGly Arg Gln Ser IleGln Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser GlyThr Asp Phe Thr Leu ThrIle Ser Ser Leu Glu Pro Glu Asp Phe Ala ValTyr Tyr Cys Gln Gln Ser Glu SerTrp Pro Leu His Phe Gly Gly Gly ThrLys Val Glu Ile Lys Arg Thr Val Ala AlaPro Ser Val Phe Ile Phe Pro ProSer Asp Glu Gln Leu Lys Ser Gly Thr Ala SerVal Val Cys Leu Leu AsnAsn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys ValAsp Asn Ala LeuGln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser LysAsp SerThr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu LysHisLys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro ValThr Lys SerPhe Asn Arg Gly Glu Cys(SEQ ID NO:16)

[0105] HC

[0106] Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr ThrMet Ser TrpVal Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala ThrIle Ser Gly Gly Gly P He Gly Gly Ser Asp Tyr Tyr ArgPhe Thr Ile Ser Arg Asp AsnAla Lys Asn Ser Leu Tyr Leu Gln Met AsnSer Leu Arg Ala Glu Asp Thr Ala ValTyr Tyr Cys Ala Arg Pro Gly TyrAsn Asn Tyr Tyr Phe Asp Ile Trp Gly Pro Glyn GlyThr Thr Val Thr Val Thr Ala Ser Val Ser Pro Val Ser Cys Ser ArgSer Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys AspTyr PhePro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly ValHisThr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser SerVal Val ThrVal Ser Thr AsnVal Asp His Lys ProSer Asn Thr Lys Val Asp Lys Arg Val Glu Ser LysTyr Gly Pro Pro Cys Pro ProCys Pro Ala Pro Glu Ala Ala Gly ProSer Val Phe Leu Phe Pro Pro Lys ProLys Asp Thr Leu Met Ile Ser ArgThr Pro GluVal Thr Cys Val Val Val Asp ValSer Gln Glu Asp Pro GluVal Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val HisAsn Ala Lys ThrLys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val SerVal LeuThr Val Leu Asp ValSer Gln Glu Lys As Glu Lys Cys Lys ValSerAsn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys GlyGln ProArg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu MetThr Lys Asn GlnVal Ser Leu Thr Cys Leu Val Gly Lys Phe Tyr Pro Gly Ser Gln Pro Glu Asn Asn Tyr LysThr Thr Pro Pro Val Leu Asp SerAsp Gly Ser Phe Phe Leu Tyr Ser ArgLeu Thr Val Asp Lys Ser Arg Trp Gln GluGly Asn Val Phe Ser Cys SerVal Met His Glu Ala Leu His Asn His Tyr Thr Gln Ser Leu Leu Ser GlyS Q NO:17)

[0107] LC

[0108] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro GlyGluArg Ala Thr Leu Ser Cys His Ala Ser Asp Ser Ile Ser Asn Ser LeuHis Trp TyrGln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr TyrAla Arg Gln Ser IleGln Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser GlyThr Asp Phe Thr Leu ThrIle Ser Ser Leu Glu Pro Glu Asp Phe Ala ValTyr Tyr Cys Gln Gln Ser Glu SerTrp Pro Leu His Phe Gly Gly Gly ThrLys Val Glu Ile Lys Arg Thr Val Ala AlaPro Ser Val Phe Ile Phe Pro ProSer Asp Glu Gln Leu Lys Ser Gly Thr Ala SerVal Val Cys Leu Leu AsnAsn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys ValAsp Asn Ala LeuGln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser LysAsp SerThr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu LysHisLys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro ValThr Lys SerPhe Asn Arg Gly Glu Cys(SEQ ID NO:18)

[0109] HC

[0110] Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly GlySerLeu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr ThrMet Ser TrpVal Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala ThrIle Ser Gly Gly Gly P He Gly Gly Ser Asp Tyr Tyr ArgPhe Thr Ile Ser Arg Asp AsnAla Lys Asn Ser Leu Tyr Leu Gln Met AsnSer Leu Arg Ala Glu Asp Thr Ala ValTyr Tyr Cys Ala Arg Pro Gly TyrHis Asn Tyr Tyr Phe Asp Ile Trp Gly Glyn GlyThr Thr Val Thr Val Thr Val Ser Ser Pro Ala Ser Cys Ser ArgSer Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys AspTyr PhePro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly ValHisThr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser SerVal Val ThrVal Ser Thr AsnVal Asp His Lys ProSer Asn Thr Lys Val Asp Lys Arg Val Glu Ser LysTyr Gly Pro Pro Cys Pro ProCys Pro Ala Pro Glu Ala Ala Gly ProSer Val Phe Leu Phe Pro Pro Lys ProLys Asp Thr Leu Met Ile Ser ArgThr Pro GluVal Thr Cys Val Val Val Asp ValSer Gln Glu Asp Pro GluVal Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val HisAsn Ala Lys ThrLys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val SerVal LeuThr Val Leu Asp ValSer Gln Glu Lys As Glu Lys Cys Lys ValSerAsn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys GlyGln ProArg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu MetThr Lys Asn GlnVal Ser Leu Thr Cys Leu Val Gly Phe Lys Pro Gly Ser Gln Pro Glu Asn Asn Tyr LysThr Thr Pro Pro Val Leu Asp SerAsp Gly Ser Phe Phe Leu Tyr Ser ArgLeu Thr Val Asp Lys Ser Arg Trp Gln GluGly Asn Val Phe Ser Cys SerVal Met His Glu Ala Leu His Asn His Tyr Thr Gln Ser Leu Leu Ser GlyS Q NO:19)

[0111] LC

[0112] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro GlyGluArg Ala Thr Leu Ser Cys His Ala Ser Asp Ser Ile Ser Asn Ser LeuHis Trp TyrGln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr TyrGly Arg Gln Ser IleGln Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser GlyThr Asp Phe Thr Leu ThrIle Ser Ser Leu Glu Pro Glu Asp Phe Ala ValTyr Tyr Cys Gln Gln Ser Ala SerTrp Pro Leu His Phe Gly Gly Gly ThrLys Val Glu Ile Lys Arg Thr Val Ala AlaPro Ser Val Phe Ile Phe Pro ProSer Asp Glu Gln Leu Lys Ser Gly Thr Ala SerVal Val Cys Leu Leu AsnAsn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys ValAsp Asn Ala LeuGln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser LysAsp SerThr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu LysHisLys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro ValThr Lys SerPhe Asn Arg Gly Glu Cys(SEQ ID NO:20)

[0113] LC

[0114] Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys His Ala Ser Asp Ser Ile Ser Asn Ser Leu His Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Tyr Ala Arg Gln Ser Glu Gln Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ala Ser Trp Pro Leu His Phe Gly Gly Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys(SEQ ID NO:21)

[0115] Human DKK1 amino acid sequence

[0116] Thr Leu Asn Ser Val Leu Asn Ser Asn Ala Ile Lys Asn Leu Pro ProProLeu Gly Gly Ala Ala Gly His Pro Gly Ser Ala Val Ser Ala Ala ProGly Ile LeuTyr Pro Gly Gly Asn Lys Tyr Gln Thr Ile Asp Asn Tyr GlnPro Tyr Pro Cys AlaGlu Asp Glu Glu Cys Gly Thr Asp Glu Tyr Cys AlaSer Pro Thr Arg Gly Gly AspAla Gly Val Gln Ile Cys Leu Ala Cys ArgLys Arg Arg Lys Arg Cys Met Arg HisAla Met Cys Cys Pro Gly AsnTyr Cys Lys Asn Gly Ile Cys Val Ser Ser Asp GlnAsn His Phe Arg GlyGlu Ile Glu Glu Thr Ile Thr Glu Ser Phe Gly Asn Asp HisSer Thr LeuAsp Gly Tyr Ser Arg Arg Thr Thr Leu Ser Ser Lys Met Tyr His ThrLysGly Gln Glu Gly Ser Val Cys Leu Arg Ser Ser Asp Cys Ala Ser Gly LeuCys CysAla Arg His Phe Trp Ser Lys Ile Cys Lys Pro Val Leu Lys GluGly Gln Val CysThr Lys His Arg Arg Lys Gly Ser His Gly Leu Glu IlePhe Gln Arg Cys Tyr CysGly Glu Gly Leu Ser Cys Arg Ile Gln Lys AspHis His Gln Ala Ser Asn Ser SerArg Leu His Thr Cys Gln Arg His(SEQ ID NO:22)

[0117] VEGF / VEGFR inhibitor

[0118] Vascular endothelial growth factor (VEGF) / vascular endothelial growth factor receptor (VEGFR) inhibitors are agents that inhibit the activity of VEGF and VEGFR (including VEGFR2). Anti-VEGF monoclonal antibodies include bevacizumab (Avastin, Genentech) or bevacizumab-awwb (Mvasi, Amgen). Bevacizumab is a vascular endothelial growth factor directed antibody. Bevacizumab is a recombinant humanized monoclonal IgG1 antibody containing human framework regions and mouse complementary determining regions (CDRs). Bevacizumab binds to VEGF and prevents VEGF from interacting with its receptors (Flt-I and KDR) on the endothelial cell surface. The interaction of VEGF with its receptors causes endothelial cell proliferation and new blood vessel formation in an in vitro model of angiogenesis. Administering bevacizumab reduces microvascular growth and inhibits metastatic disease progression.

[0119] In various embodiments of the methods of the present invention, bevacizumab can be replaced by one of its biosimilars. For example, bevacizumab can be replaced by Mvasi (bevacizumab-awwb) available from Amgen. In another example, bevacizumab can be replaced by Zirabev (bevacizumab-bvzr) available from Pfizer Inc. In another example, bevacizumab can be replaced by Alymsys (bevacizumab-maly) available from Amneal Pharmaceuticals Co., Ltd. In another example, bevacizumab can be replaced by Vegzelma (bevacizumab-adcd) available from Celltrion Healthcare.

[0120] The mechanism of action of biosimilars of bevacizumab is thought to be similar to that of bevacizumab.

[0121] In the treatment of colorectal cancer, bevacizumab is typically administered on day 1 of each 14-day cycle during the induction and maintenance phases, or once every two weeks, at a dose of between about 4 and 12 mg / kg as an intravenous injection, with dose adjustments as needed. In some embodiments, bevacizumab is administered at about 5 mg / kg. In some embodiments, bevacizumab is administered at about 10 mg / kg.

[0122] Ramucirumab (Cyramza; Eli Lily) is a human vascular endothelial growth factor receptor 2 (VEGFR2) antagonist that specifically binds to VEGFR2 and blocks the binding of the VEGFR ligands VEGF-A, VEGF-C, and VEGF-D. Thus, ramucirumab inhibits ligand-stimulated activation of VEGFR2, thereby inhibiting ligand-induced proliferation and migration of human endothelial cells. Ramucirumab is a recombinant human IgG1 monoclonal antibody. Ramucirumab has an approximate molecular weight of 147 kDa. Ramucirumab is produced in genetically engineered mammalian NSO cells. Ramucirumab injection for intravenous use is a sterile, preservative-free, clear to slightly opalescent and colorless to slightly yellow solution. Ramucirumab is provided in a concentration of 10 mg / mL in 100 mg (10 mL) or 500 mg (50 mL) single-dose vials. Ramucirumab was formulated in glycine (9.98 mg / mL), histidine (0.65 mg / mL), histidine monohydrochloride (1.22 mg / mL), polysorbate 80 (0.1 mg / mL), sodium chloride (4.383 mg / mL), and Water for Injection, USP, pH 6.0.

[0123] In the treatment of colorectal cancer, ramucirumab is typically administered on day 1 of each 14-day cycle during the induction and maintenance phases, or once every two weeks, at a dose of between about 5 and 15 mg / kg as an intravenous infusion, with dose adjustments as needed. In some embodiments, ramucirumab is administered at about 8 mg / kg as an intravenous infusion over 1 hour.

[0124] In various embodiments, the following VEGF and / or VEGFR inhibitors can be employed when practicing the methods disclosed herein: Votrient (pazopanib, Novartis), Sutent (sunitinib, Pfizer), Nexavar (sorafenib, Bayer), Stivarga (regorafenib, Bayer), Cabometyx (cabozantinib, Exelixis), Lenvima (lenvatinib, Eisai), and vedotinib. The patients were randomized to receive either of the following: 1) nab (Aspirin, Inc., Eisai), 2) Iclusig (ponatinib, Ariad), 3) Cometriq (cabozantinib, Exelixis), 4) Zaltrap (ziv-aflibercept, Regeneron), 5) Inlyta (axitinib, Pfizer), 6) Fotivda (tivozanib, Aveo), 7) Cyramza (ramucirumab, Eli Lilly and Company), 8) Caprelsa (vandetanib, Genzyme), or 9) Alymsys (bevacizumab, Amneal).

[0125] Chemotherapeutic agents

[0126] Many different classes of chemotherapy, or chemotherapeutic drugs, are used to treat cancer—either alone or in combination with other drugs or treatments. These drugs vary widely in their chemical makeup (ingredients), how they are prescribed and given, their availability for treating certain types of cancer, and their possible side effects.

[0127] Not all drugs and medicines for treating cancer work in the same way. Other drugs for treating cancer, such as targeted therapy, hormone therapy and immunotherapy, work in different ways than traditional or standard chemotherapeutics. The chemotherapeutics used in the methods of treatment described herein are traditional or standard chemotherapeutics, rather than targeted therapy, hormone therapy or immunotherapy (e.g., biological agents).

[0128] Chemotherapy drugs can target cells at different stages of the cell cycle. Chemotherapy drugs can be grouped according to how they work, their chemical structure, and their relationship to other drugs. Some drugs work in more than one way and may belong to more than one group.

[0129] Alkylating agents prevent cell proliferation by damaging the cell's DNA. Examples of alkylating agents for use as at least one chemotherapeutic agent in the methods described herein include: altretamine; bendamustine; busulfan; carboplatin; carmustine; chlorambucil; cisplatin; cyclophosphamide; dacarbazine; ifosfamide; lomustine; mechlorethamine; melphalan; oxaliplatin; temozolomide; thiotepa; and trabectedin.

[0130] Antimetabolites interfere with DNA and RNA by acting as substitutes for their normal building blocks. When this happens, DNA cannot replicate itself and cells cannot divide. Examples of antimetabolites used as at least one chemotherapeutic agent in the methods described herein include: azacitidine; 5-fluorouracil (5-FU); 6-mercaptopurine (6-MP); capecitabine; cladribine; clofarabine; cytarabine (Ara-C); decitabine; floxuridine; fludarabine; gemcitabine; hydroxyurea; methotrexate; nelarabine; pemetrexed; pentostatin; pralatrexate; thioguanine; and trifluridine / tipirimidine combinations.

[0131] Anthracyclines are antitumor antibiotics that interfere with enzymes involved in DNA replication during the cell cycle. They bind to DNA, preventing it from replicating itself and preventing cells from dividing. Examples of anthracyclines that can be used as at least one chemotherapeutic agent in the methods described herein include: daunorubicin; doxorubicin; liposomal doxorubicin; epirubicin; idarubicin; and valrubicin. Antitumor antibiotics that are not anthracyclines include: bleomycin; dactinomycin; mitomycin C; and mitoxantrone.

[0132] Topoisomerase inhibitors are drugs that interfere with enzymes called topoisomerases. Topoisomerase I inhibitors (also known as camptothecins) include: irinotecan; liposomal irinotecan; and topotecan. Topoisomerase II inhibitors (also known as epipodophyllotoxins) include: etoposide (VP-16); mitoxantrone (also used as an antitumor antibiotic); and teniposide.

[0133] Mitotic inhibitors include taxanes and vinca alkaloids. Taxanes include cabazitaxel, docetaxel, nab-paclitaxel, and paclitaxel. Vinca alkaloids include vinblastine, vincristine, liposomal vincristine, and vinorelbine.

[0134] Some chemotherapy drugs do not fit neatly into any of the categories. Examples include: all-trans retinoic acid; arsenic trioxide; asparaginase; eribulin; hydroxyurea; ixabepilone; mitotane; galactopyranoside; pegaspargase; procarbazine; romidepsin; and vorinostat.

[0135] In certain embodiments, the one or more optional chemotherapeutic agents are fluorouracil-based chemotherapy and are administered according to the methods described herein. Fluorouracil-based chemotherapy includes, but is not limited to, 5-fluorouracil (5-FU); combinations of 5-FU such as oxaliplatin plus 5-FU / leucovorin (FOLFOX), FLOT (5-FU / leucovorin, oxaliplatin, and docetaxel), and irinotecan plus 5-FU / leucovorin (FOLFIRI).

[0136] FOLFOX is a three-drug regimen consisting of fluorouracil (5-FU), folinic acid (e.g., leucovorin or leucovorin), and oxaliplatin that is commonly used to treat mCRC (National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology Colon Cancer. 2nd Edition. 2019. NCCN, Fort Washington, PA). Versions of FOLFOX include FOLFOX4, FOLFOX6, modified FOLFOX6 (mFOLFOX6), FOLFOX7, and modified FOLFOX7. Descriptions of FOLFOX versions are readily available in the literature and are well known.

[0137] In one embodiment, the FOLFOX dosing regimen consists of leucovorin or leucovorin, oxaliplatin, and 5-fluorouracil and is commercially available as part of clinical standard of care, and the modified FOLFOX6 (mFOLFOX6) administration regimen can include folinic acid 400 mg / m2 IV on Day 1 of each 14-day cycle, fluorouracil 1200 mg / m2 IV / day (Days 1 and 2), and oxaliplatin 85 mg / m2 on Day 1. Additional information can be found in the current prescribing information.

[0138] In one embodiment, the FOLFIRI dosing regimen consists of leucovorin or leucovorin, irinotecan, and 5-fluorouracil and will be commercially available as part of clinical standard of care. The FOLFIRI regimen's administration regimen may include a 90-min IV infusion of irinotecan (180 mg / m2), followed by a simplified LV5FU2 regimen (leucovorin [400 mg / m2] and a bolus of fluorouracil [400 mg / m2] on day 1 and a 46-h infusion of fluorouracil [2400 mg / m2]). Additional information can be found in the current prescribing information.

[0139] In certain embodiments, one of the plurality of chemotherapeutic agents for use in the method of treating colorectal cancer is mFOLFOX6 or FOLFIRI.

[0140] Targeted therapy (as additional therapeutic agent for co-administration)

[0141] (PI3K inhibitor)

[0142] Phosphatidylinositol 3-kinase inhibitors (PI3K inhibitors) are a class of drugs that work by inhibiting one or more phosphatidylinositol 3-kinases (PI3Ks) that are part of the PI3K / AKT / mTOR pathway. This signaling pathway regulates cell functions, such as growth and survival. It is strictly regulated in healthy cells, but is always active in many cancer cells, allowing cancer cells to survive and reproduce better. PI3K inhibitors block the PI3K / AKT / mTOR pathway and therefore slow cancer growth. They are examples of targeted therapies.

[0143] PI3K inhibitors include, but are not limited to, copanlisib (Aliqopa), duvelisib (Copiktra), idelalisib (Zydelig), and alpelisib (Piqray).

[0144] In some alternative embodiments, the methods described herein further comprise administering a PI3K inhibitor and / or an immunomodulator. Examples of PI3K inhibitors include, but are not limited to, Aliqopa, Copiktra, Zydelig, and Piqray.

[0145] Immunotherapy (as an additional therapeutic agent for co-administration)

[0146] Combination with immune checkpoint inhibitors

[0147] In some alternative embodiments, the methods described herein further include administering an immune checkpoint inhibitor or immunomodulator. Examples of immune checkpoint inhibitors and immunomodulators include, but are not limited to, PD-1 inhibitors, PD-L1 inhibitors, PD-L2 inhibitors, and CTLA-4 inhibitors. In some aspects, the immunomodulator is an antibody, such as a monoclonal antibody.

[0148] In some alternative embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor, which blocks the interaction between PD-1 and PD-L1 by binding to the PD-1 receptor, thereby inhibiting immunosuppression. In some alternative embodiments, the immune checkpoint inhibitor is a PD-1 immune checkpoint inhibitor selected from but not limited to the following: nivolumab ( Bristol-Myers Squibb), pembrolizumab, Merck (Merck), pidilizumab (formerly CT-011, Pfizer), AMP-224 (Amplimmune); sasanlimab (PF-06801591; Pfizer), spartalizumab (PDR001; Novartis), cemiplimab ( REGN2810; Regeneron Pharmaceuticals), retifanlimab (MGA012 MacroGenics and Zynyz; Incyte), tislelizumab (BeiGene), camrelizumab (SHR-1210; Jiangsu Hengrui Medicine Company), dostarlimab (TSR-042; GlaxoSmithKline), budigalimab (ABBV-181; Abbvie), and zimberelimab (AB122; Arcus).

[0149] In some alternative embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor, which blocks the interaction between PD-1 and PD-L1 by binding to the PD-L1 receptor, thereby inhibiting immunosuppression. PD-L1 inhibitors include but are not limited to atezolizumab ( Genentech), durvalumab, AstraZeneca); avelumab ( Merck & Co., Inc.), envafolimab (KN035; Alphamab), BMS-936559 (Bristol-Myers Squibb), lodapolimab (LY3300054; Eli Lilly and Company), cosibelimab (Checkpoint Therapeutics), sugemalimab (Cstone Pharmaceuticals), and adebrelimab.

[0150] In one aspect, the immune checkpoint inhibitor is a CTLA-4 immune checkpoint inhibitor that binds to CTLA-4 and inhibits immunosuppression. CTLA-4 inhibitors include but are not limited to ipilimumab, Bristol-Myers Squibb); tremelimumab (AstraZeneca / MedImmune), zalifrelimab (AGEN I 884; Agenus), and AGEN204 I (Agenus).

[0151] In certain aspects of the exemplary embodiments, the PD-1 / PD-L1 axis checkpoint inhibitor is selected from pembrolizumab, nivolumab, tislelizumab, bregmalimumab, sepalizumab, cemiplizumab, atezolizumab, avelumab, and durvalumab. For example, the PD-1 / PD-L1 axis checkpoint inhibitor is pembrolizumab, nivolumab, tislelizumab, bregmalimumab, or atezolizumab.

[0152] Administration and dosing

[0153] Administration

[0154] The DKK1 antibody and VEGF or VEGFR inhibitor (e.g., bevacizumab) used in the combinations described herein, as well as one or more optional chemotherapeutic agents (i.e., components of the combination therapy), can be formulated alone or in combination for parenteral (e.g., intravenous), oral, transdermal, sublingual, buccal, rectal, intranasal, intrabronchial, or intrapulmonary administration. In a specific embodiment, the DKK1 antibody (e.g., DKN-01) is administered intravenously. In another specific embodiment, the VEGF or VEGFR inhibitor (e.g., bevacizumab) is administered intravenously. In another embodiment, the DKK1 antibody and VEGF or VEGFR inhibitor are formulated in combination for intravenous administration.

[0155] For parenteral administration, one or more of the components of the combination therapy for use in the methods or compositions of the invention (e.g., DKK1 antibody, VEGF or VEGFR inhibitor (e.g., bevacizumab) and one or more optional chemotherapeutic agents) can be formulated for injection or infusion, such as intravenous, intramuscular or subcutaneous injection or infusion, or for administration in the form of a bolus dose and / or infusion (e.g., continuous infusion). Suspensions, solutions or emulsions in oily or aqueous vehicles, optionally containing other formulating agents (such as suspending agents, stabilizers and / or dispersants), can be used.

[0156] For oral administration, one or more of the components of the combination therapy (e.g., DKK1 antibody, VEGF or VEGFR inhibitor (e.g., bevacizumab) and one or more optional chemotherapeutic agents) can be in the form of tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binders (e.g., polyvinyl pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystalline cellulose or calcium phosphate); lubricants (e.g., magnesium stearate, talc or silicon dioxide); disintegrants (e.g., sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). If desired, tablets can be coated using suitable methods. Liquid preparations for oral administration can be in the form of solutions, syrups or suspensions. Liquid preparations can be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fats); emulsifiers (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or ethanol); and preservatives (e.g., methyl or propylparaben or sorbic acid).

[0157] For oral administration, one or more of the components of the combination therapy for use in the methods or compositions of the invention (e.g., a DKK1 antibody, a VEGF or VEGFR inhibitor (e.g., bevacizumab), and one or more optional chemotherapeutic agents) can be in the form of tablets or lozenges formulated in conventional manner.

[0158] For rectal administration, one or more of the components of the combination therapy for use in the methods or compositions of the invention (e.g., a DKK1 antibody, a VEGF or VEGFR inhibitor (e.g., bevacizumab), and one or more optional chemotherapeutic agents) can be in the form of a suppository.

[0159] For sublingual administration, tablets may be formulated in conventional manner.

[0160] For intranasal, intrabronchial or intrapulmonary administration, conventional formulations may be employed.

[0161] Furthermore, one or more of the components of the combination therapy used in the methods or compositions of the present invention (e.g., a DKK1 antibody, a VEGF or VEGFR inhibitor (e.g., bevacizumab), and one or more optional chemotherapeutic agents) can be formulated in a sustained-release formulation. For example, one or more of these components can be formulated with a suitable polymer or hydrophobic material that provides sustained-release and / or controlled-release properties to the active compound. Thus, one or more components of the combination therapy used in the methods of the present invention can be administered in the form of microparticles, for example, by injection, or in the form of a wafer or disc by implantation. Various methods for formulating controlled-release pharmaceutical formulations are known in the art.

[0162] Administration of one or more components of the combination therapies of the invention, or pharmaceutically acceptable salts thereof, or compositions comprising one or more components of the combination therapies (or pharmaceutically acceptable salts thereof), useful in practicing the methods described herein can be continuous, hourly, four times a day, three times a day, twice a day, daily, every other day, twice a week, weekly, every two weeks, monthly, or every two months, or longer, or some other intermittent dosing schedule.

[0163] Combination therapy

[0164] As used herein, "co-administration," "co-administering," "in combination with," or "in conjunction with" refers to the administration of a therapeutic modality in addition to at least one other therapeutic modality. Thus, "in combination with" or "in conjunction with" refers to the administration of a therapeutic modality before, during, or after the administration of at least one other therapeutic modality to a subject.

[0165] The DKK1 antibodies disclosed herein can be used in combination with VEGF or VEGFR inhibitors (e.g., bevacizumab) and one or more optional chemotherapeutic agents for the treatment of colorectal cancer. Such combined administration can be by a single dosage form comprising a DKK1 antibody, a VEGF or VEGFR inhibitor (e.g., bevacizumab), and one or more optional chemotherapeutic agents, such a single dosage form including tablets, capsules, sprays, inhalation powders, injectable liquids, etc. Alternatively, combined administration (e.g., co-administration) can be by administering different dosage forms, wherein one dosage form contains a DKK1 antibody, another dosage form includes a VEGF or VEGFR inhibitor (e.g., bevacizumab), and yet another dosage form includes one or more optional chemotherapeutic agents (if more than one chemotherapeutic agent is used, it should be understood that these agents can be administered using the same or different dosage forms). For example, a DKK1 antibody (e.g., DKN-01) and a VEGF or VEGFR inhibitor (e.g., bevacizumab) can be administered together in a single dosage form (e.g., a single dosage form for intravenous administration), and one or more optional chemotherapeutic agents can be administered in separate single doses by any other suitable means.

[0166] The components of the combination therapy can be administered in any order. For example, a VEGF or VEGFR inhibitor (e.g., bevacizumab) can be administered before, simultaneously with, or after the administration of the DKK1 antibody and before, simultaneously with, or after the administration of one or more optional chemotherapeutic agents. Thus, the components of the combination therapy can be administered together in a single formulation, or can be administered in separate formulations, such as simultaneously or sequentially, or both. For example, if the DKK1 antibody and VEGF or VEGFR inhibitor (e.g., bevacizumab) are administered sequentially in separate compositions, the DKK1 antibody can be administered before or after the VEGF or VEGFR inhibitor (e.g., bevacizumab). The duration between administration of the components of the combination therapy will be readily determined by the administering physician.

[0167] Furthermore, the components of the combination therapy may or may not be administered with a similar dosing schedule. For example, a DKK1 antibody and a VEGF or VEGFR inhibitor (e.g., bevacizumab) may have different half-lives and / or function on different timescales, such that the DKK1 antibody is administered at a greater frequency than a VEGF or VEGFR inhibitor (e.g., bevacizumab), or vice versa. For example, a DKK1 antibody and a VEGF or VEGFR inhibitor (e.g., bevacizumab) may be administered together in one day (e.g., in a single dose or sequentially), followed by administration of only one or more optional chemotherapeutic agents after a certain number of days. The number of days between administrations of the components of the combination therapy may be appropriately determined based on the safety and pharmacodynamics of each drug.

[0168] In a specific embodiment, the treatment period of combination therapy is 21 day cycles, and the cycle can be repeated until it is determined that the patient will not obtain any clinical benefit from combination therapy. In another specific embodiment, the treatment period of combination therapy is 14 day cycles, and the cycle can be repeated until it is determined that the patient no longer needs treatment or will not obtain any clinical benefit from combination therapy. For example, the patient can experience the treatment (for example, 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,7,18,19,20,21,22,23,24,25,26,27,28,29,30) of about one cycle to about 30 cycles (for example, 14 day cycles).

[0169] As used herein, an "effective amount" refers to an amount of a combination of therapeutic agents sufficient to treat the target disorder therapeutically or prophylactically. The effective amount will depend on the patient's age, sex, and weight, the patient's current medical condition, and the nature of the colorectal cancer being treated. One skilled in the art will be able to determine an appropriate dosage based on these and other factors.

[0170] For DKK1 antibodies, suitable dosages for each administration include dosages of about or greater than about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 625 mg, about 650 mg, about 675 mg, about 700 mg, about 725 mg, about 750 mg, about 775 mg, about 800 mg, about 825 mg, about 850 mg, about 875 mg, about 900 mg. g, about 925 mg, about 950 mg, about 975 mg, about 1000 mg, about 1025 mg, about 1050 mg, about 1075 mg, about 1100 mg, about 1125 mg, about 1150 mg, about 1175 mg, about 1200 mg, about 1225 mg, about 1250 mg, about 1275 mg, about 1300 mg, about 1325 mg, about 1350 mg, about 1375 mg, about 1400 mg, about 1425 mg, about 1450 mg, about 1475 mg, about 1500mg, about 1525mg, about 1550mg, about 1575mg, about 1600mg, about 1625mg, about 1650mg, about 1675mg, about 1700mg, about 1725mg, about 1750mg, about 1775mg, about 1800mg, about 1825mg, about 1850mg, about 1875mg, about 1900mg, about 1925mg, about 1950mg, about 1975mg, about 2000mg, about 2025mg, about 2050mg, about In some embodiments, the dosage of the present invention can be 200mg, 2075mg, 2100mg, 2125mg, 2150mg, 2175mg, 2200mg, 2225mg, 2250mg, 2275mg, 2300mg, 2325mg, 2350mg, 2375mg, 2400mg, 2425mg, 2450mg, 2475mg, 2500mg, 2525mg, 2550mg, 2575mg, 2600mg or 3,000mg. Often applicable dosage can be used in the technical staff thinking that suitable time period. For example, often applicable dosage can be used in the time period of about 30 minutes to about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours or about 8 hours.In certain embodiments, a suitable dose of a DKK1 antibody (e.g., DKN-01) can be from about 20 mg to about 1200 mg, such as from about 30 mg to about 600 mg, such as from about 50 mg to about 500 mg, such as from about 50 mg to about 300 mg (such as 50 mg, 75 mg, 100 mg, 125 mg, 150 mg, 175 mg, 200 mg, 225 mg, 250 mg, 275 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, or 1200 mg). The selected dose can be administered intravenously over a period of about 30 minutes to about 2 hours. In certain embodiments, a suitable dose of a DKK1 antibody can be about 300 mg administered over a period of about 30 minutes to about 2 hours. Another suitable dose of a DKK1 antibody may be about 600 mg administered over a period of about 30 minutes to about 2 hours. Another suitable dose of a DKK1 antibody (e.g., DKN-01) may be about 400 mg administered IV over a period of about 30 minutes to about 2 hours. Administration of these doses within the recited time periods may be accomplished using an intravenous route. For example, a DKK1 antibody (e.g., DKN-01) may be administered on Day 1 of a 21-day cycle, followed by repeated administration on Day 1 of each additional cycle. For such a 21-day cycle, administration may be the amounts described above, and in particular, a 600 mg dose may be used. In another example, a DKK1 antibody (e.g., DKNK-01) may be administered on Day 1 of a 14-day cycle, followed by repeated administration on Day 1 of each additional cycle. In a specific embodiment, during the first of multiple 14-day cycles, DKN-01 may be administered at about 400 mg on Day 1 of the cycle and at about 400 mg on Day 8 of the cycle, and then administered once in each subsequent 14-day cycle. In another specific example, during the first of a plurality of 21-day cycles, DKN-01 can be administered at about 600 mg on days 1 and 15 of a 21-day cycle, and then once in each 21-day cycle thereafter.

[0171] For VEGF or VEGFR inhibitors (for example, bevacizumab), the suitable dosage of each use can be determined based on the recommended dose known to standard treatment. For example, the suitable dosage of each use of bevacizumab is intravenous from about 5mg / kg to about 100mg / kg. Use can deliver desired dosage in a safe and effective manner within the necessary time period. In some cases, this time is at least 30 minutes time period, 60 minutes time period, 90 minutes time period, 2 hours time period, 3 hours time period etc. Each cycle can repeat this use (for example, once every 14 days of 14 day cycles). In a specific embodiment, the suitable dosage of each use is to use the about 5mg / kg of intravenous approach.

[0172] An effective amount can be achieved in the methods of the present invention by co-administering an initial amount of a DKK1 antibody (or a pharmaceutically acceptable salt, hydrate, or solvate thereof), an initial amount of a VEGF or VEGFR inhibitor (e.g., bevacizumab), and an initial amount of one or more optional chemotherapeutic agents. It should be understood that if more than one chemotherapeutic agent is included, the amount of the chemotherapeutic agent administered may vary. In one embodiment, the components of the combination (e.g., the DKK1 antibody, the VEGF or VEGFR inhibitor (e.g., bevacizumab), and the one or more optional chemotherapeutic agents) are each administered in a corresponding effective amount (e.g., an amount that would be therapeutically effective if administered alone). In another embodiment, the components of the combination (e.g., the DKK1 antibody, the VEGF or VEGFR inhibitor (e.g., bevacizumab), and the one or more optional chemotherapeutic agents) are each administered in an amount that would not provide a therapeutic effect alone (a subtherapeutic dose). In yet another embodiment, one or two components of the combination may be administered in an effective amount, while the remaining components are administered in a subtherapeutic dose. For example, the DKK1 antibody can be administered at a subtherapeutic dose, the VEGF or VEGFR inhibitor (e.g., bevacizumab) can be administered in an effective amount, and the one or more therapeutic agents can each be administered in an effective amount thereof. In yet another embodiment, the DKK1 antibody is DKN-01 and is administered at 400 mg, the VEGF or VEGFR inhibitor (e.g., bevacizumab) is administered at 5 mg / kg, and the one or more optional chemotherapeutic agents are fluorouracil-based chemotherapeutic agents selected from 5-FU (fluorouracil), FOLFIRI, and FOLFOX (e.g., modified FOLFOX6) and are administered as described herein.

[0173] In specific embodiments, subjects will receive DKN-01 (IV administration) on Day 1 of each 14-day cycle at a dose of 400 mg, with an additional 400 mg loading dose administered only on D8 of C1, in combination with either of the following two regimens on Day 1 of each 14-day cycle:

[0174] FOLFIRI plus bevacizumab: 90-minute IV infusion of bevacizumab (5 mg / kg) and 90-minute IV infusion of irinotecan (180 mg / m2), followed by a simplified LV5FU2 regimen (leucovorin [400 mg / m2] and bolus fluorouracil [400 mg / m2] on day 1 and a 46-hour infusion of fluorouracil [2400 mg / m2]).

[0175] Modified FOLFOX6 (mFOLFOX6) plus bevacizumab: 90-min IV infusion of bevacizumab (5 mg / kg) followed by mFOLFOX6 (oxaliplatin 85 mg / m2, leucovorin 400 mg / m2, and fluorouracil 400 mg / m2 IV bolus on day 1, followed by 2,400 mg / m2 as a continuous infusion over 46 hours).

[0176] Additional therapeutic agents, such as those used in targeted therapy or immunotherapy, can be administered to the subject. Such agents include, but are not limited to, PI3K inhibitors and immune checkpoint inhibitors. Additional agents can be administered according to known methods.

[0177] As used herein, the term "subject" refers to a mammal, preferably a human, but may also refer to animals in need of veterinary treatment, such as companion animals (e.g., dogs, cats, etc.), farm animals (e.g., cows, sheep, pigs, horses, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.).

[0178] As used herein, "treatment" includes achieving, partially or substantially delaying, suppressing or preventing the progress of clinical indications associated with colorectal cancer. For example, "treatment" includes detecting and reducing tumor growth or preventing further growth as determined by standard imaging methods known in the art (including, for example, computed tomography (CT) scans, magnetic resonance imaging (MRI), chest X-rays, and CT / positron emission tomography (CT / PET) scans) and evaluating according to guidelines and methods known in the art. For example, the response to treatment can be evaluated by the solid tumor efficacy evaluation criteria (RECIST) (revised RECIST guidelines version 1.1; see Eisenhauer et al., Eur..J.Cancer [European Journal of Cancer] 45 (2): 228-47, 2009). Therefore, in some embodiments, "treatment" refers to complete remission (CR), which is defined as the disappearance of all target lesions according to the RECIST guidelines; or partial remission (PR), which is defined as a reduction of at least 30% in the sum of target lesion diameters with reference to the baseline total diameter. Other means for evaluating tumor response to treatment include evaluating tumor markers and evaluating performance status (e.g., evaluating creatinine clearance; see Cockcroft and Gault, Nephron. 16:31-41, 1976, evaluating carcinoembryonic antigen (CEA) levels in the blood and evaluating cancer antigen 19-9 (CA 19-9) levels in the blood).

[0179] "Objective response" refers to a measurable response, including complete response (CR) or partial response (PR). In some embodiments, the term "objective response rate" (ORR) refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0180] As used herein, "complete remission" or "CR" refers to the disappearance of all signs of cancer (eg, disappearance of all target lesions) in response to treatment. This does not always mean that the cancer has been cured.

[0181] As used herein, "partial response" or "PR" refers to a decrease in the size of one or more tumors or lesions, or the extent of cancer in the body, in response to treatment.

[0182] For example, in some embodiments, PR refers to a decrease of at least 30% in the sum of the longest diameters (SLD) of target lesions, relative to the baseline SLD.

[0183] As used herein, "progressive disease" or "PD" refers to an increase in the SLD of a target lesion by at least 20% relative to the smallest SLD recorded since the start of treatment or the appearance of one or more new lesions.

[0184] Pharmaceutical composition

[0185] The components of the combination therapy (e.g., a DKK1 antibody, a VEGF or VEGFR inhibitor (e.g., bevacizumab), and one or more optional chemotherapeutic agents) can be incorporated into a pharmaceutical composition suitable for administration. Such compositions typically contain a DKK1 antibody, or a VEGF or VEGFR inhibitor (e.g., bevacizumab), alone or together in any combination, and one or more optional chemotherapeutic agents, and a pharmaceutically acceptable carrier. As used herein, the expression "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Unless any conventional media or agents are incompatible with the active compound, their use in the composition is contemplated.

[0186] The pharmaceutical composition of the present invention is formulated into a method compatible with the intended route of administration. Examples of routes of administration include parenteral, such as intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous applications may include the following components: a sterile diluent, such as water for injection, saline solution, fixed oil, polyethylene glycol, glycerol, propylene glycol, or other synthetic solvents; an antibacterial agent, such as benzyl alcohol or methyl paraben; an antioxidant, such as ascorbic acid or sodium bisulfite; a chelating agent, such as ethylenediaminetetraacetic acid; a buffer, such as acetate, citrate, or phosphate, and an agent for regulating tonicity, such as sodium chloride or dextrose. pH can be adjusted with an acid or base (such as hydrochloric acid or sodium hydroxide). The parenteral preparation may be packaged in an ampoule, a disposable syringe, or a multidose bottle made of glass or plastic.

[0187] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water-soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (TM) (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should have a fluidity that allows for easy injection. It must be stable under manufacturing and storage conditions and must prevent the contamination of microorganisms (such as bacteria and fungi). The carrier can be a solvent or dispersion medium containing the following substances: for example, water, ethanol, polyols (for example, glycerol, propylene glycol and liquid polyethylene glycol, etc.), and mixtures thereof that are suitable. For example, by using a coating (such as lecithin), by maintaining the size of the desired particles in the dispersion, and by using a surfactant, suitable fluidity can be maintained. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, etc.). In many cases, it will be preferred to include isotonic agents such as sugars, polyols (e.g., mannitol, sorbitol), sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including agents that delay absorption (e.g., aluminum monostearate and gelatin) in the composition.

[0188] Sterile injectable solutions can be prepared by incorporating the combination (e.g., DKK1 antibody, VEGF or VEGFR inhibitor (e.g., bevacizumab) and one or more optional chemotherapeutic agents) in the desired amount in an appropriate solvent with one or a combination of the ingredients listed above (as needed), followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the desired other ingredients from those listed above. In the case of sterile powders for the preparation of sterile injectable solutions, preferred methods of preparation are vacuum drying and freeze drying, which yield a powder of the active ingredient and any additional desired ingredients from a previously sterile-filtered solution thereof.

[0189] Oral compositions typically include an inert diluent or an edible carrier. They can be encapsulated in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared using a fluid carrier used as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binders and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. can contain any of the following ingredients or compounds with similar properties: binders, such as microcrystalline cellulose, tragacanth gum, or gelatin; excipients, such as starch or lactose; disintegrants, such as alginic acid, Primogel, or corn starch; lubricants, such as magnesium stearate or Sterotes; glidants, such as colloidal silicon dioxide; sweeteners, such as sucrose or saccharin; or flavorings, such as mint, methyl salicylate, or orange flavoring.

[0190] For administration by inhalation, the compounds are delivered in the form of an aerosol spray from a pressured container or dispenser that contains a suitable propellant, eg, a gas such as carbon dioxide, or a nebulizer.

[0191] Systemic administration can also be performed via transmucosal or transdermal routes. For transmucosal or transdermal administration, the formulation can be modified with penetrants appropriate to the barrier to be penetrated. Such penetrants are generally well known in the art and, for transmucosal administration, include, for example, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished using nasal sprays or suppositories.

[0192] For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0193] The compounds may also be prepared for rectal delivery in the form of suppositories (eg, with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas.

[0194] In one embodiment, these active compounds are prepared together with a carrier that protects the compound from rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. The method for preparing such formulations will be apparent to those skilled in the art. These materials can also be commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to antiviral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those of ordinary skill in the art, for example, as described in U.S. Patent number 4,522,811.

[0195] It is particularly advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. As used herein, dosage unit form refers to physically discrete units suitable as unitary dosages for the subject to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specifications for the dosage unit forms of the present invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved, as well as the inherent limitations of the art in compounding such active compounds for treatment of individuals.

[0196] As used herein, the term "kRas gene" refers to the human gene with the following NCBI Gene ID: 3845.

[0197] As used herein, the term "nRas gene" refers to the human gene with the following NCBI ID: 4893.

[0198] Thus, in a first embodiment, the method comprises co-administering to a subject having colorectal cancer an effective amount of a DKK1 antibody or antigen-binding fragment thereof, a VEGF or VEGFR inhibitor; and optionally one or more chemotherapeutic agents or pharmaceutically acceptable salts of any of the foregoing.

[0199] In a first aspect of the first embodiment, the VEGF inhibitor is bevacizumab.

[0200] In the second aspect of the first embodiment or any specific aspects thereof, the DKK1 antibody is DKN-01.

[0201] In the third aspect of the first embodiment, any specific aspect thereof, or the first and second aspects thereof, the one or more chemotherapeutic agents are fluorouracil-based chemotherapeutic agents selected from the group consisting of 5-FU (fluorouracil), FOLFIRI, and FOLFOX. In specific aspects, the FOLFOX is a modified FOLFOX6.

[0202] In the fourth aspect of the first embodiment, any specific aspect thereof, or the first, second, or third aspect thereof, the method further comprises administering one or more additional therapeutic agents selected from PI3K inhibitors and / or immune checkpoint inhibitors. In specific aspects, the PI3K inhibitor is selected from cupranixib, duvilisib, idelanib, and apelisib. In specific aspects, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In specific aspects, the PD-1 inhibitor is selected from nivolumab, pembrolizumab, pidilizumab, AMP-224, sazanlizumab, spartalizumab, cemiplizumab, rivulizumab, tislelizumab, carrelizumab, bruglimab, sepalizumab, and dotalizumab. In more specific aspects, the PD-1 inhibitor is pembrolizumab, nivolumab, tislelizumab, or bruglimab. In a specific aspect, the PD-L1 inhibitor is selected from atezolizumab, durvalumab, avelumab, envolimab, BMS-936559, lodalizumab, coxilimab, sugemalimab, and adebelimumab. In a more specific aspect, the PD-L1 inhibitor is atezolizumab.

[0203] In the fifth aspect of the first embodiment, any specific aspect thereof, or the first, second, third, or fourth aspect thereof, the subject's colorectal cancer has a detectable expression level of DKK-1.

[0204] In the sixth aspect of the first embodiment, any specific aspect thereof, or the first, second, third, fourth, or fifth aspect thereof, the subject's plasma has a detectable level of DKK1.

[0205] In the seventh aspect of the first embodiment, any specific aspect thereof, or the first, second, third, fourth, fifth, or sixth aspect thereof, the subject's serum has a detectable level of DKK1.

[0206] In the eighth aspect of the first embodiment, any specific aspect thereof, or the first, second, third, fourth, fifth, sixth, or seventh aspect thereof, the subject has received a prior 5-FU-based therapy for colorectal cancer.

[0207] In a ninth aspect of the first embodiment, any specific aspect thereof, or first, second, third, fourth, fifth, sixth, seventh, or eighth aspect thereof, the colorectal cancer is microsatellite stable (MSS).

[0208] In the tenth aspect of the first embodiment, any specific aspect thereof, or the first, second, third, fourth, fifth, sixth, seventh, eighth, or ninth aspect thereof, the subject's colorectal cancer does not have a BRAF V600E mutation.

[0209] In the eleventh aspect of the first or second embodiment, any specific aspect thereof, or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth or tenth aspect thereof, the colorectal cancer is advanced.

[0210] In a twelfth aspect of the first embodiment, any specific aspect thereof, or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or eleventh aspect thereof, the colorectal cancer is metastatic.

[0211] In a thirteenth aspect of the first embodiment, any specific aspect thereof, or first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, or twelfth aspect thereof, the colorectal cancer is an adenocarcinoma.

[0212] In the fourteenth aspect of the first embodiment, any specific aspect thereof, or first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth aspect thereof, treatment is administered during one or more 14-day cycles.

[0213] In the fifteenth aspect of the first embodiment, any specific aspect thereof, or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, or fourteenth aspect thereof, the DKK1 antibody or antigen-binding fragment thereof is administered in an amount of 400 mg on day 1 of a 14-day cycle.

[0214] In a sixteenth aspect of the first embodiment, any specific aspect thereof, or first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth or fifteenth aspect thereof, the VEGF inhibitor is bevacizumab and is administered at 5 mg / kg on day 1 of a 14-day cycle.

[0215] In the seventeenth aspect of the first embodiment, any specific aspect thereof, or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, or sixteenth aspect thereof, the DKKl antibody or antigen-binding fragment thereof comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3, wherein LCDR1 has the amino acid sequence of SEQ ID NO: 1, LCDR2 has the amino acid sequence of SEQ ID NO: 2, LCDR3 has the amino acid sequence of SEQ ID NO: 3, HCDR1 has the amino acid sequence of SEQ ID NO: 4, HCDR2 has the amino acid sequence of SEQ ID NO: 5, and HCDR3 has the amino acid sequence of SEQ ID NO: 6. In a specific aspect of the seventeenth aspect, the LCVR comprises the amino acid sequence of SEQ ID NO: 7, and the HCVR comprises the amino acid sequence of SEQ ID NO: 8. In another specific aspect, the LCVR and HCVR comprise an amino acid sequence selected from the group consisting of: (i) a LCVR comprising the amino acid sequence of SEQ ID NO: 9 and a HCVR comprising the amino acid sequence of SEQ ID NO: 10; (ii) a LCVR comprising the amino acid sequence of SEQ ID NO: 11 and a HCVR comprising the amino acid sequence of SEQ ID NO: 12; (iii) a LCVR comprising the amino acid sequence of SEQ ID NO: 13 and a HCVR comprising the amino acid sequence of SEQ ID NO: 10; and (iv) a LCVR comprising the amino acid sequence of SEQ ID NO: 14 and a HCVR comprising the amino acid sequence of SEQ ID NO: 10. In yet another specific aspect of the seventeenth aspect, the LCVR comprises the amino acid sequence of SEQ ID NO: 11, and the HCVR comprises the amino acid sequence of SEQ ID NO: 12.

[0216] In the eighteenth aspect of the first or second embodiment, any specific aspect thereof, or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth or seventeenth aspect thereof, the DKK1 antibody comprises heavy and light chain amino acid sequences selected from the group consisting of a) a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and a light chain comprising the amino acid sequence of SEQ ID NO: 16, b) a heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and a light chain comprising the amino acid sequence of SEQ ID NO: 18, c) a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and a light chain comprising the amino acid sequence of SEQ ID NO: 20, and d) a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and a light chain comprising the amino acid sequence of SEQ ID NO: 21.

[0217] In the nineteenth aspect of the first embodiment, any specific aspect thereof, or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, or seventeenth aspect thereof, the DKK1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain comprising the amino acid sequence of SEQ ID NO: 18.

[0218] In the twentieth aspect of the first embodiment, any specific aspect thereof, or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, sixteenth, seventeenth, eighteenth, or nineteenth aspect thereof, the DKK1 antibody is DKN-01.

[0219] In the 21st aspect of the 1st embodiment or any aspect thereof, the subject is a human.

[0220] In the 22nd aspect of the 1st embodiment or any aspect thereof, the subject is a rapid progressor.

[0221] In the 23rd aspect of the first embodiment or any aspect thereof, the subject carries a mutation in the kRas gene or the nRas gene.

[0222] In the 24th aspect of the first embodiment or any aspect thereof, the subject has liver metastasis.

[0223] In the 25th aspect of the 1st embodiment or any aspect thereof, the colorectal cancer is rectal cancer.

[0224] In a second embodiment, the present invention relates to a pharmaceutical composition comprising: a DKK1 antibody or an antigen-binding fragment thereof; bevacizumab; and optionally one or more chemotherapeutic agents or a pharmaceutically acceptable salt of any of the foregoing.

[0225] In various aspects, the second embodiment has been described above with respect to the first to twenty-fifth aspects of the first embodiment.

[0226] In a third embodiment, the present invention is a pharmaceutical composition comprising: a DKK1 antibody or an antigen-binding fragment thereof; a VEGF or VEGFR inhibitor; and one or more chemotherapeutic agents or a pharmaceutically acceptable salt of any of the foregoing.

[0227] In various aspects, the third embodiment has been described above with respect to the first to twenty-fifth aspects of the first embodiment.

[0228] In a fourth embodiment, the present invention is a kit comprising a DKK1 antibody or an antigen-binding fragment thereof; a VEGF or VEGFR inhibitor; one or more chemotherapeutic agents or pharmaceutically acceptable salts thereof; and instructions for use.

[0229] In various aspects, the third embodiment has been described above with respect to the first to twenty-fifth aspects of the first embodiment.

[0230] In a fifth embodiment, the present invention is a kit comprising a DKK1 antibody or an antigen-binding fragment thereof; bevacizumab; one or more chemotherapeutic agents or pharmaceutically acceptable salts thereof; and instructions for use.

[0231] In various aspects, the fifth embodiment is described above with respect to the first to twenty-fifth aspects of the first embodiment.

[0232] Examples

[0233] Example I: Clinical Protocol

[0234] This study describes a phase 2, randomized, open-label, two-part, multicenter study with a safety run-in period to evaluate the efficacy and safety of DKN-01 plus [FOLFIRI or FOLFOX and bevacizumab] versus standard of care (SOC) [FOLFIRI or FOLFOX and bevacizumab] as second-line treatment for patients with advanced CRC. (See NCT05480306 at clinicaltrials.gov)

[0235] In Parts A and B, approximately 150 evaluable adult patients with advanced CRC with measurable disease (RECIST v1.1) who have radiographic progression during or after 1 line of systemic therapy will be enrolled.

[0236] The study consists of a screening period, a treatment period, a safety follow-up period (SFUP), and a long-term follow-up period (LTFU). After the last dose of study drug, patients will be followed for approximately 30 days (+7 days) in the SFUP and then enter the LTFU period to track survival and subsequent therapy. In addition, patients who discontinue study treatment for reasons unrelated to progressive disease (PD) will also be followed for disease progression during the LTFU period.

[0237] Part A: Safety Introduction Period

[0238] Enroll enough patients in Part A to ensure at least 20 patients are evaluable for Safety Review Committee (SRC) review. Evaluable patients are defined as those who complete all treatment doses (a total of 3 DKN-01 doses) in C1 and C2 and all safety assessments by the end of C2; dose delays are allowed. Unevaluable patients may be replaced.

[0239] Subjects determined to be eligible will be enrolled (ie, included) in Part A. Enrollment may occur up to 3 days prior to C1D1.

[0240] Part A patients will receive DKN-01 (also known as LY2812176) administered intravenously (IV) (30-min infusion) at a dose of 400 mg on Day (D)1 of each 14-day cycle, with an additional 400 mg loading dose administered only on Day (C)1, in combination with any of the following regimens also on Day (D)1, as recommended below or according to institutional standard practice, at the investigator's option:

[0241] FOLFIRI plus bevacizumab: 90-minute IV infusion of bevacizumab (5 mg / kg), followed by a 90-minute IV infusion of irinotecan (180 mg / m²), followed by a simplified LV5FU2 regimen (leucovorin [400 mg / m²] and bolus fluorouracil [400 mg / m²] on Day 1 and a 46-hour infusion of fluorouracil [2400 mg / m²]).

[0242] Modified FOLFOX6 (mFOLFOX6) plus bevacizumab: 90-minute IV infusion of bevacizumab (5 mg / kg) followed by mFOLFOX6 (D1: oxaliplatin 85 mg / m2, leucovorin 400 mg / m2, and fluorouracil 400 mg / m2 IV bolus, followed by a continuous infusion of fluorouracil 2,400 mg / m2 over 46 hours).

[0243] Treatment continued in repeated 14-day cycles until the patient met discontinuation criteria or no longer experienced clinical benefit.

[0244] After monitoring all evaluable patients for two cycles (minimum 28 days), the Safety Review Committee (SRC) will review the overall safety profile of patients in Part A and determine whether the 400 mg dose of DKN-01 plus FOLFIRI / FOLFOX plus bevacizumab is safe and tolerable to initiate Part B.

[0245] Section B

[0246] Approximately 130 patients will be randomly assigned to the experimental or control group in a 1:1 ratio. Randomization can be performed up to 3 days before C1D1.

[0247] Patients in the experimental arm received IV-administered DKN-01 at a dose of 400 mg on Day 1 of each 14-day cycle, with an additional 400 mg loading dose administered only on Day 8 of C1, in combination with either of the following two regimens on Day 1 of each cycle, as recommended below or according to institutional standard practice, at the investigator's option:

[0248] -FOLFIRI plus bevacizumab: 90-minute IV infusion of bevacizumab (5 mg / kg), followed by 90-minute IV infusion of irinotecan (180 mg / m²), followed by a simplified LV5FU2 regimen (leucovorin [400 mg / m²] and bolus fluorouracil [400 mg / m²] on day 1 and a 46-hour infusion of fluorouracil [2400 mg / m²]); or

[0249] - Modified FOLFOX6 (mFOLFOX6) plus bevacizumab: A 90-minute IV infusion of bevacizumab (5 mg / kg) followed by a 2-hour infusion of mFOLFOX6 (day 1: oxaliplatin 85 mg / m2, leucovorin 400 mg / m2, and fluorouracil 400 mg / m2 IV bolus, followed by a continuous infusion of 2,400 mg / m2 over 46 hours).

[0250] Patients in the control group received standard of care (SOC) alone, ie, FOLFIRI or mFOLFOX6 (investigator's choice) plus bevacizumab, where this SOC regimen was administered as described above for the experimental group.

[0251] Treatment continued in repeated 14-day cycles until the patient met discontinuation criteria or no longer experienced clinical benefit.

[0252] The research design is shown in the figure below. Figure 1 Presented in

[0253] Endpoint definition

[0254] Time to event and duration endpoint

[0255] Progression-free survival (PFS) is the primary outcome measure and is defined for each patient as the interval from the date of randomization (or enrollment date for patients in Part A) to radiographically assessed tumor progression or death from any cause, whichever occurs first. Patients who discontinue the study for reasons other than disease progression will be censored at the time of their last radiographic scan. Patients who start alternative anticancer therapy (other than palliative radiotherapy for pre-existing non-target lesions without PD) will be censored at this time. Patients lost to follow-up will be censored at the time of their last known contact. PFS for DKN-01 plus SOC relative to SOC alone will be determined by the investigator according to RECIST v1.1.

[0256] Overall survival (OS) for each patient was defined as the time from the date of randomization (or enrollment date for patients in Part A) to death due to any cause. If a patient was alive at the time of data analysis or was lost to follow-up, OS data were censored at the last date the patient was known to be alive or the data cutoff date, whichever was earlier.

[0257] Duration of response (DoR) was defined only for responders (patients with BOR of CR or PR) as the time from initial response (CR or PR) to radiographically documented progressive disease or death from any cause, whichever came first. Patients who did not experience PD or death at the time of analysis were censored using the same rules as described for PFS.

[0258] Duration of complete remission (DoCR) was defined as the time from initial CR to radiographically documented progressive disease or death from any cause, whichever occurred first. Patients who did not experience PD or death at the time of analysis were censored using the same rules as described for PFS.

[0259] Duration of clinical benefit (DoCB) is defined as the time from the date of randomization (or enrollment date for patients in Part A) to the time of progressive disease or death from any cause, whichever occurs first. Patients who have not experienced PD or death at the time of analysis will be censored using the same rules as described for PFS.

[0260] For patients with a BOR of CR or PR, the time to best response (TTRBest) was defined as the time from the date of randomization (or enrollment date for patients in Part A) to the assessment date of the first BOR of CR or PR.

[0261] The time to first response (TTRFirst) was defined as the time from the date of randomization (or enrollment date for patients in Part A) to the date of the first assessment of an overall response of CR or PR.

[0262] Time to response (TTR) was defined as the time from the date of randomization (or enrollment date for patients in Part A) to the date of the first assessment of overall response, either complete response (CR) or partial response (PR).

[0263] Best overall response (BOR) was defined as the patient's best response documented from the start of study drug treatment to the end of treatment (taking into account any confirmation requirements).

[0264] Response rate endpoint

[0265] The objective response rate (ORR) was defined as the proportion of patients who achieved a best overall response (BOR) of complete response (CR) or partial response (PR), as assessed by the investigator according to RECIST v1.1. Responses evaluated after switching to another anticancer therapy were excluded.

[0266] The durable clinical benefit (DCB) rate was defined as the proportion of patients with a duration of clinical benefit (DoCB) ≥ 180 days from randomization (or enrollment date for patients in Part A). Patients with a best overall response (BOR) to PD or patients with clinical benefit but DoCB lasting < 180 days were considered non-DCBs.

[0267] The disease control rate (DCR) was defined as the proportion of patients who achieved a best overall response (BOR) of complete response, partial response, or stable disease of at least 8 weeks duration from randomization (or enrollment date for patients in Part A), as assessed by the investigator using RECIST v1.1.

[0268] Analysis of the Primary Efficacy End Point

[0269] The primary efficacy analysis will be a comparison of investigator-assessed PFS between the two treatment arms in the ITT population using a one-sided stratified log-rank test. In other words, PFS of DKN-01 plus SOC versus SOC as determined by the investigator according to RECIST v1.1.

[0270] Data will be summarized in Kaplan-Meier curves along with medians and 95% confidence intervals for these medians. Hazard ratios and their 95% confidence intervals will be obtained using Cox proportional hazards models stratified for the randomization factors.

[0271] Analysis of Secondary Efficacy End Points

[0272] The ORR will be compared between the two treatment arms in Part B in the ITT population based on investigator assessment using the same Cochran-Mantel-Haenzel test as for the PFS analysis. Results will be reported as odds ratios and associated 95% confidence intervals. In other words, the ORR for DKN-01 plus SOC versus SOC will be as determined by the investigator according to RECIST v1.1.

[0273] Using the same analysis method as for PFS, OS will be compared between the two treatment arms in Part B in the ITT population. In other words, OS for DKN-01 plus SOC versus SOC.

[0274] There will be no formal statistical testing for DoR as this is not a randomized comparison, and these data will be evaluated by presentation of descriptive statistics including Kaplan-Meier curves. In other words, the DoR of DKN-01 plus SOC relative to SOC as determined by the investigator according to RECIST v1.1.

[0275] Tumor response endpoints, including ORR, DOR, PFS, and time to event, will be analyzed based on investigator review according to RECIST 1.1. Independent data review will provide RECIST measurements for each patient at each visit. Centrally reviewed data, if completed, will be considered exploratory. The results of this independent review will not be communicated to the investigator during the study. Patient management will be based solely on the results of the investigator-performed RECIST 1.1 assessment.

[0276] Inclusion criteria:

[0277] Histologically confirmed diagnosis of advanced colorectal adenocarcinoma (by local laboratory and local clinical guidelines) with documented objective radiographic or symptomatic disease progression after first-line systemic therapy with any fluoropyrimidine-based regimen for advanced disease (except FOLFOXIRI, see list of exclusion criteria).

[0278] a) Patients may have received prior neoadjuvant or adjuvant therapy, which may have included irinotecan or oxaliplatin. If progression occurs within 6 months of the last dose of neoadjuvant or adjuvant therapy, this regimen will be considered a line of systemic therapy for advanced disease.

[0279] i. If assigned to receive FOLFIRI, patients must not receive irinotecan as part of first-line systemic therapy.

[0280] ii. If assigned to receive FOLFOX, patients must not receive oxaliplatin as part of first-line systemic therapy.

[0281] iii. Prior treatment with anti-VEGF or anti-EGFR therapy as first-line and / or maintenance systemic therapy was allowed.

[0282] b) The presence of at least one measurable lesion as assessed by CT and / or MRI according to RECIST v1.1. (Lesions in areas that have undergone prior locoregional therapy (including prior radiation therapy) are not considered measurable unless there has been demonstrated progression of the disease since therapy, as defined by RECIST v1.1.)

[0283] Exclusion criteria

[0284] The following is a list of exclusion criteria used in the study. The list of exclusion criteria is exemplary and other criteria may be used during the study.

[0285] Microsatellite instability-high (MSI-H) / mismatch repair deficient (dMMR) and / or BRAF V600E mutation-positive colorectal cancer. Prior therapy with an anti-DKK1 agent. Prior therapy with FOLFOXIRI. Prior therapy with anti-programmed cell death protein ligand-1 (PD-(L)1) or anti-programmed cell death protein ligand-2 (PD-L2), or any other antibody or drug that specifically targets T-cell co-stimulatory or co-inhibitory checkpoint pathways, in any treatment setting (including adjuvant / neoadjuvant). Systemic anticancer therapy within 28 days prior to the first dose of study drug. Major surgery within 28 days prior to the first dose of study drug. Treatment with radiation therapy within 14 days prior to the first dose of study drug.

[0286] Enrollment in Part A of this study has been completed, and treatment and follow-up are ongoing. 33 patients followed the experimental group according to the above-described protocol. The results are described in Example II below.

[0287] Case II: Preliminary Results from Part A of the DeFianCe Study

[0288] In Part A of the DeFianCe study, 33 patients with colorectal cancer (CRC) were treated according to the Part A protocol described above. Figure 4 Shown is a consort diagram of Part A of the DeFianCe study. The results described herein are based on preliminary data from Part A of the study, and final data are not yet available.

[0289] The patient population in Part A is characterized as follows:

[0290] -76% left colon (primary tumor location)

[0291] -52% prior bevacizumab treatment

[0292] -68% KRAS mutation

[0293] -70% have liver metastases

[0294] -45% 1L PD ≤ 6 months

[0295] Further characterization of the patients is shown in Table 1 . Figure 5 Shown in.

[0296] Twenty-six of 33 patients were determined to be response evaluable (RE). Assessed by best overall response (BOR, best % change from baseline (target lesions, sum of measurements (SoM)) Figure 4 The treatment regimen shown in . Figure 6 (The results are based on preliminary data from Part A of the study, and final data are not yet available.) It can be seen that at the time of preliminary data evaluation, the objective response rate (ORR) of RE patients was 6 / 26 = 23%, while the disease control rate (DCR) of RE patients was 24 / 26 = 92%. The results are summarized in Table 2.

[0297] Table 2

[0298]

[0299] The 26 response-evaluable patients were also categorized by the presence of KRAS mutations. Figure 7A The data are presented in Table 3 and summarized in Table 3. As can be seen, the majority of patients carrying KRAS mutations (who would be expected to have a worse outcome than patients carrying KRAS wild-type tumors) showed PR or SD.

[0300] Table 3

[0301]

[0302] The 26 response-evaluable patients were also categorized by the presence of liver metastases. Figure 7B The data are presented in Table 4 and summarized in Table 4. As can be seen, the majority of patients with liver metastases (patients expected to have a poor prognosis) showed PR or SD.

[0303] Table 4

[0304]

[0305] The 26 response-evaluable patients were also categorized according to whether they had received prior bevacizumab (anti-VEGF) therapy. Figure 7C Presented in.

[0306] Each of the 26 response-evaluable patients was also evaluated to determine whether the patient was identified as a "rapid progressor." As used herein, the phrase "rapid progressor" refers to a patient who shows evidence of progressive disease (PD) on or within 6 months of the last dose of one prior line of systemic anticancer therapy (e.g., 5-FU + / - oxaliplatin or irinotecan) for metastatic disease. Figure 7D Presented in.

[0307] Preliminary data showed that 22 of the 33 patients remained on study therapy at the time of data evaluation. Figure 8 As can be seen in the results, the majority of patients still on study showed stable disease ( Figure 8 SD) or partial remission (in Figure 8 PRs are marked with the number 1 in the Figure 8 The categories of “response” are: “EoS,” which refers to end of study, and “EoT,” which refers to end of treatment.

[0308] Example III: PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha) inhibitor BYL719 and DKN-01 combination

[0309] A study was initiated to evaluate the growth of the human colorectal cancer cell line HCT116 with and without the PI3KCA H1047R mutation in mice treated with DKN-01, the PI3KCA inhibitor BYL719 (Apellix), or a combination. Female BALB / c scid mice were inoculated subcutaneously with HCT116 human colon cancer cells with or without the H1047R mutation on day 0. On day 17, when tumor volumes reached 50-75 mm3, animals were randomly assigned to treatment groups and dosing began.

[0310] In HCT116 PIK3CA+ / - vaccinated mice (HCT116 cell line without PIK3CA H1047 mutation), DKN-01 and BYL719 monotherapy resulted in 45% (p = 0.03) and 50% (p = 0.005) tumor growth inhibition (TGI) on day 52 (treatment day 36) compared to IgG4 controls. When compared to IgG4 controls, the combination therapy resulted in an overall TGI of 68% (p < 0.0001) on day 52. ​​In addition, when compared to DKN-01 and BYL719 monotherapy, the combination therapy resulted in a TGI of 42% (p = 0.02) and 36% (p = 0.04), respectively (Figure 2A).

[0311] In mice vaccinated with HCT116 H1047 / - (HCT116 cell line with the H1047R mutation), DKN-01 and BYL719 monotherapy resulted in 49% (p = 0.03) and 56% (p = 0.001) TGI, respectively, on day 48 (treatment day 36) compared to IgG4 controls. Combination therapy resulted in an overall TGI of 77% (p < 0.0001) on day 48 compared to IgG4 controls. Furthermore, combination therapy resulted in TGIs of 54% (p = 0.03) and 48% (p = 0.01) compared to mDKN-01 and BYL719 monotherapy ( FIG. 2B ).

[0312] The detailed scheme of the model used is as follows:

[0313] Female BALB / cscid mice (10 / group) were inoculated subcutaneously in the right flank with 10 x 10^6 HCT116 human colorectal cancer cells on day 0. Human IgG4 control (10 mg / kg) and DKN-01 (5 mg / kg) were administered intraperitoneally (IP) twice a week. BYL719 (25 mg / kg) was administered orally (PO) daily (QD) during the study. Tumor measurements were taken with a caliper every 3-4 days, and the volume (V) was calculated using the following formula: V = 1 = / 2 (length [mm] × width [mm]^2). Data are presented as mean tumor volume (MTV) ± SEM. #, p < 0.05. Percent TGI = [(control MTV) - (treated MTV)] / (control MTV). Comparisons between groups were performed at study termination using one-way repeated measures ANOVA with Tukey's multiple comparison test.

[0314] Example IV: Activity of DKN-01 in combination with anti-PD-1 antibody in a colorectal cancer model

[0315] The activity of mDKN-01 and anti-PD-1 antibodies alone and in combination was evaluated in the CT26 syngeneic tumor xenograft model ( Figure 3). Female BALB / c mice were subcutaneously inoculated with CT26 mouse colon cancer cells on day 0. On day 7, when the tumor volume reached 50-65 mm3, the animals were randomly assigned to treatment groups and dosing began. Compared with IgG2a controls, mDKN-01 and anti-PD-1 monotherapy resulted in 71% (p<0.0001) and 29% (p=0.20) tumor growth inhibition (TGI), respectively, on day 21 (day 14 of treatment). When compared to IgG2a controls, the combination therapy resulted in an overall TGI of 88% (p<0.0001) on day 21. In addition, when compared to mDKN-01 and anti-PD-1 monotherapy, the combination therapy resulted in TGIs of 58% (p=0.03) and 83% (p=0.0004), indicating that additive efficacy was observed.

[0316] The detailed scheme of the model used is as follows:

[0317] Female BALB / c mice (15 / group) were inoculated subcutaneously in the right flank with 0.5 x 10^6 CT26 mouse colon cancer cells on day 0. IgG2a control (10 mg / kg) and mouse surrogate IgG2a DKN-01 (mDKN-01) antibody (10 mg / kg) with a D265A mutation that reduces effector function were administered intraperitoneally (IP) twice a week. Anti-PD-1 (10 mg / kg) was administered IP every four days (Q4D). Tumor measurements were performed with a caliper every 3-4 days, and the volume (V) was calculated using the following formula: V = 1 = / 2 (length [mm] × width [mm]^2). Data are shown as mean tumor volume (MTV) ± SEM. #, p < 0.05; ***, p < 0.0001. Percent TGI = [(control MTV) - (treated MTV)] / (control MTV). Comparisons between groups were performed using a two-way repeated-measures ANOVA with a Tukey-Kramer post hoc test.

[0318] While the invention has been particularly shown and described with reference to example embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention as encompassed by the appended claims.

Claims

1. A method of treating colorectal cancer in a subject in need of treatment, the method comprising co-administering to the subject an effective amount of: a) DKK1 antibody or an antigen-binding fragment thereof; b) VEGF or VEGFR inhibitors; and c) optionally one or more chemotherapeutic agents Or a pharmaceutically acceptable salt of any one of the foregoing.

2. The method of claim 1, wherein the VEGF or VEGFR inhibitor is selected from pazopanib, sunitinib, sorafenib, regorafenib, cabozantinib, lenvatinib, ponatinib, cabozantinib, ziv-aflibercept, axitinib, tivozanib, ramucirumab, vandetanib or bevacizumab.

3. The method of claim 1, wherein the VEGF or VEGFR inhibitor is bevacizumab or a biosimilar of bevacizumab.

4. The method of claim 3, wherein the VEGF or VEGFR inhibitor is a biosimilar of bevacizumab selected from mvasi, zirabev, alymsys and vegzelma.

5. The method of any one of claims 1-4, wherein the DKK1 antibody is DKN-01.

6. The method of any one of claims 1-5, wherein the one or more chemotherapeutic agents are fluorouracil-based chemotherapeutic agents selected from the group consisting of 5-FU (fluorouracil), FOLFIRI, FOLFOX.

7. The method of claim 6, wherein the FOLFOX is a modified FOLFOX6.

8. The method of any one of claims 1-7, further comprising administering one or more additional therapeutic agents selected from a PI3K inhibitor and / or an immune checkpoint inhibitor.

9. The method of claim 8, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor.

10. The method of claim 9, wherein the PD-1 inhibitor is selected from nivolumab, pembrolizumab, pidilizumab, AMP-224, sazanalizumab, spartalizumab, cemiplizumab, rivolimab, tislelizumab, carrelizumab, bruglimumab, sepalizumab, and dotalimumab.

11. The method of claim 10, wherein the PD-1 inhibitor is pembrolizumab, nivolumab, tislelizumab, or brigatinib.

12. The method of claim 9, wherein the PD-L1 inhibitor is selected from atezolizumab, durvalumab, avelumab, envolimab, BMS-936559, lodalimab, cocilimab, sugemalimab, and adebelimumab.

13. The method of claim 8, wherein the PI3KCA inhibitor is selected from the group consisting of cupanisib, duvilisib, idranib and apellisib.

14. The method of any one of claims 1-13, wherein the subject's colorectal cancer is determined to have a detectable tumor DKK-1 expression level.

15. The method of any one of claims 1-13, wherein the subject's plasma has detectable levels of DKK1.

16. The method of any one of claims 1-13, wherein the subject's serum has detectable levels of DKK1.

17. The method of any one of claims 1-16, wherein the subject has received a prior 5-FU-based therapy for the colorectal cancer.

18. The method of any one of claims 1-17, wherein the subject's colorectal cancer is microsatellite stable (MSS).

19. The method of any one of claims 1-17, wherein the subject's colorectal cancer does not have a BRAFV600E mutation.

20. The method of any one of claims 1-19, wherein the subject's colorectal cancer is advanced colorectal cancer.

21. The method of any one of claims 1-20, wherein the subject's colorectal cancer is metastatic cancer.

22. The method of any one of claims 1-21, wherein the subject's colorectal cancer is adenocarcinoma.

23. The method of any one of claims 1-22, wherein the treatment is administered over the course of one or more 14-day cycles.

24. The method of claim 23, wherein 400 mg of the DKK1 antibody or antigen-binding fragment thereof is administered on day 1 of the 14-day cycle.

25. The method of claim 23 or 24, wherein the VEGF inhibitor is bevacizumab and is administered at 5 mg / kg on day 1 of the 14-day cycle.

26. The method of any one of claims 23-25, wherein the 14 day cycle is repeated.

27. The method of any one of claims 1-26, wherein the DKK1 antibody or antigen-binding fragment thereof comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), wherein the LCVR comprises complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3, wherein LCDR1 has the amino sequence of SEQ ID NO: 1, LCDR2 has the amino sequence of SEQ ID NO: 2, LCDR3 has the amino sequence of SEQ ID NO: 3, HCDR1 has the amino sequence of SEQ ID NO: 4, HCDR2 has the amino sequence of SEQ ID NO: 5, and HCDR3 has the amino sequence of SEQ ID NO:

6.

28. The method of claim 27, wherein the LCVR comprises the amino acid sequence of SEQ ID NO:7 and the HCVR comprises the amino acid sequence of SEQ ID NO:

8.

29. The method of claim 27 or 28, wherein the LCVR and HCVR comprise an amino acid sequence selected from the group consisting of: (i) a LCVR comprising the amino acid sequence of SEQ ID NO:9 and a HCVR comprising the amino acid sequence of SEQ ID NO:10; (ii) a LCVR comprising the amino acid sequence of SEQ ID NO:11 and a HCVR comprising the amino acid sequence of SEQ ID NO:12; (iii) a LCVR comprising the amino acid sequence of SEQ ID NO:13 and a HCVR comprising the amino acid sequence of SEQ ID NO:10; (iv) a LCVR comprising the amino acid sequence of SEQ ID NO:14 and a HCVR comprising the amino acid sequence of SEQ ID NO:

10.

30. The method of claim 29, wherein the LCVR comprises the amino acid sequence of SEQ ID NO: 11 and the HCVR comprises the amino acid sequence of SEQ ID NO:

12.

31. The method of claim 30, wherein the DKK1 antibody comprises heavy chain and light chain amino acid sequences selected from the group consisting of: a) a heavy chain comprising the amino acid sequence of SEQ ID NO:19 and a light chain comprising the amino acid sequence of SEQ ID NO:16, b) a heavy chain comprising the amino acid sequence of SEQ ID NO:17 and a light chain comprising the amino acid sequence of SEQ ID NO:18, c) a heavy chain comprising the amino acid sequence of SEQ ID NO:19 and a light chain comprising the amino acid sequence of SEQ ID NO:20, and d) a heavy chain comprising the amino acid sequence of SEQ ID NO:19 and a light chain comprising the amino acid sequence of SEQ ID NO:

21.

32. The method of claim 31, wherein the DKK1 antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17 and a light chain comprising the amino acid sequence of SEQ ID NO:

18.

33. The method of any one of claims 1-32, wherein the DKK1 antibody is DKN-01.

34. The method of any one of claims 1-33, wherein the subject is a human.

35. The method of any one of claims 1-34, wherein the subject is a rapid progressor.

36. The method of any one of claims 1-35, wherein the subject carries a mutation in the kRas gene or the nRas gene.

37. The method of any one of claims 1-36, wherein the subject has liver metastases.

38. The method of any one of claims 1-37, wherein the colorectal cancer is rectal cancer.

39. A pharmaceutical composition comprising: a) DKK1 antibody or an antigen-binding fragment thereof; b) VEGF or VEGFR inhibitors; and c) one or more chemotherapeutic agents or a pharmaceutically acceptable salt of any of the foregoing.

40. A pharmaceutical composition comprising: a) DKK1 antibody or an antigen-binding fragment thereof; b) bevacizumab; and c) one or more chemotherapeutic agents or a pharmaceutically acceptable salt of any of the foregoing.

41. A kit comprising: a) DKK1 antibody or an antigen-binding fragment thereof; b) VEGF or VEGFR inhibitors; c) one or more chemotherapeutic agents or pharmaceutically acceptable salts thereof; and d) Instructions for use.

42. A kit comprising: a) DKK1 antibody or an antigen-binding fragment thereof; b) Bevacizumab; c) one or more chemotherapeutic agents or pharmaceutically acceptable salts thereof; and d) Instructions for use.

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