Combination therapy

Abstract

The present invention relates to a method of treating colorectal cancer in a subject in need thereof. The method comprises simultaneously administering to the subject in an effective amount: a) a DKK1 antibody, or an antigen-binding fragment thereof; b) a VEGF or 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

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 388,553, filed Jul. 12, 2022. The entire teachings of the above application are incorporated herein by reference.

Background Art

[0002] 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 death, 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). Contrary to the favorable contribution of screening programs, 25% of patients have disease progression at diagnosis, while an additional 50% will later progress to metastasis, which accounts for the increased mortality (Van Cutsem et al, 2011). Advanced colorectal cancer (CRC) remains a life-threatening serious disease. As such, there is a continuing need for new and improved treatments for cancer patients.

Summary of the Invention

Means for Solving the Problems

[0003] The invention described herein relates to a method of treating colorectal cancer in a subject in need of treatment.

[0004] In a first embodiment, the method comprises co-administering, in an effective amount, a DKK1 antibody, or an antigen-binding fragment thereof, a VEGF or VEGFR inhibitor; and optionally, one or more chemotherapeutic agents or a pharmaceutically acceptable salt of any of the foregoing, to a subject suffering from colorectal cancer.

[0005] 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.

[0006] 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.

[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 for use in the treatment of colorectal cancer.

[0008] 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 for use in the preparation of a medicament for treating colorectal cancer.

[0009] 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 a pharmaceutically acceptable salt thereof; and d) instructions for use.

[0010] 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 a pharmaceutically acceptable salt thereof; and d) instructions for use.

[0011] The foregoing will become apparent from the following more particular description of example embodiments of the invention, as illustrated in the accompanying drawings in which like reference characters refer to the same parts throughout the different views. The drawings are not necessarily to scale, emphasis instead being placed upon illustrating embodiments of the invention.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0013] The description of the exemplary embodiments of the present invention is as follows.

[0014] The teachings of all patents, published applications, and references cited herein are incorporated by reference in their entirety.

[0015] Although the present invention has been particularly shown and described with reference to its exemplary embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the invention as defined by the appended claims.

[0016] Colorectal cancer 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 about 1 in 21 men and 1 in 23 women in the United States will develop colorectal cancer during their lifetime.

[0017] Colorectal cancer can be stage 0, stage I, stage IIA, stage IIB, stage IIC, stage IIIA, 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).

[0018] 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 outside the primary site). The methods described herein are directed to treating a subject having 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 proficient (pMMR) mCRC. In some embodiments, the CRC is not pMMR. In some embodiments, the CRC is deficient mismatch repair (dMMR). 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 / MSS mCRC. In some embodiments, the colorectal cancer is pMMR / MSS mCRC and has not responded to prior treatment with a single-agent checkpoint inhibitor. In some embodiments, the CRC is wild-type KRAS and has not responded to prior treatment with an EGFR inhibitor. In some embodiments, the colorectal cancer is 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 includes both the colon and the rectum.

[0019] MSS CRC: Each of our cells contains DNA with genes that provide instructions to our cells on how to grow, perform certain activities, divide, or die. Also, the DNA within our cells contains segments of short repetitive DNA sequences called microsatellites. This microsatellite DNA serves as a biomarker for how stable our DNA is. The DNA is considered stable when the number of microsatellite repeats is the same in all cells of the body, as is also referred to as microsatellite stability or MSS.

[0020] DNA mismatch repair (MMR) is a quality control, a "spell-check" process that is involved in 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 depends on four major proteins, MLH1, MSH2, MSH6, and PMS2, which function together to repair errors in DNA.

[0021] In some cancer patients, the MMR process functions adequately. In these people, the cancer cells are mismatch repair proficient (pMMR), and their tumor cells have the same number of repeats as their healthy cells. This is also referred to as microsatellite stability or MSS.

[0022] In some embodiments, colorectal cancer has mutations, chromosomal changes, or translocations that affect one or more of the WNT / β-catenin pathway, MAPK / PBK pathway, TGF-β pathway, or TP53 pathway. In some embodiments, colorectal cancer has mutations in genes selected from c-MYC, KRAS, NRAS, HRAS, BRAF, PIK3CA, PTEN, SMAD2, or SMAD4. In some embodiments, mCRC has BRAF or KRAS mutations. In some embodiments, the BRAF mutation is a V600E substitution mutation. In some embodiments, the KRAS mutation is a V9, Gl2, Gl3, Vl4, Ll9, Q22, D33, A59, G60, Q61 R68, Kll7, Al46, Rl64, Kl76, or Kl80 substitution. In some embodiments, the KRAS mutation is a G12D, G12V, G12C, G12A, G12S or other G12 variant. In other embodiments, the KRAS mutation is a 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 Gl2, Gl3, G60, Q61, El23, 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 Gl2, Gl3, Q61, Kl17, Rl64, or Pl67. 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 the BRAFV600E mutation.

[0023] Evaluation of DKK1 levels in tumor, serum, and plasma In a further embodiment, the subject's tumor (a subject suffering from colorectal cancer) has a detectable level of DKK1 expression when measured 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.

[0024] H score and % positive value The expression level of the gene product of interest, e.g., the expression level of DKK1, can be evaluated by methods of immunohistochemistry or chromogenic in situ hybridization techniques. A convenient semi - quantitative measure of the expression level is the calculation of the % positive value (the % of tumor cells stained by the DKK - 1 RNA detection reagent) or the assignment of an H - score (or "histo" score) to the tumor sample. For the H - score, the amount or intensity of staining (0, 1+, 2+, or 3+) is determined for each cell within a fixed field. For in situ hybridization techniques, the number of dots per cell can be determined where 0 is no dots detected per cell, 1+ is 1 - 3 dots per cell, 2+ is 4 - 9 dots per cell, and 3+ is 10+ dots per cell. Next, the H - score may be based on the predominant amount of staining (or number of dots per cell), or more complexly, may include the sum of the individual percentages for each recognized level of the amount of staining (or number of dots per cell). By one method, the percentage of cells at each level of staining intensity (or number of dots per cell) is calculated and ultimately, the H - score is assigned using the following formula: H - score = [1×(% cells 1+) + 2×(% cells 2+) + 3×(% cells 3+)]

[0025] The final H score in the range of 0 to 300 gives relative weight to higher intensity or amount of staining (e.g., dots per cell) in a given tumor sample. The sample can then be considered positive or negative based on specific characteristic thresholds. See, for example, 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 M-S: 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.

[0026] In various embodiments, the H score (e.g., a predetermined value of the H score) can be from 0 to 300, such as 0, 1, 2, 3, etc. Example predetermined values of the H score are 1, 2, 3, 4, and 5. In certain embodiments, the predetermined value of the H score is 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, 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, 138, 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, 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, 248It is 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.

[0027] In other embodiments, the measure of DKK1 expression can be the value of the percentage of tumor cells in which DKK1 staining is positive (% positive or tumor percentage score (TPS)). First, the amount of staining (0, 1+, 2+, or 3+) based on the number of dots in the cells is determined for each tumor cell within a fixed field. After all tumor cells are assigned as "positive" (e.g., detecting a single staining dot for RNAscope chromogenic in situ hybridization), the percentage of positive tumor cells is determined by summing all tumor cells with staining and dividing by the total number of tumor cells. % positive can range from 0 to 100.

[0028] In various embodiments, the % positive value (e.g., a predetermined value of % positive) can be from 0% to 50%, such as 1% to 5%. Examples of predetermined values for % positive are 1% or more, 2% or more, 3% or more, 4% or more, or 5% or more. In certain embodiments, the predetermined value of % positive is 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, or 100%.

[0029] RNAscope assay One way to calculate the H score of a sample is, for example, the RNAscope® in situ hybridization technology developed and commercially available from Advanced Cell Diagnostics, Inc. and Flagship Biosciences, Broomfield, CO, as described at, for example, the URL https: / / acdbio.com / . This technique relies on the optical signals from hybridization probes that are homologous to the mRNA of interest. The signals can be detected by either brightfield or fluorescence microscopy. This technique enables 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.

[0030] Enzyme-linked immunosorbent assay Serum and plasma DKK1 levels can be measured using an improved immunoassay by electrochemiluminescence detection. In this assay, a serum or plasma sample containing DKK1 is captured by anti-hDKK1 (MAB1096) coated on a microtiter plate. Unbound material is washed away, leaving only the captured DKK1. DKK1 is released from the MAB1096-coated plate by acidification (the MAB1096 bound to the plate remains). The released DKK1 is transferred to an MSD plate, where it can bind directly to the plate. The bound DKK1 is detected by the addition of biotinylated anti-hDKK1 (MAB1096) and subsequent development with streptavidin-ruthenium and MSD read buffer. Results are reported as the concentration (ng / mL) of DKK1 in the serum sample or plasma sample.

[0031] DKK1 and DKK1 antibody Dickkopf-1 (DKK1) is a secreted modulator of the Wnt signaling pathway that affects several biological processes such as stem cell maintenance, cell fate determination, cell proliferation, survival, migration, and polarity determination during development and adult tissue homeostasis. DKK1 is most widely characterized as an inhibitor of canonical Wnt / β-catenin-dependent signaling, which is associated with contributing to an immunosuppressive tumor microenvironment. DKK1 is also involved in promoting tumor growth and metastasis through activation of non-canonical (β-catenin-independent Wnt signaling) pathways and the PI3K / AKT signaling pathway. DKK1 also controls bone homeostasis in developing and adult organisms. DKK1 inhibits osteoblast formation, or the differentiation of mesenchymal stem cells into osteoblasts (OBs) (a process promoted by Wnt signaling). As a result, the OB (bone formation) / osteoclast (OC) (bone resorption) balance is shifted towards increased bone resorption, ultimately leading to osteolytic lesions. Certain tumors are associated with the development of osteolytic bone diseases mediated by increased OC bone resorption and impaired OB bone formation.

[0032] In a further embodiment, colorectal cancer has increased levels of DKK1 expression when measured by one or more of the various standard mRNA or protein detection methods known in the art, such as chromogenic in situ hybridization or immunohistochemistry.

[0033] 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 of the present disclosure are therapeutically useful DKK1 antagonists having several desirable properties. For example, the DKK1 antibodies reduce the DKK1-mediated inhibition of alkaline phosphatase, a marker of osteoblast activity, and treat various types of cancer (e.g., non-small cell lung cancer).

[0034] Full-length antibodies are immunoglobulin molecules that, as they exist in nature, contain two heavy (H) chains and two light (L) chains that are interconnected by disulfide bonds. The amino-terminal portion of each chain contains a variable region of about 100-110 amino acids that is primarily responsible for antigen recognition via the complementarity-determining regions (CDRs) contained therein. The carboxy-terminal portion of each chain determines the constant region that is primarily responsible for effector function.

[0035] Between the CDRs are more conserved regions named framework regions ("FRs"). Each light chain variable region (LCVR) and heavy chain variable region (HCVR) is composed of three CDRs and four FRs and are arranged in the following order from amino-terminus to carboxy-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The three CDRs of the light chain are called "LCDR1, LCDR2, and LCDR3," and the three CDRs of the heavy chain are called "HCDR1, HCDR2, and HCDR3." The 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.

[0036] Light chains are classified as kappa or lambda and are characterized by specific constant regions as is known in the art. Heavy chains are classified as gamma, mu, alpha, delta, or epsilon and determine the antibody isotype as IgG, IgM, IgA, IgD, or IgE, respectively. IgG antibodies can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, IgG4. Each type of heavy chain is characterized by a specific constant region having a sequence well known in the art.

[0037] As used herein, the term "monoclonal antibody" (Mab) refers to an antibody derived from a single copy or clone, including, for example, any eukaryotic, prokaryotic, or phage clone, rather than the method by which the antibody is produced. The Mabs of the present invention preferably exist in a homogeneous or substantially homogeneous population. A complete Mab contains two heavy chains and two light chains.

[0038] Unless otherwise specified, the term "DKK1 antibody" encompasses both full-length antibodies and antigen-binding fragments of DKK1 antibodies.

[0039] Examples of such "antigen-binding fragments" of monoclonal antibodies include Fab fragments, Fab' fragments, F(ab')2 fragments, single-chain Fv fragments, and bispecific antibodies and / or multivalent antibodies that use DKK1 antibody CDRs. Monoclonal antibodies and their antigen-binding fragments can be produced, for example, by recombinant techniques, phage display techniques, synthetic techniques such as CDR grafting, or combinations of such techniques or other techniques known in the art. For example, mice can be immunized with human DKK1 or fragments thereof, and the resulting antibodies can be recovered and purified, and whether they have binding and functional properties similar to or the same as the antibody compounds disclosed herein can be determined by methods known in the art. 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, N.Y., chapters 5-8 and 15, ISBN 0-87969-314-2.

[0040] The monoclonal DKK1 antibodies disclosed herein are modified to include a framework region that is substantially or fully human and surrounds the CDRs derived from non-human antibodies. "Antigen-binding fragments" of such humanized 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 1-4. The humanized antibodies and antigen-binding fragments thereof encompassed by the antibodies disclosed herein include molecules in which any one or more of framework regions 1-4 are substantially or fully human, i.e., any of the possible combinations of individual framework regions 1-4 that are substantially or fully human. 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. A substantially human framework has at least about 80% sequence identity with a known human germline framework sequence. Preferably, a substantially human framework has at least about 85%, about 90%, about 95%, or about 99% sequence identity with a known human germline framework sequence.

[0041] In addition to the antibodies disclosed herein, humanized antibodies that exhibit similar functional characteristics can be generated using several different methods. The specific antibody compounds disclosed herein can be used as templates or parental antibody compounds for preparing additional antibody compounds. In one approach, the CDRs of the parental antibody compound are grafted into a human framework that has a high sequence identity with the parental 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 parental antibody compound. This grafting may result in a decrease in binding affinity as compared to the binding affinity of the parental antibody. In this case, the framework can be reverted to the parental framework at certain positions based on specific criteria disclosed by Queen et al. (1991) Proc. Natl. Acad. Sci. USA 88:2869. Additional references that describe methods useful for humanizing mouse antibodies include U.S. Patent No. 4,816,397; U.S. Patent No. 5,225,539 and U.S. Patent No. 5,693,761; the computer programs ABMOD and ENCAD described in Levitt (1983) J. Mol. Biol. 168:595-620; and the methods 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 for which reversion mutations should be considered are known in the art (see, for example, U.S. Patent No. 8,148,498).

[0042] The DKK1 antibody administered by the treatment method described herein includes a light chain variable region (LCVR) and a heavy chain variable region (HCVR), the LCVR includes complementarity determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR includes CDRs HCDR1, HCDR2, and HCDR3.

[0043] In one embodiment, the DKK1 antibody comprises LCDR1 having the amino acid sequence of SEQ ID NO: 1, LCDR2 having the amino acid sequence of SEQ ID NO: 2, LCDR3 having the amino acid sequence of SEQ ID NO: 3, HCDR1 having the amino acid sequence of SEQ ID NO: 4, HCDR2 having the amino acid sequence of SEQ ID NO: 5, and HCDR3 having the amino acid sequence of SEQ ID NO: 6.

[0044] In another embodiment, the DKK1 antibody comprises LCVR having the amino acid sequence of SEQ ID NO: 7 and HCVR having the amino acid sequence of SEQ ID NO: 8. In certain embodiments, LCVR comprises the amino acid sequence of SEQ ID NO: 11 and HCVR comprises the amino acid sequence of SEQ ID NO: 12.

[0045] In a further 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 an antigen-binding fragment thereof comprising the HC amino acid sequence and LC amino acid sequence of SEQ ID NO: 17 and SEQ ID NO: 18, respectively, is referred to herein as DKN-01. In particular, DKN-01 has a molecular / empirical formula of C 6394 H 9810 N 1698 O 2012 S 42 and a molecular weight (uncharged) of 144,015 daltons.

[0046] In one embodiment, the DKK1 antibody disclosed herein is an IgG4 antibody or a fragment thereof having neutralizing activity against human DKK1 comprising the sequence shown in SEQ ID NO: 22. For example, classical Wnt signaling is crucial for osteoblast differentiation and activity. Wnt-3a bound to BMP-4 induces pluripotent mouse C2C12 cells to differentiate into osteoblasts with a measurable endpoint of alkaline phosphatase (「AP」), a marker of osteoblast activity. DKK1 is an inhibitor of classical Wnt signaling and inhibits differentiation and AP production. Neutralizing DKK1 antibodies prevent DKK1-mediated inhibition of AP. Antibodies that block DKK1 inhibitory activity prevent loss of AP activity (see U.S. Patent No. 8,148,498). In certain embodiments, the DKK1 antibody having neutralizing activity is DKN-01, which is an IgG4 antibody.

[0047] The DKK1 antibody disclosed herein has 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 antibody of the present invention has a Kd between 0.5×10 -12 M and 3.0×10 -11 M at 37°C.

[0048] Sequence The following are the sequences of the DKN-01 antibody that can be used in the practice of various exemplary embodiments described herein. LCDR1 His Ala Ser Asp Ser Ile Ser Asn Ser Leu His (SEQ ID NO: 1) LCDR2 Tyr Xaa Arg Gln Ser Xaa Gln (SEQ ID NO: 2) Xaa at position 2 is Gly or Ala, and Xaa at position 6 is Ile or Glu. LCDR3 Gln Gln Ser Xaa Ser Trp Pro Leu His (SEQ ID NO: 3) The 4-position Xaa is Glu or Ala. HCDR1 Gly Phe Thr Phe Ser Ser Tyr Thr Met Ser (SEQ ID NO: 4) HCDR2 Thr Ile Ser Gly Gly Gly Phe Gly Thr Tyr Tyr Pro Asp Ser Val Lys (SEQ ID NO: 5) HCDR3 Pro Gly Tyr Xaa Asn Tyr Tyr Phe Asp Ile (SEQ ID NO: 6) The 4-position Xaa is His or Asn. LCVR

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[0049] VEGF / VEGFR inhibitor Vascular endothelial growth factor (VEGF) inhibitors / vascular endothelial growth factor receptor (VEGFR) inhibitors are agents that inhibit the activities 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-specific antibody. Bevacizumab is a recombinant humanized monoclonal IgG1 antibody containing human framework regions and mouse complementarity-determining regions (CDRs). Bevacizumab binds to VEGF and blocks the interaction of VEGF with its receptors (Flt-I and KDR) on the surface of endothelial cells. The interaction between VEGF and its receptors causes endothelial cell proliferation and neovascularization in in vitro models of angiogenesis. Administration of bevacizumab causes a decrease in microvascular growth and inhibition of metastatic disease progression.

[0050] In various embodiments of the methods of the present invention, bevacizumab can be replaced with one of its biosimilars. For example, bevacizumab can be replaced with Mvasi (bevacizumab-awwb), which is available from Amgen Inc. In another example, bevacizumab can be replaced with Zirabev (bevacizumab-bvzr), which is available from Pfizer Inc. In another example, bevacizumab can be replaced with Alymsys (bevacizumab-maly), which is available from Amneal Pharmaceuticals LLC. In another example, bevacizumab can be replaced with Vegzelma (bevacizumab-adcd), which is available from Celltrion Healthcare.

[0051] The mechanism of action of bevacizumab biosimilars is thought to be similar to that of bevacizumab.

[0052] In the treatment of colorectal cancer, bevacizumab is generally administered as an intravenous injection at a dose of about 4-12 mg / kg, with dose adjustment as needed, on the first day of each 14-day cycle of the induction and maintenance phases, or once every two weeks. In some embodiments, bevacizumab is administered at about 5 mg / kg. In some embodiments, bevacizumab is administered at about 10 mg / kg.

[0053] 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 VEGFR ligands, VEGF-A, VEGF-C, and VEGF-D. As a result, ramucirumab inhibits the activation of VEGFR2 by ligand stimulation, thereby inhibiting ligand-induced proliferation and the migration of human endothelial cells. Ramucirumab is a recombinant human IgG1 monoclonal antibody. Ramucirumab has a molecular weight of approximately 147 kDa. Ramucirumab is produced in genetically modified mammalian NSO cells. The ramucirumab injection for intravenous use is a sterile, preservative-free, clear to slightly opalescent and colorless to slightly yellow solution. Ramucirumab is supplied at a concentration of 10 mg / mL in single-dose vials of either 100 mg (10 mL) or 500 mg (50 mL). Ramucirumab is formulated with 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.

[0054] In the treatment of colorectal cancer, ramucirumab is generally administered as an intravenous infusion at a dose of about 5-15 mg / kg, with dose adjustment as needed, on the first day of each 14-day cycle of the induction and maintenance phases, or once every two weeks. In some embodiments, ramucirumab is administered as an intravenous infusion at about 8 mg / kg over 1 hour.

[0055] In various embodiments, the following VEGF inhibitors and / or VEGFR inhibitors can be used when practicing the methods disclosed herein: Votrient (pazopanib, Novartis), Sutent (sunitinib, Pfizer), Nexavar (sorafenib, Bayer), Stivarga (regorafenib, Bayer), Cabometyx (cabozantinib, Exelixis), Lenvima (lenvatinib, Eisai), Iclusig (ponatinib, Ariad), Cometriq (cabozantinib, Exelixis), Zaltrap (ziv-aflibercept, Regeneron), Inlyta (axitinib, Pfizer), Fotivda (tivozanib, Aveo), Cyramza (ramucirumab, Eli Lilly), Caprelsa (vandetanib, Genzyme), or Alymsys (bevacizumab, Amneal).

[0056] Chemotherapeutic agent Multiple different chemotherapeutic agents or chemical agents are used to treat cancer, either alone or in combination with other agents or treatments. These agents vary widely in their chemical composition (what they are made of), how they are formulated and administered, how useful they are in treating specific types of cancer, and the side effects they may have.

[0057] Not all medical and pharmaceutical agents for treating cancer act in the same manner. Other agents for treating cancer, such as targeted therapies, hormonal therapies, and immunotherapies, work in different ways from traditional or standard chemotherapeutic agents. The chemotherapeutic agents used in the treatment methods described herein are traditional or standard chemotherapeutic agents and are not targeted therapies, hormonal therapies, or immunotherapies (e.g., biologics).

[0058] Chemotherapeutic drugs can target cells at different phases of the cell cycle. Chemotherapeutic drugs can be broadly classified by how they act, their chemical structure, and their relationship to other drugs. Some drugs act in more than one way and can belong to more than one group.

[0059] Alkylating agents prevent cells from proliferating by damaging their 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.

[0060] Antimetabolites interfere with DNA and RNA by acting as substitutions for normal components of RNA and DNA. When this occurs, DNA cannot make a copy of itself and cells cannot divide. Examples of antimetabolites for use 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 the combination of trifluridine / tipiracil.

[0061] Anthracyclines are antitumor antibiotics that interfere with enzymes involved in replicating DNA during the cell cycle. By binding to DNA, it cannot make a copy of itself and cells cannot divide. Examples of anthracyclines for use as at least one chemotherapeutic agent in the methods described herein include daunorubicin; doxorubicin; doxorubicin liposome; epirubicin; idarubicin; and valrubicin. Antitumor antibiotics that are not anthracyclines include bleomycin; dactinomycin; mitomycin C; and mitoxantrone.

[0062] Topoisomerase inhibitors are agents that interfere with an enzyme called topoisomerase. Topoisomerase I inhibitors (also called camptothecins) include irinotecan; irinotecan liposomes; and topotecan. Topoisomerase II inhibitors (also called epipodophyllotoxins) include etoposide (VP-16); mitoxantrone (which also acts as an antitumor antibiotic); and teniposide.

[0063] Mitotic inhibitors include taxanes and vinca alkaloids. Taxanes include cabazitaxel; docetaxel; Nab-paclitaxel; and paclitaxel. Vinca alkaloids include vinblastine; vincristine; vincristine liposomes; and vinorelbine.

[0064] Some chemotherapeutic agents do not fit neatly into any of the listed categories. Examples include all-trans retinoic acid; arsenic trioxide; asparaginase; eribulin; hydroxyurea; ixabepilone; mitotane; omacetaxine; pegaspargase; procarbazine; romidepsin; and vorinostat.

[0065] In certain embodiments, one or more optional chemotherapeutic agents are fluorouracil-based chemotherapy administered according to the methods described herein. Fluorouracil-based chemotherapy includes, but is not limited to, 5-fluorouracil (5-FU); combinations such as 5-FU + 5-FU / leucovorin (FOLFOX), FLOT (5-FU / leucovorin, oxaliplatin, and docetaxel), irinotecan + 5-FU / leucovorin (FOLFIRI), for example, oxaliplatin.

[0066] FOLFOX is a three-drug dosing regimen consisting of fluorouracil (5-FU), folinic acid (e.g., leucovorin or levoleucovorin), and oxaliplatin, which is often used in the treatment of mCRC (National Comprehensive Cancer Network. NCCN Clinical Practice Guidelines in Oncology Colon Cancer. Version 2.2019. 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.

[0067] In one embodiment, the FOLFOX dosing regimen consists of leucovorin or leucovorin, oxaliplatin, and 5-fluorouracil and is a commercial source as part of the standard of clinical care. The dosing regimen for modified FOLFOX6 (mFOLFOX6) may include 400 mg / m2 IV of folinic acid on day 1, 1200 mg / m2 IV / day of fluorouracil (days 1 and 2), and 85 mg / m2 of oxaliplatin on day 1 of each 14-day cycle. Additional information can be found in current prescribing information.

[0068] In one embodiment, the FOLFIRI dosing regimen consists of leucovorin or leucovorin, irinotecan, and 5-fluorouracil and is a commercial source as part of the standard of clinical care. The dosing regimen for FOLFIRI may include a 90-minute IV infusion of irinotecan (180 mg / m2), followed by a simplified LV5FU2 dosing regimen (leucovorin [400 mg / m2] and bolus fluorouracil [400 mg / m2] on day 1 and a 46-hour infusion of fluorouracil [2400 mg / m2]). Additional information can be found in current prescribing information. In a specific embodiment, one or more chemotherapeutic agents used in the method for treating colorectal cancer are mFOLFOX6 or FOLFIRI.

[0069] Targeted therapy (as an additional therapeutic agent for co - administration) (PI3K inhibitor) Phosphoinositide 3 - kinase inhibitors (PI3K inhibitors) are a class of agents that function by inhibiting one or more of the phosphoinositide 3 - kinase (PI3K) enzymes, which are part of the PI3K / AKT / mTOR pathway. This signaling pathway controls cellular functions such as proliferation and survival. It is tightly regulated in healthy cells but is constitutively active in many cancer cells, enabling cancer cells to survive and proliferate more efficiently. PI3K inhibitors block the PI3K / AKT / mTOR pathway and thus slow cancer growth. They are examples of targeted therapies.

[0070] Examples of PI3K inhibitors include, but are not limited to, copanlisib (Aliqopa), duvelisib (Copiktra), idelalisib (Zydelig), and alpelisib (Piqray).

[0071] In some alternative embodiments, the methods described herein further comprise the administration of a PI3K inhibitor and / or an immunomodulatory agent. Examples of PI3K inhibitors include, but are not limited to, copanlisib (Aliqopa), duvelisib (Copiktra), idelalisib (Zydelig), and alpelisib (Piqray).

[0072] Immunotherapy (as an additional therapeutic agent for co - administration) Combination with immune checkpoint inhibitors In some alternative embodiments, the methods described herein further comprise the administration of an immune checkpoint inhibitor or an immunomodulatory agent. Examples of immune checkpoint inhibitors and immunomodulatory agents include, but are not limited to, PD - 1 inhibitors, PD - L1 inhibitors, PD - L2 inhibitors, CTLA - 4 inhibitors. In certain embodiments, the immunomodulatory substance is an antibody, such as a monoclonal antibody.

[0073] In some alternative embodiments, the immune checkpoint inhibitor is a PD-1 inhibitor that blocks the interaction of PD-1 and PD-L1 by binding to the PD-1 receptor and then inhibits immunosuppression. In some alternative embodiments, the immune checkpoint inhibitor is, but is not limited to, nivolumab (Opdivo®; Bristol-Myers Squibb), pembrolizumab (Keytruda®; Merck), pidilizumab (Pfizer), which was previously CT-011, AMP-224 (Amplimmune); south rimab (PF-06801591; Pfizer), spartalizumab (PDR001; Novartis), cemiplimab (Libtayo®; REGN2810; Regeneron), 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).

[0074] In some alternative embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor that blocks the interaction of PD-1 and PD-L1 by binding to the PD-L1 receptor, thereby inhibiting immunosuppression. Examples of PD-L1 inhibitors include, but are not limited to, atezolizumab (Tecentriq®; Genentech), durvalumab (Imfinzi®; AstraZeneca); avelumab (Bavencio®; Merck), enoblituzumab (KN035; Alphamab), BMS-936559 (Bristol-Myers Squibb), rodaplimab (LY3300054; Eli Lilly), cosibelimab (Checkpoint Therapeutics), sugemalimab (Cstone Pharmaceuticals), and adebrelimab.

[0075] In one aspect, the immune checkpoint inhibitor is a CTLA-4 immune checkpoint inhibitor that binds to CTLA-4 and inhibits immunosuppression. Examples of CTLA-4 inhibitors include, but are not limited to, ipilimumab (Yervoy®, Bristol Myers Squibb); tremelimumab (AstraZeneca / Medlmmune), zalifrelimab (AGEN1884; Agenus) and AGEN2041 (Agenus).

[0076] In certain aspects of the exemplary embodiments, the PD-1 / PD-L1-based checkpoint inhibitor is selected from pembrolizumab, nivolumab, tislelizumab, budigalimab, zimerelimab, semaprimab, atezolizumab, avelumab, and durvalumab. For example, the PD-1 / PD-L1-based checkpoint inhibitor is pembrolizumab, nivolumab, tislelizumab, budigalimab or atezolizumab.

[0077] Administration and Dosage Mode of Administration In the combinations described herein, the DKK1 antibody and VEGF or VEGFR inhibitor (e.g., bevacizumab) and one or more optional chemotherapeutic agents (i.e., components of the combination therapy) used can be formulated separately or in combination for parenteral (e.g., intravenous), oral, transdermal, sublingual, buccal, rectal, intranasal, intratracheal or intrapulmonary administration. In certain embodiments, the DKK1 antibody (e.g., DKN-01) is administered intravenously. In another certain embodiment, the VEGF or VEGFR inhibitor (e.g., bevacizumab) is administered intravenously. In a further embodiment, the DKK1 antibody and VEGF or VEGFR inhibitor are formulated in combination for intravenous administration.

[0078] For parenteral administration, one or more of the components of the combination therapy used 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, for example, for intravenous, intramuscular or subcutaneous injection or infusion, or for administration and / or infusion in bolus administration (e.g., continuous infusion). Optionally, suspensions, solutions or emulsions in oily or aqueous media containing other excipients such as suspending agents, stabilizers and / or dispersing agents can be used.

[0079] For oral administration, one or more of 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) may be in the form of tablets or capsules prepared by conventional means using pharmaceutically acceptable excipients such as binders (e.g., polyvinylpyrrolidone or hydroxypropylmethylcellulose); fillers (e.g., lactose, microcrystalline cellulose, or calcium phosphate); lubricants (e.g., magnesium stearate, talc, or silica); disintegrants (e.g., sodium starch glycolate); or wetting agents (e.g., sodium lauryl sulfate). Optionally, the tablets can be coated using suitable methods. Liquid formulations for oral administration can be in the form of solutions, syrups, or suspensions. Liquid formulations can be prepared by conventional means using pharmaceutically acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous media (e.g., almond oil, oily esters, or ethyl alcohol); and preservatives (e.g., methyl or propyl p-hydroxybenzoate esters or sorbic acid).

[0080] For buccal administration, one or more of the components of the combination therapy used 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) may be in the form of tablets or lozenges formulated in a conventional manner.

[0081] For rectal administration, one or more of the components of the combination therapy used 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) may be in the form of suppositories.

[0082] For sublingual administration, the tablets can be formulated in a conventional manner.

[0083] For nasal, bronchial, or pulmonary administration, conventional formulations can be used.

[0084] Moreover, one or more of the components of the combination therapy used in the method or composition of the present invention (e.g., DKK1 antibody, VEGF or VEGFR inhibitor (e.g., bevacizumab), and one or more optional chemotherapeutic agents) can be formulated into sustained-release preparations. For example, one or more of the components can be formulated with a polymer or hydrophobic material suitable for providing the active pharmaceutical compound with sustained and / or controlled release properties. Thus, one or more of the components of the combination therapy used in the method of the present invention can be administered, for example, in the form of microparticles by injection or in the form of an oblate or disk by implantation. Various methods for formulating controlled-release drug preparations are known in the art.

[0085] Administration of one or more of the components of the combination therapy, or a pharmaceutically acceptable salt thereof, or a composition comprising one or more of the components of the combination therapy of the present invention (or a pharmaceutically acceptable salt thereof) useful for carrying out the methods described herein can be continued at once per hour, four times a day, three times a day, twice a day, once a day, every other day, twice a week, once a week, once every two weeks, once a month, or once every two months or longer, or in some other intermittent dosing regimen.

[0086] Combination therapy As used herein, "co-administration," "co-administer," "in combination with," or "in conjunction with" refers to the administration of at least one other therapeutic modality in addition to one therapeutic modality. Thus, "in combination with" or "in conjunction with" refers to the administration of one therapeutic modality before, during, or after the administration of at least one other therapeutic modality to an individual.

[0087] The DKK1 antibodies disclosed herein can be used in combination with a VEGF or VEGFR inhibitor (e.g., bevacizumab) and one or more optional chemotherapeutic agents to treat colorectal cancer. Such combination administration can be by a single dosage form comprising the DKK1 antibody, the VEGF or VEGFR inhibitor (e.g., bevacizumab), and one or more optional chemotherapeutic agents, and such single dosage form can include tablets, capsules, sprays, inhalable powders, injectable liquids, or the like. Alternatively, the combination administration (e.g., co-administration) can be by administration of different dosage forms, where one dosage form contains the DKK1 antibody, another dosage form contains the VEGF or VEGFR inhibitor (e.g., bevacizumab), and yet another dosage form contains one or more optional chemotherapeutic agents (it is understood that when two or more chemotherapeutic agents are used, these agents can be administered using the same or different dosage forms). For example, the DKK1 antibody (e.g., DKN-01) and the 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 the one or more optional chemotherapeutic agents can be administered in different single doses by any other suitable means.

[0088] The components of the combination therapy can be administered in any order. For example, the VEGF or VEGFR inhibitor (e.g., bevacizumab) can be administered before, simultaneously with, or after administration of the DKK1 antibody, and before, simultaneously with, or after administration of the one or more optional chemotherapeutic agents. Thus, the components of the combination therapy can be administered together in a single formulation or in separate formulations, e.g., simultaneously or sequentially or both. For example, if the DKK1 antibody and the 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 time between administrations of the components of the combination therapy will be readily determined by the treating physician.

[0089] Moreover, the components of the combination therapy may or may not be administered on a similar dosing schedule. For example, the DKK1 antibody and the VEGF or VEGFR inhibitor (e.g., bevacizumab) may have different half-lives and / or act on different time scales such that the DKK1 antibody is administered more frequently than the VEGF or VEGFR inhibitor (e.g., bevacizumab) or the VEGF or VEGFR inhibitor (e.g., bevacizumab) is administered more frequently than the DKK1 antibody. For example, the DKK1 antibody and the VEGF or VEGFR inhibitor (e.g., bevacizumab) can be administered together (e.g., in a single dose or sequentially) on a given day, followed by administration of only one or more optional chemotherapeutic agents after a set number of days. The number of days between administrations of the components of the combination therapy can be appropriately determined according to the safety and pharmacodynamics of each drug.

[0090] In certain embodiments, the treatment period for the combination treatment is a 21-day cycle, which can be repeated until it is determined that the patient is not deriving any clinical benefit from this combination therapy. In another specific embodiment, the treatment period for the combination treatment is a 14-day cycle, which can be repeated until it is determined that the patient no longer requires treatment or is not deriving any clinical benefit from the combination therapy. For example, a patient can receive treatment (e.g., 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) for about 1 cycle to about 30 cycles (e.g., 14-day cycles).

[0091] As used herein, "effective amount" refers to an amount of a combination of therapeutic agents that is therapeutically or prophylactically sufficient to treat a target disorder. The effective amount will depend on the age, sex, and weight of the patient, the patient's current medical condition, and the nature of the colorectal cancer being treated. Those skilled in the art will be able to determine an appropriate dosage depending on these and other factors.

[0092] Suitable dosages per administration of the DKK1 antibody include dosages of 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, 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 1500 mg, about 1525 mg, about 1550 mg, about 1575 mg, about 1600 mg, about 1625 mg, about 1650 mg, about 1675 mg, about 1700 mg, about 1725 mg, about 1750 mg, about 1775 mg, about 1800 mg, about 1825 mg, about 1850 mg, about 1875 mg, about 1900 mg, about 1925 mg, about 1950 mg, about 1975 mg, about 2000 mg, about 2025 mg, about 2050 mg, about 2075 mg, about 2100 mg, about 2125 mg, about 2150 mg, about 2175 mg, about 2200 mg, about 2225 mg, about 2250 mg, about 2275 mg, about 2300 mg, about 2325 mg, about 2350 mg, about 2375 mg, about 2400 mg, about 2425 mg, about 2450 mg, about 2475 mg, about 2500 mg, about 2525 mg, about 2550 mg, about 2575 mg, about 2600 mg, or about 3,000 mg or more. Each suitable dosage can be administered over a period of time considered appropriate by those skilled in the art. For example, each suitable dosage can be administered over a period of about 30 minutes and within 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 a specific embodiment, suitable dosages for 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, etc.). The selected dosage can be administered intravenously over a period of about 30 minutes to about 2 hours. In a particular embodiment, a suitable dosage for the DKK1 antibody can be about 300 mg administered over a period of about 30 minutes to about 2 hours. Another suitable dosage for the DKK1 antibody can be about 600 mg administered over a period of about 30 minutes to about 2 hours. Another suitable dosage for a DKK1 antibody (e.g., DKN-01) can be about 400 mg administered intravenously over a period of about 30 minutes to about 2 hours. Administration of these dosages over the recited periods can be accomplished using the intravenous route. For example, a DKK1 antibody (e.g., DKN-01) can be administered on day 1 of a 21-day cycle and then repeated on day 1 of any additional cycles. For such 21-day cycles, the dosing can be within the amounts described above, and in particular a dosage of 600 mg can be used. In another example, a DKK1 antibody (e.g., DKNK-01) can be administered on day 1 of a 14-day cycle and then repeated on day 1 of any additional cycles. In a particular embodiment, during the first cycle of a plurality of 14-day cycles, DKN-01 can be administered on both day 1 of the cycle at about 400 mg and day 8 of the cycle at about 400 mg, and then once within each subsequent 14-day cycle. In another particular embodiment, within the first cycle of a plurality of 21-day cycles, DKN-01 can be administered on both day 1 and day 15 of the 21-day cycle at about 600 mg, and then once within each subsequent 21-day cycle.

[0093] The appropriate dosage per administration for a VEGF or VEGFR inhibitor (e.g., bevacizumab) can be determined based on the recommended dosage known as the standard of care. For example, the appropriate dosage per administration of bevacizumab is about 5 mg / kg to about 100 mg / kg intravenously. Administration can be over a period necessary to deliver the desired dosage in a safe and effective manner. In some cases, the time is for a period of at least 30 minutes, 60 minutes, 90 minutes, 2 hours, 3 hours, etc. This administration can be repeated for each cycle (e.g., once every 14 days in a 14-day cycle). In certain embodiments, the appropriate dosage per administration is about 5 mg / kg using the intravenous route.

[0094] 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 can be simultaneously administered in combination to achieve an effective amount in the method of the present invention. It is understood that when two or more chemotherapeutic agents are included, the amount of chemotherapeutic agent administered can vary. In one embodiment, the components of the combination (e.g., DKK1 antibody, VEGF or VEGFR inhibitor (e.g., bevacizumab) and one or more optional chemotherapeutic agents) are each administered in an individual effective amount (e.g., each in an amount therapeutically effective when administered alone). In another embodiment, each of the components of the combination (e.g., DKK1 antibody, VEGF or VEGFR inhibitor (e.g., bevacizumab) and one or more optional chemotherapeutic agents) is administered in an amount that does not provide a therapeutic effect alone (less than a therapeutic dose). In yet another embodiment, one or two components of the combination can be administered in an effective amount while the remaining components are administered in less than a therapeutic dose. For example, the DKK1 antibody can be administered in less than a therapeutic dose, the VEGF or VEGFR inhibitor (e.g., bevacizumab) can be administered in an effective amount, and one or more therapeutic agents can be each administered in their effective amounts. 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 one or more optional chemotherapeutic agents are fluorouracil-based chemotherapy selected from 5-FU (fluorouracil), FOLFIRI and FOLFOX (e.g., modified FOLFOX6) and are administered as described herein.

[0095] In certain embodiments, the subject receives DKN-01 (administered IV) at a dose of 400 mg on day 1 of each 14-day cycle, and in addition, an additional loading dose of 400 mg is administered only on D8 of C1 in combination with either of the following two dosing regimens on day 1 of each 14-day cycle: FOLFIRI + 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 dosing regimen (leucovorin [400 mg / m2] and bolus fluorouracil [400 mg / m2] on day 1 and 46-hour infusion of fluorouracil [2400 mg / m2]). Modified FOLFOX6 (mFOLFOX6) + Bevacizumab: 90-minute IV infusion of bevacizumab (5 mg / kg), followed by mFOLFOX6 (day 1: IV bolus of oxaliplatin [85 mg / m2], folinic acid [400 mg / m2], and fluorouracil [400 mg / m2], then 46-hour continuous infusion of 2,400 mg / m2).

[0096] Additional therapeutic agents, such as agents 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. The additional agents can be administered according to known methods.

[0097] As used herein, the term "subject" refers to a mammal, preferably a human, but can also mean an animal in need of veterinary treatment, such as a companion animal (e.g., dog, cat, etc.), a livestock animal (e.g., female cow, sheep, pig, horse, etc.), and a laboratory animal (e.g., rat, mouse, guinea pig, etc.).

[0098] As used herein, "treating" includes partially or substantially achieving delaying, inhibiting, or preventing the progression of clinical symptoms associated with colorectal cancer. For example, "treating" includes reducing tumor growth or preventing further growth as detected by standard imaging methods known in the art, such as computerized tomography (CT) scans, magnetic resonance imaging (MRI), chest X-rays, and CT / positron emission tomography (CT / PET) scans, and evaluated according to guidelines and methods known in the art. For example, the response to treatment can be evaluated through Response Evaluation Criteria in Solid Tumors (RECIST) (see Revised RECIST Guideline version 1.1; Eisenhauer et al., Eur. J. Cancer 45(2):228-47, 2009). Thus, in some embodiments, "treating" refers to a complete response (CR) defined according to the RECIST guidelines as the disappearance of all target lesions or a partial response (PR) defined as at least a 30% decrease in the sum of the diameters of target lesions based on the baseline sum of diameters. Other means for evaluating the tumor response to treatment include evaluation of tumor markers and evaluation of performance status (e.g., evaluation of creatinine clearance; see Cockcroft and Gault, Nephron. 16:31-41, 1976, evaluation of carcinoembryonic antigen (CEA) levels in the blood, and evaluation of cancer antigen 19-9 (CA19-9) levels in the blood).

[0099] "Objective response" refers to a measurable response that includes a complete response (CR) or a partial response (PR). In some embodiments, the term "overall response rate" (ORR) refers to the sum of the complete response (CR) rate and the partial response (PR) rate.

[0100] "Complete response" or "CR," as used herein, means the disappearance of all signs of cancer that have responded to treatment (e.g., the disappearance of all target lesions). This does not always mean that the cancer has been cured.

[0101] As used herein, "partial response" or "PR" refers to a decrease in the size of one or more tumors or lesions, or in the extent of cancer in the body, in response to treatment.

[0102] For example, in some embodiments, PR refers to at least a 30% decrease in the sum of the longest diameters (SLD) of target lesions, taking the baseline SLD as a reference.

[0103] As used herein, "disease progression" or "PD" refers to at least a 20% increase in the SLD of target lesions, taking the minimum SLD recorded from the start of treatment or the presence of one or more new lesions as a reference.

[0104] Pharmaceutical composition The components of the combination therapy (e.g., DKK1 antibody, VEGF or VEGFR inhibitor (e.g., bevacizumab) and one or more chemotherapeutic agents) can be incorporated into a pharmaceutical composition suitable for administration. Such compositions generally contain a DKK1 antibody, or a VEGF or VEGFR inhibitor (e.g., bevacizumab), and one or more optional chemotherapeutic agents, either separately or in any combination together, as well as a pharmaceutically acceptable carrier. As used herein, the term "pharmaceutically acceptable carrier" is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are compatible with the administration of a medicament. The use of such media and agents for pharmaceutically active substances is well known in the art. The use of any conventional media or agent in the composition is contemplated, except where it is incompatible with the active compound.

[0105] The pharmaceutical composition of the present invention is formulated to be compatible with its 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. Aqueous solutions or suspensions used for parenteral, intradermal, or subcutaneous application can contain the following components: sterile diluents such as water for injection, physiological saline, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methylparaben; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetic acid, citric acid, or phosphoric acid; and agents for adjusting tonicity such as sodium chloride or dextrose. The pH can be adjusted with an acid or a base such as hydrochloric acid or sodium hydroxide. Parenteral preparations can be sealed in ampoules, disposable syringes, or multi-dose vials made of glass or plastic.

[0106] A pharmaceutical composition suitable for injectable use includes a sterile aqueous solution (water-soluble) or a dispersant and a sterile powder for the immediate preparation of a sterile injectable solution or dispersant. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL (trademark) (BASF, Parsippany, N.J), or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy passage through a hypodermic needle is observed. It must be stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyols (such as glycerol, propylene glycol, and liquid polyethylene glycol, and others of the same kind), and suitable mixtures thereof. Suitable fluidity can be maintained, for example, by the use of coatings such as lecithin, in the case of dispersants, by the maintenance of the required particle size, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and others of the same kind. In many cases, it will be preferable to include in the composition isotonic agents, such as sugars, polyhydric alcohols such as mannitol, sorbitol, and sodium chloride. Prolonged absorption of the injectable composition can be brought about by including in the composition agents that delay absorption, such as aluminum monostearate and gelatin.

[0107] The sterile aqueous injection can be prepared by incorporating a combination (e.g., a DKK1 antibody, a VEGF or VEGFR inhibitor (e.g., bevacizumab), and one or more optional chemotherapeutic agents) in a required amount into a suitable solvent having one or a combination of the ingredients listed above as needed, and then filter sterilizing. Generally, the dispersant is prepared by incorporating the active compound into a sterile medium containing a basic dispersion medium from the ingredients listed above and other necessary ingredients. In the case of a sterile powder for the preparation of a sterile aqueous injection, the preferred preparation methods are vacuum drying and lyophilization, and the powder of the active ingredient + any desired additional ingredients is obtained from its previously filter-sterilized solution.

[0108] Oral compositions generally contain an inert diluent or an edible carrier. They can be enclosed 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, troches, or capsules. Oral compositions can also be prepared using a liquid carrier for use as a mouthwash, and the compound in the liquid carrier is applied orally, quickly rinsed, spat out, or swallowed. Pharmaceutically compatible binders and / or auxiliary materials can be included as part of the composition. Tablets, pills, capsules, troches, and other like items can contain any of the following ingredients or compounds of similar nature: binders such as crystalline 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; sweetening agents such as sucrose or saccharin; or flavoring agents such as peppermint, methyl salicylate, or orange flavoring.

[0109] For administration by inhalation, the compound is delivered in the form of an aerosol spray from a pressurized container, dispenser, or nebulizer containing a suitable propellant, such as a gas like carbon dioxide.

[0110] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, suitable penetrants for the permeation barrier are used in the formulation. Such penetrants are generally known in the art and include, for example, detergents, bile salts, and fusidic acid derivatives for transmucosal administration. Transmucosal administration can be achieved through the use of nasal sprays or suppositories.

[0111] For transdermal administration, the active compound is formulated in an ointment, balm, gel, or cream generally known in the art.

[0112] The compound can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0113] In one embodiment, the active compound is prepared with a carrier that can protect the compound from rapid elimination from the body, such as a controlled-release formulation including implants and microencapsulated delivery systems. Biodegradable and biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid can be used. Methods for the preparation of such formulations will be apparent to those skilled in the art. Materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposome suspensions (including liposomes targeted to infected cells with monoclonal antibodies against viral antigens) can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, as described, for example, in U.S. Patent No. 4,522,811.

[0114] 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, a dosage unit form refers to physically discrete units suitable for single dosing of the subject to be treated, each unit containing a predetermined quantity of an 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 invention are necessarily determined by and directly dependent on the unique characteristics of the active compound, the particular therapeutic effect to be achieved, and the inherent limitations in the art of compounding such active compounds for the treatment of individuals.

[0115] As used herein, the term "kRas gene" refers to the human gene having NCBI Gene ID: 3845.

[0116] As used herein, the term "nRas gene" refers to the human gene having NCBI ID: 4893.

[0117] Accordingly, in a first embodiment, the method comprises co-administering, to a subject suffering from colorectal cancer, a DKK1 antibody, or an antigen-binding fragment thereof, a VEGF or VEGFR inhibitor; and optionally, one or more chemotherapeutic agents or a pharmaceutically acceptable salt of any of the foregoing, in an effective amount.

[0118] In a first aspect of the first embodiment, the VEGF inhibitor is bevacizumab.

[0119] In a second aspect of the first embodiment or any particular aspect thereof, the DKK1 antibody is DKN-01.

[0120] In the third aspect of the first embodiment, any specific aspect thereof, or the first and second aspects thereof, one or more chemotherapeutic agents are fluorouracil-based chemotherapeutic agents selected from 5-FU (fluorouracil), FOLFIRI, and FOLFOX. In a specific aspect, FOLFOX is modified FOLFOX6.

[0121] In the fourth aspect of the first embodiment, any specific aspect thereof, or the first, second, or third aspects thereof, the method further comprises administering one or more additional therapeutic agents selected from PI3K inhibitors and / or immune checkpoint inhibitors. In a specific aspect, the PI3K inhibitor is selected from copanlisib, duvelisib, idelalisib, and alpelisib. In a specific aspect, the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. In a specific aspect, the PD-1 inhibitor is selected from nivolumab, pembrolizumab, pidilizumab, AMP-224, sasanlimab, spartalizumab, semiprimab, retilimab, tislelizumab, camrelizumab, budigalimab, ginberlimab, and dostarlimab. In a further specific aspect, the PD-1 inhibitor is pembrolizumab, nivolumab, tislelizumab, or budigalimab. In a specific aspect, the PD-L1 inhibitor is selected from atezolizumab, durvalumab, avelumab, enoblituzumab, BMS-936559, rodaplimab, cosibelimab, sugemalimab, and adebrelimab. In a further specific aspect, the PD-L1 inhibitor is atezolizumab.

[0122] In the fifth aspect of the first embodiment, any specific aspect thereof, or the first, second, third, or fourth aspects thereof, the subject's colorectal cancer has a detectable level of DKK-1 expression.

[0123] In the sixth aspect of the first embodiment, any specific aspect thereof, or the first, second, third, fourth, or fifth aspects thereof, the subject's plasma has a detectable level of DKK1.

[0124] In the seventh aspect of the first embodiment, any particular aspect thereof, or the first, second, third, fourth, fifth, or sixth aspect thereof, the serum of the subject has a detectable level of DKK1.

[0125] In the eighth aspect of the first embodiment, any particular aspect thereof, or the first, second, third, fourth, fifth, sixth, or seventh aspect thereof, the subject has received one prior 5-FU-based therapy for colorectal cancer.

[0126] In the ninth aspect of the first embodiment, any particular aspect thereof, or the first, second, third, fourth, fifth, sixth, seventh, or eighth aspect thereof, the colorectal cancer is microsatellite stable (MSS).

[0127] In the tenth aspect of the first embodiment, any particular 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.

[0128] In the eleventh aspect of the first or second embodiment, any particular aspect thereof, or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspect thereof, the colorectal cancer is progressive.

[0129] In the twelfth aspect of the first embodiment, any particular aspect thereof, or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or eleventh aspect thereof, the colorectal cancer is metastatic.

[0130] In the thirteenth aspect of the first embodiment, any particular aspect thereof, or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, or twelfth aspect thereof, the colorectal cancer is adenocarcinoma.

[0131] In the fourteenth aspect of the first embodiment, any particular aspect thereof, or the first, second, third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, or thirteenth aspect thereof, the therapeutic agent is administered over the course of one or more 14-day cycles.

[0132] In the 15th aspect of the first embodiment, any specific aspect thereof, or in its 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th or 14th aspect, the DKK1 antibody, or an antigen-binding fragment thereof, is administered in an amount of 400 mg on the first day of a 14-day cycle.

[0133] In the 16th aspect of the first embodiment, any specific aspect thereof, or in its 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th or 15th aspect, the VEGF inhibitor is bevacizumab and is administered at 5 mg / kg on the first day of a 14-day cycle.

[0134] In the 17th aspect of the first embodiment, any specific aspect thereof, or its 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th or 16th aspect, the DKK1 antibody, or an 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, where 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 17th 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 a further specific aspect, the LCVR and HCVR are (i) an LCVR comprising the amino acid sequence of SEQ ID NO: 9 and an HCVR comprising the amino acid sequence of SEQ ID NO: 10; (ii) an LCVR comprising the amino acid sequence of SEQ ID NO: 11 and an HCVR comprising the amino acid sequence of SEQ ID NO: 12; (iii) an LCVR comprising the amino acid sequence of SEQ ID NO: 13 and an HCVR comprising the amino acid sequence of SEQ ID NO: 10; (iv) an LCVR comprising the amino acid sequence of SEQ ID NO: 14 and an HCVR comprising the amino acid sequence of SEQ ID NO: 10, and comprise an amino acid sequence selected from the group consisting of. In yet another specific aspect of the 17th 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.

[0135] In the 18th aspect of the first or second embodiment, any particular aspect thereof, or its 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th aspect, the DKK1 antibody comprises a heavy chain and a light chain amino acid sequence 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.

[0136] In the 19th aspect of the first embodiment, any particular aspect thereof, or its 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th aspect, 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.

[0137] In the 20th aspect of the first embodiment, any particular aspect thereof, or its 1st, 2nd, 3rd, 4th, 5th, 6th, 7th, 8th, 9th, 10th, 11th, 12th, 13th, 14th, 15th, 16th, 17th, 18th, 19th aspect, the DKK1 antibody is DKN-01.

[0138] In the 21st aspect of the first embodiment, or any aspect thereof, the subject is human.

[0139] In the 22nd aspect of the first embodiment, or any aspect thereof, the subject is a rapid onset patient.

[0140] In the 23rd aspect of the first embodiment, or any aspect thereof, the subject has a mutation in the kRas gene or the nRas gene.

[0141] In the 24th aspect of the first embodiment, or any aspect thereof, the subject has liver metastasis.

[0142] In the 25th aspect of the first embodiment, or any aspect thereof, the colorectal cancer is rectal cancer.

[0143] 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.

[0144] In various aspects, the second embodiment is described above in relation to aspects 1 to 25 of the first embodiment.

[0145] 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.

[0146] In various aspects, the third embodiment is described above in relation to aspects 1 to 25 of the first embodiment.

[0147] 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 a pharmaceutically acceptable salt thereof; and instructions for use.

[0148] In various aspects, the third embodiment is described above in relation to aspects 1 to 25 of the first embodiment.

[0149] 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 a pharmaceutically acceptable salt thereof; and instructions for use.

[0150] In various aspects, the fifth embodiment is described above in relation to aspects 1 to 25 of the first embodiment.

Example

[0151] Illustration Example I: Clinical Protocol As a second-line treatment for patients with advanced CRC, a Phase 2 randomized, open-label, two-part, multi-center trial with a safety run-in to evaluate the efficacy and safety of DKN-01 + [FOLFIRI or FOLFOX and bevacizumab] versus standard of care (SOC) [FOLFIRI or FOLFOX and bevacizumab] is described (see NCT05480306 on clincaltrials.gov).

[0152] In Parts A and B, approximately 150 evaluable adult patients with advanced CRC who have measurable disease (RECIST v1.1) that has progressed radiologically during or after first-line systemic therapy are enrolled in the trial.

[0153] The trial consists of a screening period, a treatment period, a safety follow-up period (SFUP), and a long-term follow-up period (LTFU). Patients enter the SFUP for approximately 30 days (+7 days) after the last dose of the study drug and then enter the LTFU period during which they are to follow up for survival and subsequent treatment. In addition, patients who discontinue the study treatment for reasons unrelated to disease progression [PD] also follow up for disease progression during the LTFU period.

[0154] Part A: Safety Run-in To ensure that at least 20 patients are evaluable for review by the Safety Review Committee (SRC), sufficient patients are enrolled in Part A. An evaluable patient is defined as one who has completed all treatment doses (the entire three DKN-01 doses) at C1 and C2 and has completed all safety assessments up to the end of C2; dosing delays are allowed. Patients who are not evaluable may be replaced.

[0155] Subjects determined to be eligible are entered (i.e., enrolled) in Part A. Enrollment can be done up to 3 days before C1D1.

[0156] Patients in Part A receive intravenous (IV) administration (30-minute infusion) of DKN-01 (also known as LY2812176) at a dose of 400 mg on Day 1 (D) of each 14-day cycle, as indicated below or as suggested by the facility's standard of care, and receive administration in combination with either of the following regimens on D1, at the discretion of the treating investigator, of an additional loading dose of 400 mg on D8 of Cycle (C) 1: · FOLFIRI + 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] and 46-hour infusion of fluorouracil [2400 mg / m2] on D1). · Modified FOLFOX6 (mFOLFOX6) + bevacizumab: 90-minute IV infusion of bevacizumab (5 mg / kg), followed by mFOLFOX6 (D1: IV bolus of 85 mg / m2 oxaliplatin, 400 mg / m2 folinic acid, and 400 mg / m2 fluorouracil, then continuous infusion of 2,400 mg / m2 fluorouracil (lurorouracil) over 46 hours).

[0157] Treatment is continued with 14-day cycles until the patient meets the criteria for discontinuation or no longer derives a clinical benefit.

[0158] After monitoring all evaluable patients for 2 cycles (minimum 28 days), the Safety Review Committee (SRC) reviews the overall safety profile of the Part A patients and determines whether the 400 mg DKN-01 dose + FOLFIRI / FOLFOX + bevacizumab is safe and acceptable to initiate Part B.

[0159] Part B Approximately 130 patients are randomized 1:1 to either the experimental or control arm. Randomization may be done up to 3 days prior to C1D1.

[0160] Patients in the experimental arm received IV administration of DKN-01 at a dose of 400 mg on Day 1 of each 14-day cycle, either as follows or as suggested by the facility's standard medical care, and received administration in combination with either of the following two regimens on D1 of each cycle, at the discretion of the study physician, of an additional loading dose of 400 mg of C1 D8 only: - FOLFIRI + bevacizumab: a 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] and a 46-hour infusion of fluorouracil [2400 mg / m2] on Day 1); or - Modified FOLFOX6 (mFOLFOX6) + bevacizumab: a 90-minute IV infusion of bevacizumab (5 mg / kg), followed by a 2-hour infusion of mFOLFOX6 (Day 1: an IV bolus of 85 mg / m2 of oxaliplatin, 400 mg / m2 of folinic acid, and 400 mg / m2 of fluorouracil, followed by a continuous infusion of 2,400 mg / m2 over 46 hours).

[0161] Patients in the control arm received only standard of care (SOC) with FOLFIRI or mFOLFOX6 (at the discretion of the study physician) + bevacizumab, and this SOC regimen was administered as described above for the experimental arm.

[0162] Treatment continued with 14-day cycles until the patient met the criteria for discontinuation or no longer derived a clinical benefit.

[0163] Figure 1 shows a schematic diagram of the study design.

[0164] Definition of Endpoints Event Time and Duration Endpoints Progression-free survival (PFS) is the primary endpoint and is defined as the interval from the date of randomization (or date of record for Part A patients) for each patient until either radiological tumor progression or death from any cause first occurs. Patients for whom the trial is interrupted for reasons other than disease progression are censored at the time of the last radiological scan. (With the exception of palliative radiotherapy for existing non-target lesions without PD) Patients who initiate alternative anti-cancer therapy are treated as being censored at that point. Patients with follow-up deficiencies are censored at the time of the last known contact. PFS is determined by the investigator according to RECIST v1.1 for DKN-01 + SOC versus SOC alone.

[0165] Overall survival (OS) is defined as the time from the date of randomization (or date of record for Part A patients) for each patient until death from any cause. If the patient is alive or has follow-up deficiencies at the time of data analysis, OS data are censored at the earliest of the date the patient is known to be alive or the data cut-off date.

[0166] Duration of response (DoR) is defined only for responders (patients with BOR of CR or PR) as the time from initial response (CR or PR) until either radiologically documented disease progression or death from any cause, whichever is earlier. Patients who have not experienced PD or death at the time of analysis are censored using the same rules as described for PFS.

[0167] Duration of complete response (DoCR) is defined as the time from initial CR until either radiologically documented disease progression or death from any cause, whichever is earlier. Patients who have not experienced PD or death at the time of analysis are censored using the same rules as described for PFS.

[0168] The duration of clinical benefit (DoCB) is defined as the time from the date of randomization (or the date of record for Part A patients) to the earlier of the time of disease progression or death for any reason. Patients who have not experienced PD or death at the time of analysis are censored using the same rules as described for PFS.

[0169] The time to the best response (TTRBest) for patients with a BOR of CR or PR is defined as the time from the date of randomization (or the date of record for Part A patients) to the date of assessment of the first BOR of either CR or PR.

[0170] The time to the first response (TTRFirst) is defined as the time from the date of randomization (or the date of record for Part A patients) to the date of assessment of the first instance of the combined effect of CR or PR.

[0171] The time to response (TTR) is defined as the time from the date of randomization (or the date of record for Part A patients) to the date of assessment of the first instance of the combined effect of complete response (CR) or partial response (PR).

[0172] The best overall response (BOR) is defined as the best response recorded for a patient, taking into account all requirements for confirmation, from the start of treatment with the investigational product to the end of treatment.

[0173] Response rate endpoint The objective response rate (ORR) is defined as the proportion of patients who achieve a best overall response (BOR) of complete response (CR) or partial response (PR) as evaluated by the study physician in accordance with RECIST v1.1. Responses evaluated after conversion to another anti-cancer therapy are excluded.

[0174] The durable clinical benefit (DCB) rate is defined as the proportion of patients who demonstrate a duration of clinical benefit (DoCB) of ≥ 180 days from randomization (or the date of record for Part A patients). Patients with the best overall response (BOR) of PD or who have a clinical benefit but whose DoCB lasts < 180 days are considered "non-DCB".

[0175] The disease control rate (DCR) is defined as the proportion of patients who demonstrate the best overall response (BOR) of complete response, partial response, or stable disease for a duration of at least 8 weeks from randomization (or the date of record for Part A patients) when evaluated by the study investigator using RECIST v1.1.

[0176] Analysis of the primary efficacy endpoint The primary efficacy analysis will be a comparison of PFS evaluated by the study investigator between the two treatment arms within the ITT population using a one-sided stratified log-rank test. In other words, PFS is when determined by the study investigator according to RECIST v1.1 for DKN-01 + SOC vs SOC.

[0177] Summarize the data with Kaplan-Meier curves together with the median and 95% confidence intervals for those medians. By using a Cox proportional hazards model stratified by the randomization factor, the hazard ratio is obtained together with its 95% confidence interval.

[0178] Analysis of secondary efficacy endpoints The ORR is compared between the two treatment arms in Part B within the ITT population based on the study investigator evaluation using the Cochran-Mantel-Haenszel test for the analysis of PFS. The results are reported in terms of the odds ratio and the associated 95% confidence interval. In other words, the ORR is when determined by the study investigator according to RECIST v1.1 for DKN-01 + SOC vs SOC.

[0179] The OS is compared between the two treatment arms in Part B within the ITT population using the same analysis method for PFS. In other words, the OS is in the case of DKN-01 + SOC vs SOC.

[0180] Formal statistical tests for DoR are not performed because this is not a randomized comparison, and these data are evaluated through the presentation of descriptive statistics, including the Kaplan-Meier curve. In other words, the DoR is in the case where it is determined by the investigator in charge of the clinical trial according to RECIST v1.1 for DKN-01 + SOC vs SOC.

[0181] Endpoint based on tumor response, including ORR, DOR, PFS, and time to event, is analyzed based on the results reviewed by the investigator in charge of the clinical trial according to RECIST 1.1. The independent data review provides RECIST measurements for each patient at each visit. Central review data, if conducted, is considered exploratory. The results of this independent review are not communicated to the investigator in charge of the clinical trial during the trial. Patient management will be based only on the results of the RECIST 1.1 evaluation performed by the investigator in charge of the clinical trial.

[0182] Inclusion criteria: Histologically proven diagnosis of advanced colorectal adenocarcinoma with documented objective radiographic or symptomatic disease progression (by standard laboratory and standard clinical guidelines) following any first-line systemic therapy with a fluoropyrimidine-based regimen for disease progression (see list of exclusion criteria, excluding FOLFOXIRI) a) Patients may have received prior neoadjuvant or adjuvant therapy that may have included irinotecan or oxaliplatin. If progression occurred within 6 months of the last dose of neoadjuvant or adjuvant therapy, this regimen is considered first-line systemic therapy for disease progression. i. If assigned to receive FOLFIRI, the patient must not have received irinotecan as part of first-line systemic therapy. ii. If assigned to receive FOLFOX, the patient must not have received oxaliplatin as part of first-line systemic therapy. iii. Prior treatment with anti-VEGF or anti-EGFR therapy is permitted as first-line and / or maintenance systemic therapy. b) The presence of at least one measurable lesion as evaluated by CT and / or MRI according to RECIST 1.1 (lesions within the area treated with prior locoregional therapy, including prior radiotherapy, are not considered measurable unless progression of the lesion has been demonstrated by the therapy defined by RECIST v1.1).

[0183] Exclusion Criteria The following is a list of exclusion criteria to be used in the trial. The list of exclusion criteria is illustrative and other criteria may be used during the trial.

[0184] High microsatellite instability (MSI-H) / deficient mismatch repair (dMMR) and / or BRAF V600E mutation-positive colorectal cancer. Prior therapy with an anti-DKK1 agent. Prior therapy with FOLFOXIRI. Prior therapy with any 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 agent that specifically targets the T cell co-stimulatory or co-inhibitory checkpoint pathway, under any treatment setting (including adjuvant / neoadjuvant). Systemic anti-cancer therapy within 28 days prior to the first dose of the investigational drug. Major surgery within 28 days prior to the first dose of the investigational drug. Treatment with radiotherapy within 14 days prior to the first dose of the investigational drug.

[0185] Enrollment in Part A of this trial is complete and treatment and follow-up are ongoing. For 33 patients, the experimental arm of the above protocol continued. The results are described below in Example II.

[0186] Example II: Preliminary Results of Part A of the DeFianCe Trial In Part A of the DeFianCe trial, 33 colorectal cancer (CRC) patients were treated according to the above Part A protocol. Figure 4 shows the CONSORT diagram for Part A of the DeFianCe trial. The results described herein are based on preliminary data from Part A of the trial, and the final data are not yet available. The patient population in Part A was characterized as follows: - 76% left colon (primary tumor location) - 52% prior bevacizumab treatment - 68% KRAS mutation - 70% with liver metastases - 45% 1L PD ≤ 6 months

[0187] Further characterization of the patients is found in Table 1, shown in Figure 5.

[0188] Twenty-six of the 33 patients were determined to be response evaluable (RE). The treatment regimen shown in Figure 4 was evaluated for best overall response (BOR, best % change from baseline, target lesions, sum of measurements (SoM)). The results are shown in Figure 6 (the results are preliminary data from Part A of the trial, and the final data are not yet available). The response rate (ORR) in RE patients at the time of preliminary data evaluation was 6 / 26 = 23%, and the disease control rate (DCR) in RE patients was 24 / 26 = 92%. The results are summarized in Table 2.

[0189] [Table 1]

[0190] Also, the 26 response evaluable patients were categorized by the presence of a KRAS mutation. The results are shown in Figure 7A and summarized in Table 3. As is apparent, the majority of patients with a KRAS mutation (patients expected to have a worse outcome than those with a KRAS wild-type tumor) showed either a PR or an SD.

[0191]

Table 2

[0192] In addition, 26 evaluable patients for efficacy were categorized according to the presence of liver metastases. The results are shown in Figure 7B and summarized in Table 4. As is clear, the majority of patients with liver metastases (patients expected to have poor prognosis) showed either PR or SD.

[0193]

Table 3

[0194] In addition, 26 evaluable patients for efficacy were categorized according to whether the patients had received prior bevacizumab (anti-VEGF) treatment. The results are shown in Figure 7C.

[0195] In addition, each of the 26 evaluable patients for efficacy was 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 disease progression (PD) at or within 6 months after the last administration of one of the first-choice systemic anti-cancer therapies (e.g., 5-FU + / - oxaliplatin or irinotecan) administered for metastatic disease. The results are shown in Figure 7D.

[0196] Preliminary data indicate that 22 out of 33 patients were continuing the test therapy at the time of data evaluation. As is clear in Figure 8, the majority of patients who continued the test showed either stable (SD identified as number 2 in Figure 8) or partial response (PR identified as number 1 in Figure 8). In Figure 8, the "response" category is "EoS" indicating the end of the test and "EoT" indicating the end of treatment.

[0197] Example III: Combination of BYL719 and DKN-01, inhibitors of PIK3CA (phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α) A study was initiated to evaluate the growth of the human colorectal cancer cell line HCT116 in the presence and absence of the PI3KCA H1047R mutation in mice treated with DKN-01, the PI3KCA inhibitor BYL719 (alpelisib), or a combination thereof. On day 0, female BALB / c scid mice were subcutaneously inoculated with HCT116 human colorectal cancer cells with or without the H1047R mutation. On day 17, when the tumor volume reached 50 - 75 mm3, the animals were randomized into treatment groups and dosing was initiated.

[0198] In HCT116 PIK3CA+ / - inoculated mice (HCT116 cell line without the PIK3CA H1047 mutation), single-agent DKN-01 and BYL719 resulted in 45% (p = 0.03) and 50% (p = 0.005) tumor growth inhibition (TGI) respectively on day 52 (day 36 of treatment), compared to the IgG4 control. The combination therapy resulted in 68% total TGI (p < 0.0001) on day 52 compared to the IgG4 control. Additionally, the combination therapy resulted in 42% (p = 0.02) and 36% (p = 0.04) TGI compared to single-agent DKN-01 and BYL719 respectively (Figure 2A).

[0199] In HCT116 H1047 / - (HCT116 cell line with the H1047R mutation inoculated into mice), single-agent DKN-01 and BYL719 resulted in 49% (p = 0.03) and 56% (p = 0.001) TGI respectively on day 48 (day 36 of treatment), compared to the IgG4 control. The combination therapy resulted in 77% total TGI (p < 0.0001) on day 48 compared to the IgG4 control. Additionally, the combination therapy resulted in 54% (p = 0.03) and 48% (p = 0.01) TGI compared to single-agent mDKN-01 and BYL719 respectively (Figure 2B).

[0200] The detailed protocol for the model used is as follows: On day 0, 10×10 was placed on the right flank of female BALB / c scid mice (10 mice per group)6 Individual HCT116 human colorectal cancer cells were subcutaneously inoculated. 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) throughout the duration of the study. Tumor measurements were performed every 3 - 4 days with calipers, 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. Percent TGI = [(MTV control) - (treated MTV)] / (MTV control). Intergroup comparisons at the end of the study were performed using one-way repeated measures ANOVA by Tukey's multiple comparison test.

[0201] Example IV: DKN-01 Activity in Combination with Anti-PD-1 Antibody in a Colorectal Cancer Model The activities of mDKN-01 and anti-PD-1 antibody alone and in combination were evaluated in a CT26 syngeneic model (Figure 3). On day 0, female BALB / c mice were subcutaneously inoculated with CT26 murine colon cancer cells. On day 7, when the tumor volume reached 50 - 65 mm3, the animals were randomized into treatment groups and dosing was initiated. By mDKN-01 and anti-PD-1 monotherapy, on day 21 (day 14 of treatment), 71% (p < 0.0001) and 29% (p = 0.20) tumor growth inhibition (TGI) were respectively brought about compared to IgG2a control. By combination therapy, on day 21, 88% (p < 0.0001) total TGI was brought about compared to IgG2a control. In addition, by combination therapy, compared to mDKN-01 and anti-PD-1 monotherapy, 58% (p = 0.03) and 83% (p = 0.0004) TGI were respectively brought about, indicating that the effectiveness of the additive was recognized.

[0202] The detailed protocol for the model used is as follows: On day 0, 0.5 × 10 6CT26 mouse colon cancer cells were subcutaneously inoculated. IgG2a control (10 mg / kg) and mouse surrogate IgG2a DKN-01 (mDKN-01) antibody with D265A mutation to reduce effector function (10 mg / kg) were administered intraperitoneally (IP) twice a week. Anti-PD-1 (10 mg / kg) was administered IP every 4 days (Q4D). Tumor measurements were performed with calipers 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 = [(MTV control) - (treated MTV)] / (MTV control). Intergroup comparisons were performed using two-way repeated measures ANOVA with Tukey-Kramer post hoc test.

[0203] The present invention has been particularly shown and described with reference to its exemplary embodiments, but 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

**Claim 1** A method for treating colorectal cancer in a subject in need of treatment, comprising: a) a DKK1 antibody, or an antigen-binding fragment thereof; b) a VEGF or VEGFR inhibitor; and c) optionally, one or more chemotherapeutic agents or a pharmaceutically acceptable salt of any of the foregoing, in an effective amount co-administering to the subject. **Claim 2** The method according to 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. **Claim 3** The method according to claim 1, wherein the VEGF or VEGFR inhibitor is bevacizumab or a biosimilar of bevacizumab. **Claim 4** The method according to claim 3, wherein the VEGF or VEGFR inhibitor is a biosimilar of bevacizumab selected from mvas i, zirabev, alymsys, and vegzelma. **Claim 5** The method according to any one of claims 1 to 4, wherein the DKK1 antibody is DKN-01. **Claim 6** The method according to any one of claims 1 to 5, wherein the one or more chemotherapeutic agents are a fluorouracil-based chemotherapy selected from 5-FU (fluorouracil), FOLFIRI, and FOLFOX. **Claim 7** The method according to claim 6, wherein the FOLFOX is modified FOLFOX6. **Claim 8** The method according to any one of claims 1 to 7, further comprising administering one or more additional therapeutic agents selected from PI3K inhibitors and / or immune checkpoint inhibitors. **Claim 9** The method according to claim 8, wherein the immune checkpoint inhibitor is a PD-1 inhibitor or a PD-L1 inhibitor. **Claim 10** The method according to claim 9, wherein the PD-1 inhibitor is selected from nivolumab, pembrolizumab, pidilizumab, AMP-224, southern rimab, spartalizumab, semiprimab, retilfanlimab, tislelizumab, camrelizumab, budigalimab, ginberlimab, and dostarlimab. **Claim 11** The method according to claim 10, wherein the PD-1 inhibitor is pembrolizumab, nivolumab, tislelizumab, or budigalimab. **Claim 12** The method according to claim 9, wherein the PD-L1 inhibitor is selected from atezolizumab, durvalumab, avelumab, enoblituzumab, BMS-936559, rodaplimab, cosibelimab, sugemalimab, and adebrelimab.

13. The method according to claim 8, wherein the PI3KCA inhibitor is selected from copanlisib, duvelisib, idelalisib, and alpelisib.

14. The method according to any one of claims 1 to 13, wherein the colorectal cancer of the subject is determined to have detectable levels of tumorigenic DKK-1 expression.

15. The method according to any one of claims 1 to 13, wherein the plasma of the subject has detectable levels of DKK1.

16. The method according to any one of claims 1 to 13, wherein the serum of the subject has detectable levels of DKK1.

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

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

19. The method according to any one of claims 1 to 17, wherein the colorectal cancer of the subject does not have a BRAF V600E mutation.

20. The method according to any one of claims 1 to 19, wherein the colorectal cancer of the subject is advanced colorectal cancer.

21. The method according to any one of claims 1 to 20, wherein the colorectal cancer of the subject is metastatic cancer.

22. The method according to any one of claims 1 to 21, wherein the colorectal cancer of the subject is adenocarcinoma.

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

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

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

27. The method according to any one of claims 1 to 26, wherein the DKK1 antibody or an antigen-binding fragment thereof comprises a light chain variable region (LCVR) and a heavy chain variable region (HCVR), the LCVR comprises complementarity-determining regions (CDRs) LCDR1, LCDR2, and LCDR3, and the HCVR comprises CDRs HCDR1, HCDR2, and HCDR3, 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.

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

10.

30. The method according to 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 according to claim 30, wherein the anti-DKK1 antibody comprises a heavy chain and a light chain amino acid sequence 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 according to claim 31, wherein the anti-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 according to any one of claims 1 to 32, wherein the DKK1 antibody is DKN-01.

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

35. The method according to any one of claims 1 to 34, wherein the subject is a rapid-onset patient.

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

37. The method according to any one of claims 1 to 36, wherein the subject is suffering from liver metastasis.

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

39. 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 A pharmaceutical composition comprising.

40. a) 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 A pharmaceutical composition comprising.

41. a) a DKK1 antibody, or an antigen-binding fragment thereof; b) a VEGF or VEGFR inhibitor; c) one or more chemotherapeutic agents or a pharmaceutically acceptable salt thereof; and d) instructions for use A kit comprising.

42. a) a DKK1 antibody, or an antigen-binding fragment thereof; b) bevacizumab; c) one or more chemotherapeutic agents or a pharmaceutically acceptable salt thereof; and d) instructions for use A kit comprising.

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