Anti-periostin antibody and its uses

Abstract

This document describes antibodies that block the function of periostin. This document also describes the use of said antibodies in treating cancer and altering the immune properties of tumors.

Description

[0001] Cross - Reference to Related Applications

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 779,996, filed on December 14, 2018, and U.S. Provisional Application Serial No. 62 / 899,075, filed on September 11, 2019, which are hereby incorporated by reference in their entirety. Background of the Invention

[0003] Periostin (POSTN) is a matricellular protein that is involved in many physiological processes, including epithelial - mesenchymal transition (EMT), cell - matrix interactions, and inflammation. POSTN is overexpressed in several pathological settings, including inflammation, fibrotic diseases, and cancer, in which it is associated with poor prognosis. In cancer, POSTN is typically expressed by stromal cells such as cancer - associated fibroblasts (CAFs), although POSTN expression has also been reported in cancer - initiating cells (CICs) and MDSCs. POSTN regulates extracellular remodeling by binding to other matricellular proteins such as fibronectin and collagen, and acts as an integrin receptor ligand to promote cell survival, migration / invasion, EMT, angiogenesis, and immune cell recruitment. It is hypothesized that POSTN drives tumor growth and progression by acting on multiple aspects of cancer biology, including suppressing anti - tumor immunity by promoting immune exclusion and increasing immunosuppression through tumor - infiltrating myeloid cells. Summary of the Invention

[0004] Antibodies that inhibit periostin function, such as integrin - mediated cell attachment, are described herein. Such antibodies can be used to treat cancer. The anti - periostin antibodies described herein reduce the collagen content of tumors, decrease the infiltration of suppressive myeloid cell populations such as granulocytes and tumor - associated macrophages, while increasing the polarization of macrophages towards the M1 phenotype, and increasing the accumulation and anti - tumor properties of tumor - infiltrating T cells.

[0005] This text describes a recombinant antibody or an antigen-binding fragment of the recombinant antibody that binds to periostin, wherein the antibody or the antigen-binding fragment of the antibody comprises: immunoglobulin heavy chain CDR1 (CDR-H1), which comprises the amino acid sequence shown in SEQ ID NO: 1 (GYTFTSYG); immunoglobulin heavy chain CDR2 (CDR-H2), which comprises the amino acid sequence shown in any one of SEQ ID NO: 2 (ISAYNGNT), 3 (ISAYSGNT), 4 (ISAYQGNT), 5 (ISAYTGNT), or 6 (ISAYDGNT); immunoglobulin heavy chain CDR3 (CDR-H3), which comprises the amino acid sequence shown in any one of SEQ ID NO: 7 (DILVVPFDY), 8 (DVLVVPFDY), or 9 (DMLVVPFDY); immunoglobulin light chain CDR1 (CDR-L1), which comprises the amino acid sequence shown in SEQ ID NO: 10 (SSDIGSNR); immunoglobulin light chain CDR2 (CDR-L2), which comprises the amino acid sequence shown in SEQ ID NO: 11 (SND); and immunoglobulin light chain CDR3 (CDR-L3), which comprises the amino acid sequence shown in SEQ ID NO: 12 (AAWDDSLSTYV). In some embodiments, the antibody or the antigen-binding fragment of the antibody comprises: immunoglobulin heavy chain CDR1 (CDR-H1), which comprises the amino acid sequence shown in SEQ ID NO: 1 (GYTFTSYG); immunoglobulin heavy chain CDR2 (CDR-H2), which comprises the amino acid sequence shown in SEQ ID NO: 2 (ISAYNGNT); immunoglobulin heavy chain CDR3 (CDR-H3), which comprises the amino acid sequence shown in SEQ ID NO: 9 (DMLVVPFDY); immunoglobulin light chain CDR1 (CDR-L1), which comprises the amino acid sequence shown in SEQ ID NO: 10 (SSDIGSNR); immunoglobulin light chain CDR2 (CDR-L2), which comprises the amino acid sequence shown in SEQ ID NO: 11 (SND); and immunoglobulin light chain CDR3 (CDR-L3), which comprises the amino acid sequence shown in SEQ ID NO: 12 (AAWDDSLSTYV). In some embodiments, the recombinant antibody or the antigen-binding fragment of the recombinant antibody is human, chimeric, or humanized. In some embodiments, the recombinant antibody or the antigen-binding fragment of the recombinant antibody is an IgG antibody. In certain embodiments, the recombinant antibody or the antigen-binding fragment of the recombinant antibody comprises one or more mutations to reduce one or more effector functions of the recombinant antibody or the antigen-binding fragment of the recombinant antibody.In certain embodiments, the one or more mutations that reduce one or more effector functions of the recombinant antibody or antigen-binding fragment thereof include one or more mutations or groups of mutations selected from: N434A, N434H, T307A / E380A / N434A, M252Y / S254T / T256E, 433K / 434F / 436H, T250Q, T250F, M428L, M428F, T250Q / M428L, N434S, V308W, V308Y, V308F, M252Y / M428L, D259I / V308F, M428L / V308F, Q311V / N434S, T307Q / N434A, E258F / V427T, S228P, L235E, S228P / L235E / R409K, S228P / L235E, K370Q, K370E, G446 deletion, K447 deletion, and combinations thereof according to the EU numbering system of IgG4. In certain embodiments, the one or more mutations that reduce one or more effector functions of the recombinant antibody or antigen-binding fragment thereof include the S228P, F234A, and L235A mutations of IgG4 according to the EU numbering system. In some embodiments, the recombinant antibody or antigen-binding fragment thereof is a Fab, F(ab)2, single-domain antibody, or single-chain variable fragment (scFv). In some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy-chain variable region and an immunoglobulin light-chain variable region: wherein the immunoglobulin heavy-chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:13; and wherein the immunoglobulin light-chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO:14, wherein the amino acid at amino acid residue number 55 of SEQ ID NO:13 is asparagine, serine, glutamine, threonine, or aspartic acid, and wherein the amino acid at amino acid residue number 100 of SEQ ID NO:13 is methionine, isoleucine, or valine. In some embodiments, the recombinant antibody or antigen-binding fragment thereof requires at least one of the following residues of SEQ ID NO:15 for specific binding to periostin: N276, R284, E288, L287, V295, or K302. In some embodiments, the recombinant antibody or antigen-binding fragment thereof requires at least two, three, four, or five of the following residues of SEQ ID NO:15 for specific binding to periostin: N276, R284, E288, L287, V295, or K302.In some embodiments, the recombinant antibody or the antigen-binding fragment of the recombinant antibody requires at least all of the following residues of SEQ ID NO: 15 for specific binding to periostin: N276, R284, E288, L287, V295, or K302. Also described herein is a pharmaceutical composition comprising the recombinant antibody or the antigen-binding fragment of the recombinant antibody and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for intratumoral administration. Also described herein is the recombinant antibody or the antigen-binding fragment of the recombinant antibody or the pharmaceutical composition for use in reducing the collagen content in a tumor. In some embodiments, the recombinant antibody or the antigen-binding fragment of the recombinant antibody or the pharmaceutical composition is for use in treating cancer. In some embodiments, the cancer includes glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, skin cancer, gastric cancer, mesothelioma, liver cancer, endometrial cancer, colon cancer, cervical cancer, prostate cancer, or lung cancer. Also described herein is a method of reducing the collagen content in a tumor in an individual, which comprises administering to the individual the recombinant antibody or the antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. Also described herein is a method of increasing the M1 macrophage phenotype and / or reducing the M2 macrophage phenotype in a tumor in an individual, which comprises administering to the individual the recombinant antibody or the antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. Also described herein is a method of reducing the accumulation of immunosuppressive granulocytic myeloid cells and / or tumor-associated macrophages in an individual, which comprises administering to the individual the recombinant antibody or the antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. Also described herein is a method of increasing the frequency of CD4+ and / or CD8+ T cells in a tumor of an individual, which comprises administering to the individual the recombinant antibody or the antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. Also described herein is a method of increasing the function of CD8+ T cells in a tumor as measured by interferon-γ expression and / or release by CD8+ T cells in an individual, which comprises administering to the individual the recombinant antibody or the antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. Also described herein is a method of treating cancer in an individual, which comprises administering to the individual a therapeutically effective amount of the recombinant antibody or the antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. In some embodiments, the cancer includes glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, skin cancer, gastric cancer, mesothelioma, liver cancer, endometrial cancer, colon cancer, cervical cancer, prostate cancer, or lung cancer.The present invention also describes a method for preparing a composition for reducing the collagen content in a tumor, which comprises mixing the recombinant antibody or the antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier or diluent. The present invention also describes a method for preparing a composition for increasing the M1 macrophage phenotype and / or reducing the M2 macrophage phenotype in a tumor, which comprises mixing the recombinant antibody or the antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier or diluent. The present invention also describes a method for preparing a composition for reducing the accumulation of suppressive granulocytic myeloid cells and / or tumor-associated macrophages in an individual, which comprises mixing the recombinant antibody or the antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier or diluent. The present invention also describes a method for preparing a composition for increasing the frequency of CD4+ and / or CD8+ T cells in a tumor of an individual, which comprises mixing the recombinant antibody or the antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier or diluent. The present invention also describes a method for preparing a composition for increasing the function of CD8+ T cells in a tumor as measured by interferon-γ expression and / or release by CD8+ T cells in an individual, which comprises mixing the recombinant antibody or the antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier or diluent. The present invention also describes a method for preparing a composition for treating cancer, which comprises mixing the recombinant antibody or the antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier or diluent. In some embodiments, the cancer comprises glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, skin cancer, gastric cancer, mesothelioma, liver cancer, endometrial cancer, colon cancer, cervical cancer, prostate cancer or lung cancer.

[0006] The present invention also describes a recombinant antibody or an antigen-binding fragment of the recombinant antibody that binds periostin, the recombinant antibody or the antigen-binding fragment of the recombinant antibody comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region: wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 13; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 14, wherein amino acid residue number 55 of SEQ ID NO: 13 is asparagine, serine, glutamine, threonine, or aspartic acid, and wherein amino acid residue number 100 of SEQ ID NO: 13 is methionine, isoleucine, or valine. In some embodiments, the recombinant antibody or the antigen-binding fragment of the recombinant antibody is a human antibody. In some embodiments, the recombinant antibody or the antigen-binding fragment of the recombinant antibody is an IgG antibody. In certain embodiments, the recombinant antibody or the antigen-binding fragment of the recombinant antibody comprises one or more mutations to reduce one or more effector functions of the recombinant antibody or the antigen-binding fragment of the recombinant antibody. In certain embodiments, the one or more mutations that reduce one or more effector functions of the recombinant antibody or the antigen-binding fragment of the recombinant antibody comprise one or more mutations or groups of mutations selected from the following: N434A, N434H, T307A / E380A / N434A, M252Y / S254T / T256E, 433K / 434F / 436H, T250Q, T250F, M428L, M428F, T250Q / M428L, N434S, V308W, V308Y, V308F, M252Y / M428L, D259I / V308F, M428L / V308F, Q311V / N434S, T307Q / N434A, E258F / V427T, S228P, L235E, S228P / L235E / R409K, S228P / L235E, K370Q, K370E, G446 deletion, K447 deletion, and combinations thereof according to the EU numbering system of IgG4. In certain embodiments, the one or more mutations that reduce one or more effector functions of the recombinant antibody or the antigen-binding fragment of the recombinant antibody comprise the S228P, F234A, and L235A mutations of IgG4 according to the EU numbering system. In some embodiments, the recombinant antibody or the antigen-binding fragment of the recombinant antibody is a Fab, F(ab)2, single domain antibody, or single chain variable fragment (scFv).The present invention also describes a pharmaceutical composition comprising the recombinant antibody or an antigen-binding fragment of the recombinant antibody and a pharmaceutically acceptable excipient, carrier or diluent. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for intratumoral administration. The present invention also describes the recombinant antibody or an antigen-binding fragment of the recombinant antibody or the pharmaceutical composition for use in reducing the collagen content in a tumor. In some embodiments, the recombinant antibody or an antigen-binding fragment of the recombinant antibody or the pharmaceutical composition is for use in treating cancer. In some embodiments, the cancer includes glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, skin cancer, gastric cancer, mesothelioma, liver cancer, endometrial cancer, colon cancer, cervical cancer, prostate cancer or lung cancer. The present invention also describes a method for reducing the collagen content in a tumor in an individual, which comprises administering to the individual the recombinant antibody or an antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. The present invention also describes a method for increasing the M1 macrophage phenotype and / or reducing the M2 macrophage phenotype in a tumor in an individual, which comprises administering to the individual the recombinant antibody or an antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. The present invention also describes a method for reducing the accumulation of immunosuppressive granulocytic myeloid cells and / or tumor-associated macrophages in an individual, which comprises administering to the individual the recombinant antibody or an antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. The present invention also describes a method for increasing the frequency of CD4+ and / or CD8+ T cells in a tumor of an individual, which comprises administering to the individual the recombinant antibody or an antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. The present invention also describes a method for increasing the function of CD8+ T cells in a tumor as measured by interferon-γ expression and / or release by CD8+ T cells in an individual, which comprises administering to the individual the recombinant antibody or an antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. The present invention also describes a method for treating cancer in an individual, which comprises administering to the individual a therapeutically effective amount of the recombinant antibody or an antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. In some embodiments, the cancer includes glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, skin cancer, gastric cancer, mesothelioma, liver cancer, endometrial cancer, colon cancer, cervical cancer, prostate cancer or lung cancer. The present invention also describes a method for preparing a composition for reducing the collagen content in a tumor, which comprises mixing the recombinant antibody or an antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier or diluent.The present disclosure also describes a method of preparing a composition for increasing the M1 macrophage phenotype and / or decreasing the M2 macrophage phenotype in a tumor, comprising mixing the recombinant antibody or an antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier, or diluent. The present disclosure also describes a method of preparing a composition for decreasing the accumulation of suppressive granulocytic myeloid cells and / or tumor-associated macrophages in an individual, comprising mixing the recombinant antibody or an antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier, or diluent. The present disclosure also describes a method of preparing a composition for increasing the frequency of CD4+ and / or CD8+ T cells in a tumor in an individual, comprising mixing the recombinant antibody or an antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier, or diluent. The present disclosure also describes a method of preparing a composition for increasing the function of CD8+ T cells in a tumor as measured by interferon-γ expression and / or release by CD8+ T cells in an individual, comprising mixing the recombinant antibody or an antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier, or diluent. The present disclosure also describes a method of preparing a composition for treating cancer, comprising mixing the recombinant antibody or an antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the cancer comprises glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, skin cancer, gastric cancer, mesothelioma, liver cancer, endometrial cancer, colon cancer, cervical cancer, prostate cancer, or lung cancer.

[0007] The present disclosure also describes a recombinant antibody that binds periostin or an antigen-binding fragment of the recombinant antibody, wherein when bound to periostin, the recombinant antibody or the antigen-binding fragment of the recombinant antibody binds to the fasciclin 2 (FAS2) domain of periostin. In some embodiments, the recombinant antibody or the antigen-binding fragment of the recombinant antibody binds to any residue of periostin (SEQ ID NO: 15) between amino acid residues 276 and 302 (inclusive of amino acid residues 276 and 302). In some embodiments, when bound to periostin, the recombinant antibody or the antigen-binding fragment of the recombinant antibody binds to at least one of the following residues of periostin (SEQ ID NO: 15): N276, R284, E288, L287, V295, or K302. In some embodiments, when bound to periostin, the recombinant antibody or the antigen-binding fragment of the recombinant antibody binds to two, three, four, or five of the following residues of periostin (SEQ ID NO: 15): N276, R284, E288, L287, V295, or K302. In some embodiments, when bound to periostin, the recombinant antibody or the antigen-binding fragment of the recombinant antibody binds to all of the following residues of periostin (SEQ ID NO: 15): N276, R284, E288, L287, V295, or K302. In some embodiments, the antibody or the antigen-binding fragment of the antibody comprises an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region: wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 13; and wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence shown in SEQ ID NO: 14, wherein amino acid residue number 55 of SEQ ID NO: 13 is asparagine, serine, glutamine, threonine, or aspartic acid, and wherein amino acid residue number 100 of SEQ ID NO: 13 is methionine, isoleucine, or valine.In some embodiments, the antibody or antigen-binding fragment of the antibody comprises: an immunoglobulin heavy chain CDR1 (CDR-H1) that comprises the amino acid sequence shown in SEQ ID NO: 1 (GYTFTSYG); an immunoglobulin heavy chain CDR2 (CDR-H2) that comprises the amino acid sequence shown in any one of SEQ ID NO: 2 (ISAYNGNT), 3 (ISAYSGNT), 4 (ISAYQGNT), 5 (ISAYTGNT), or 6 (ISAYDGNT); an immunoglobulin heavy chain CDR3 (CDR-H3) that comprises the amino acid sequence shown in any one of SEQ ID NO: 7 (DILVVPFDY), 8 (DVLVVPFDY), or 9 (DMLVVPFDY); an immunoglobulin light chain CDR1 (CDR-L1) that comprises the amino acid sequence shown in SEQ ID NO: 10 (SSDIGSNR); an immunoglobulin light chain CDR2 (CDR-L2) that comprises the amino acid sequence shown in SEQ ID NO: 11 (SND); and an immunoglobulin light chain CDR3 (CDR-L3) that comprises the amino acid sequence shown in SEQ ID NO: 12 (AAWDDSLSTYV). In some embodiments, the antibody has an IC50 of less than about 50 nanomolar in a cell adhesion assay performed with human lung fibroblasts and / or mouse fibroblasts. Also described herein is a pharmaceutical composition that comprises the recombinant antibody or antigen-binding fragment of the recombinant antibody and a pharmaceutically acceptable excipient, carrier, or diluent. In some embodiments, the pharmaceutical composition is formulated for intravenous administration. In some embodiments, the pharmaceutical composition is formulated for subcutaneous administration. In some embodiments, the pharmaceutical composition is formulated for intratumoral administration. Also described herein is the recombinant antibody or antigen-binding fragment of the recombinant antibody or the pharmaceutical composition for use in reducing the collagen content in a tumor. In some embodiments, the recombinant antibody or antigen-binding fragment of the recombinant antibody or the pharmaceutical composition is for use in treating cancer. In some embodiments, the cancer includes glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, skin cancer, gastric cancer, mesothelioma, liver cancer, endometrial cancer, colon cancer, cervical cancer, prostate cancer, or lung cancer. Also described herein is a method of reducing the collagen content in a tumor in an individual, which comprises administering to the individual the recombinant antibody or antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. Also described herein is a method of increasing the M1 macrophage phenotype and / or reducing the M2 macrophage phenotype in a tumor in an individual, which comprises administering to the individual the recombinant antibody or antigen-binding fragment of the recombinant antibody or the pharmaceutical composition.The present invention also describes a method for reducing the accumulation of suppressive granulocytic myeloid cells and / or tumor-associated macrophages in an individual, which comprises administering to the individual the recombinant antibody or an antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. The present invention also describes a method for increasing the frequency of CD4+ and / or CD8+ T cells in a tumor in an individual, which comprises administering to the individual the recombinant antibody or an antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. The present invention also describes a method for increasing the function of CD8+ T cells in a tumor as measured by interferon-γ expression and / or release by CD8+ T cells in an individual, which comprises administering to the individual the recombinant antibody or an antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. The present invention also describes a method for treating cancer in an individual, which comprises administering to the individual a therapeutically effective amount of the recombinant antibody or an antigen-binding fragment of the recombinant antibody or the pharmaceutical composition. In some embodiments, the cancer comprises glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, skin cancer, gastric cancer, mesothelioma, liver cancer, endometrial cancer, colon cancer, cervical cancer, prostate cancer or lung cancer. The present invention also describes a method for preparing a composition for reducing the collagen content in a tumor, which comprises mixing the recombinant antibody or an antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier or diluent. The present invention also describes a method for preparing a composition for increasing the M1 macrophage phenotype and / or reducing the M2 macrophage phenotype in a tumor, which comprises mixing the recombinant antibody or an antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier or diluent. The present invention also describes a method for preparing a composition for reducing the accumulation of suppressive granulocytic myeloid cells and / or tumor-associated macrophages in an individual, which comprises mixing the recombinant antibody or an antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier or diluent. The present invention also describes a method for preparing a composition for increasing the frequency of CD4+ and / or CD8+ T cells in a tumor of an individual, which comprises mixing the recombinant antibody or an antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier or diluent. The present invention also describes a method for preparing a composition for increasing the function of CD8+ T cells in a tumor in an individual as measured by interferon-γ expression and / or release by CD8+ T cells, which comprises mixing the recombinant antibody or an antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier or diluent. The present invention also describes a method for preparing a composition for treating cancer, which comprises mixing the recombinant antibody or an antigen-binding fragment of the recombinant antibody with a pharmaceutically acceptable excipient, carrier or diluent.In some embodiments, the cancer includes glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, skin cancer, gastric cancer, mesothelioma, liver cancer, endometrial cancer, colon cancer, cervical cancer, prostate cancer, or lung cancer.

[0008] Also described herein is a nucleic acid encoding any one of the recombinant antibodies or antigen-binding fragments of the recombinant antibodies described above.

[0009] Also described herein is a cell line comprising the nucleic acid described above. In some embodiments, the cell line is a Chinese hamster ovary cell line. Also described herein is a method of producing the recombinant antibody or the antigen-binding fragment of the recombinant antibody, which comprises incubating the cell line in a cell culture medium under conditions sufficient to permit expression and secretion of any one of the recombinant antibody or the antigen-binding fragment of the recombinant antibody. BRIEF DESCRIPTION OF THE DRAWINGS

[0010] The novel features described herein are particularly pointed out in the appended claims. The features and advantages of the features described herein will be better understood by reference to the following detailed description and the accompanying drawings that illustrate illustrative embodiments in which the principles of the features described herein are utilized, in which:

[0011] Figure 1 shows the inhibition of periostin (POSTN)-mediated cell attachment by 78 sequence-unique IgG tested at a single concentration of 500 nM.

[0012] Figure 2 shows tumor growth in a murine MB49 bladder cancer model after treatment with NB0828 or vehicle control.

[0013] Figure 3 shows the effect of NB0828 treatment on intratumoral myeloid cell accumulation. Mice bearing MB49 tumors were treated with NB0828 or vehicle as described in Figure 2. Data are presented as the percentage of total CD45+ immune infiltrate.

[0014] Figure 4 shows the change in total tumor collagen content after treatment with NB0828. Mice bearing MB49 tumors were treated as described in Figure 2, and the total tumor collagen content of the endpoint MB49 tumors was evaluated as described in the methods.

[0015] Figure 5 shows tumor growth in a murine CT26 colon cancer model after treatment with NB0828 or vehicle control.

[0016] Figure 6 shows the reduced intratumoral accumulation of granulocytes / TAMs (tumor-associated macrophages) and the skewing of macrophages towards the M1 phenotype in NB0828-treated mice bearing CT26 tumors.

[0017] Figure 7 shows the increased accumulation of CD8+ and CD4+ tumor-infiltrating lymphocytes (TILs) and enhanced CD8+ TIL function in CT26 tumor-bearing mice treated with NB0828.

[0018] Figure 8 shows tumor growth in the murine MC38 colon cancer model after treatment with NB0828 or vehicle control.

[0019] Figures 9A - 9D illustrate that in the MC38 colon cancer model, NB0828 reduced the total amount of tumor-associated macrophages (9A), while increasing the frequency of pro-inflammatory type I macrophages (9B) and CD8+ T cells (9C), and the antitumor efficacy of NB0828 was dependent on CD8+ T cells (9D).

[0020] Figure 10 shows a schematic diagram for generating a transforming growth factor-β-induced protein (BIGH3) / periostin chimera for epitope mapping studies.

[0021] Figures 11A - 11C show the binding of NB0828 to the FAS2 domain of periostin. 11A shows the binding of NB0828 to the chimeric protein generated in Figure 10, while Figures 11B and 11C show the binding of NB0828 to alanine mutants in the FAS2 domain of POSTN EMI-FAS4.

[0022] Figure 12 shows the crystal structure of dimeric POSTN EMI-FAS4, where the position of the NB0828 epitope is boxed and magnified in the lower half.

[0023] Figures 13A and 13B show the binding EC80 of human tenascin C to periostin and the functional blocking activity of NB0828 (13A), as well as the binding EC80 of human type I collagen to periostin and the functional blocking activity of NB0828 (13B).

[0024] Figure 14A depicts the status of periostin expression in various tumor types as measured by immunohistochemistry.

[0025] Figure 14B shows a representative depiction of IHC staining on breast cancer samples with low, medium, and high periostin expression. Detailed Description

[0026] The present invention describes a recombinant antibody that binds periostin or an antigen-binding fragment of the recombinant antibody, wherein the antibody or the antigen-binding fragment of the antibody comprises: (a) an immunoglobulin heavy chain CDR1 (CDR-H1) that comprises the amino acid sequence shown in SEQ ID NO: 1 (GYTFTSYG); (b) an immunoglobulin heavy chain CDR2 (CDR-H2) that comprises the amino acid sequence shown in any one of SEQ ID NO: 2 (ISAYNGNT), 3 (ISAYSGNT), 4 (ISAYQGNT), 5 (ISAYTGNT), or 6 (ISAYDGNT); (c) an immunoglobulin heavy chain CDR3 (CDR-H3) that comprises the amino acid sequence shown in any one of SEQ ID NO: 7 (DILVVPFDY), 8 (DVLVVPFDY), or 9 (DMLVVPFDY); (d) an immunoglobulin light chain CDR1 (CDR-L1) that comprises the amino acid sequence shown in SEQ ID NO: 10 (SSDIGSNR); (e) an immunoglobulin light chain CDR2 (CDR-L2) that comprises the amino acid sequence shown in SEQ ID NO: 11 (SND); and (f) an immunoglobulin light chain CDR3 (CDR-L3) that comprises the amino acid sequence shown in SEQ ID NO: 12 (AAWDDSLSTYV).

[0027] The present invention describes a recombinant antibody that binds periostin or an antigen-binding fragment of the recombinant antibody, wherein the antibody or the antigen-binding fragment of the antibody comprises any one, two, three, four, five, or six of the following: (a) an immunoglobulin heavy chain CDR1 (CDR-H1) that comprises the amino acid sequence shown in SEQ ID NO: 1 (GYTFTSYG); (b) an immunoglobulin heavy chain CDR2 (CDR-H2) that comprises the amino acid sequence shown in SEQ ID NO: 16 (ISAYXGNT), wherein X is any amino acid residue; (c) an immunoglobulin heavy chain CDR3 (CDR-H3) that comprises the amino acid sequence shown in SEQ ID NO: 17 (DXLVVPFDY), wherein X is any amino acid residue; (d) an immunoglobulin light chain CDR1 (CDR-L1) that comprises the amino acid sequence shown in SEQ ID NO: 10 (SSDIGSNR); (e) an immunoglobulin light chain CDR2 (CDR-L2) that comprises the amino acid sequence shown in SEQ ID NO: 11 (SND); and (f) an immunoglobulin light chain CDR3 (CDR-L3) that comprises the amino acid sequence shown in SEQ ID NO: 12 (AAWDDSLSTYV).

[0028] The present disclosure describes a recombinant antibody that binds periostin or an antigen-binding fragment of the recombinant antibody, the recombinant antibody or the antigen-binding fragment of the recombinant antibody comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region: (a) wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13; and (b) wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 14; wherein amino acid residue number 55 of SEQ ID NO: 13 is asparagine, serine, glutamine, threonine, or aspartic acid, or wherein amino acid residue number 100 of SEQ ID NO: 13 is methionine, isoleucine, or valine.

[0029] The present disclosure describes a recombinant antibody that binds periostin or an antigen-binding fragment of the recombinant antibody, the recombinant antibody or the antigen-binding fragment of the recombinant antibody comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region: (a) wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13; and (b) wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 14.

[0030] The present disclosure describes a recombinant antibody that binds periostin or an antigen-binding fragment of the recombinant antibody, the recombinant antibody or the antigen-binding fragment of the recombinant antibody comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region: (a) wherein the immunoglobulin heavy chain variable region comprises an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 13; and (b) wherein the immunoglobulin light chain variable region comprises an amino acid sequence that is identical to the amino acid sequence set forth in SEQ ID NO: 14.

[0031] This document describes a recombinant antibody or an antigen-binding fragment of the recombinant antibody that binds to the fasciclin 2 (FAS2) domain of periostin. In certain embodiments, the recombinant antibody or the antigen-binding fragment of the recombinant antibody contacts amino acid residues 276 to 302 of SEQ ID NO:15 when binding to periostin. In certain embodiments, the recombinant antibody or the antigen-binding fragment of the recombinant antibody contacts one of the following amino acid residues of SEQ ID NO:15: N276, R284, E288, L287, V295, or K302.

[0032] In the following description, certain specific details are set forth in order to provide a thorough understanding of the various embodiments. However, those skilled in the art will understand that the provided embodiments may be practiced without these details. Unless the context otherwise requires, throughout the specification and the following claims, the word "comprise" and its variants such as "comprises" and "comprising" shall be interpreted in an open, inclusive sense, i.e., "including but not limited to". Unless the context clearly dictates otherwise, as used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents. It should also be noted that, unless the content clearly dictates otherwise, the term "or" is generally used in its meaning that includes "and / or". In addition, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0033] As used herein, the term "about" refers to an amount that is within 10% or less of the stated amount.

[0034] As used herein, the terms "individual", "patient", or "subject" refer to an individual who has been diagnosed with, suspected of having, or at risk of developing at least one disease for which the compositions and methods described herein can be used for treatment. In certain embodiments, the individual is a mammal. In certain embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. In certain embodiments, the individual is a human.

[0035] As used herein, the term "treat" or "treating" refers to an intervention in the physiological or disease state of an individual that is designed or intended to alleviate at least one sign or symptom associated with the physiological or disease state. Skilled artisans will recognize that, given the heterogeneous population of individuals suffering from a disease, not all individuals will respond equally or completely to a given treatment. Regardless of any objective response criteria, an individual is considered to have been treated.

[0036] The provided antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies and polyreactive antibodies), and antibody fragments. Antibodies include antibody conjugates and molecules comprising antibodies (such as chimeric molecules). Thus, antibodies include, but are not limited to, full-length and native antibodies, as well as fragments and portions thereof that retain their binding specificity, such as any of their specific binding portions, including those having any number of immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM); and biologically relevant (antigen-binding) fragments or specific binding portions, including but not limited to Fab, F(ab')2, Fv, and scFv (single-chain or related entities). Monoclonal antibodies are typically a single one within a composition of substantially homogeneous antibodies; thus, any individual antibody contained within a monoclonal antibody composition is identical, except for possible naturally occurring mutations that may be present in small amounts. Polyclonal antibodies are preparations that include diverse antibodies that typically target two or more different determinants (epitopes). Monoclonal antibodies can comprise a human IgG1 constant region. Monoclonal antibodies can comprise a human IgG4 constant region.

[0037] The term "antibody" as used herein is used in the broadest sense and includes polyclonal and monoclonal antibodies, including intact antibodies and their functional (antigen-binding) antibody fragments, including fragment antigen-binding (Fab) fragments, F(ab')2 fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single-chain antibody fragments (including single-chain variable fragments (sFv or scFv)), and single-domain antibody (e.g., sdAb, sdFv, nanobody) fragments. The term encompasses genetically engineered and / or otherwise modified forms of immunoglobulins, such as intracellular antibodies, peptide antibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific (e.g., bispecific) antibodies, diabodies, triabodies, and tetra-bodies, tandem di-scFv, tandem tri-scFv. Unless otherwise indicated, the term "antibody" should be understood to encompass its functional antibody fragments. The term also encompasses intact or full-length antibodies, including antibodies of any class or subclass (including IgG and its subclasses, IgM, IgE, IgA, and IgD). Antibodies can comprise a human IgG1 constant region. Antibodies can comprise a human IgG4 constant region.

[0038] The terms "complementary determining region" and "CDR" (which are synonymous with "hypervariable region" or "HVR") are known in the art to refer to non-contiguous sequences of amino acids within the variable regions of antibodies that confer antigen specificity and / or binding affinity. Typically, there are three CDRs (CDR-H1, CDR-H2, CDR-H3) in each heavy chain variable region, and three CDRs (CDR-L1, CDR-L2, CDR-L3) in each light chain variable region. "Framework region" and "FR" are known in the art to refer to the non-CDR portions of the variable regions of heavy and light chains. Typically, there are four FRs (FR-H1, FR-H2, FR-H3, and FR-H4) in each full-length heavy chain variable region, and four FRs (FR-L1, FR-L2, FR-L3, and FR-L4) in each full-length light chain variable region.The exact amino acid sequence boundaries of a given CDR or FR can be readily determined using any of a number of well-known schemes, including those described in the following: Kabat et al. (1991), “Sequences of Proteins of Immunological Interest,” 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (“Kabat” numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (“Chothia” numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996), “Antibody-antigen interactions: Contact analysis and binding site topography,” J. Mol. Biol. 262, 732-745. (“Contact” numbering scheme); Lefranc MP et al., “IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, January 2003; 27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A, “Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” J Mol Biol, June 8, 2001; 309(3):657-70 (“Aho” numbering scheme); and Whitelegg NR and Rees AR, “WAM: an improved algorithm for modelling antibodies on the WEB,” Protein Eng. December 2000; 13(12):819-24 (“AbM” numbering scheme).

[0039] The boundaries of the given CDRs or FRs may vary according to the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The numbering of both the Kabat and Chothia schemes is based on the most common antibody region sequence lengths, where insertions are provided by inserting letters (e.g., "30a"), and deletions occur in some antibodies. These two schemes place certain insertions and deletions ("indels") in different positions, resulting in different numberings. The Contact scheme is based on the analysis of complex crystal structures and is similar to the Chothia numbering scheme in many respects.

[0040] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that participates in the binding of the antibody to an antigen. The variable domains of the heavy and light chains of a native antibody (V H and V L ) generally have similar structures, where each domain contains four conserved framework regions (FRs) and three CDRs (see, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., p. 91 (2007)). A single V H or V L domain may be sufficient to confer antigen-binding specificity. In addition, V H or V L domains from antibodies that bind a particular antigen can be used to isolate antibodies that bind the antigen, to separately screen libraries of complementary V L or V H domains (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991)).

[0041] The provided antibodies include antibody fragments. An "antibody fragment" refers to a molecule different from a full antibody that contains a portion of the full antibody that binds the antigen to which the full antibody binds. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv or sFv); and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single-chain antibody fragment that comprises a variable heavy chain region and / or a variable light chain region, such as scFv.

[0042] As used herein, the term "specifically binds" or "binds" refers to binding mediated by one or more amino acid residues of the CDRs of the recited antibody or fragment, or one or more variable region amino acid residues of the recited antibody or fragment. As used herein, the terms "contact" or "contacts" with respect to antibody binding or binding to a particular target refer to amino acid residues of the variable region or CDRs being within 5, 4, 3 or fewer angstroms of the recited contact residues. Contact includes hydrogen bonding, van der Waals interactions, and salt bridge formation between amino acid residues of the variable region or CDRs of the antibody and the recited residues.

[0043] Antibody fragments can be prepared by a variety of techniques including, but not limited to, proteolytic digestion of intact antibodies and production by recombinant host cells. In some embodiments, the antibody is a recombinantly produced fragment, such as those that contain arrangements not found in nature (e.g., those having two or more antibody domains or chains linked by synthetic linkers (e.g., polypeptide linkers)), and / or those not produced by enzymatic digestion of intact antibodies found in nature. In some aspects, the antibody fragment is a scFv.

[0044] A "humanized" antibody is one in which all or substantially all of the CDR amino acid residues are derived from non-human CDRs and all or substantially all of the FR amino acid residues are derived from human FRs. A humanized antibody optionally may include at least a portion of the antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody refers to a variant of the non-human antibody that has been humanized to generally reduce its immunogenicity in humans while retaining the specificity and affinity of the parental non-human antibody. In some embodiments, some FR residues in the humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), for example to restore or improve antibody specificity or affinity.

[0045] The provided antibodies include human antibodies. A "human antibody" is an antibody having an amino acid sequence corresponding to an amino acid sequence of an antibody produced by a human or human cell or a non-human source that utilizes a human antibody library or other human antibody-encoding sequences (including human antibody libraries). The term does not include humanized forms of non-human antibodies that contain non-human antigen-binding regions, e.g., those in which all or substantially all of the CDRs are non-human CDRs.

[0046] Human antibodies can be prepared by administering an immunogen to a transgenic animal that has been modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen stimulation. Such animals typically contain all or part of the human immunoglobulin locus, which replaces the endogenous immunoglobulin locus, or which is present episomally or randomly integrated into the chromosomes of the animal. In such transgenic animals, the endogenous immunoglobulin locus is typically inactivated. Human antibodies can also be derived from or selected from human antibody libraries, including phage display and cell-free libraries, which contain antibody-encoding sequences derived from a human repertoire. In certain embodiments, human antibodies can be subjected to successive rounds of selection by methods such as phage display to remove sequence susceptibility or increase their affinity.

[0047] The terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides (including the provided antibodies and antibody chains and other peptides (e.g., linkers and binding peptides)) can include amino acid residues containing natural and / or non-natural amino acid residues. The term also includes post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. In some aspects, polypeptides can contain modifications with respect to the native or natural sequence, so long as the protein retains the desired activity. These modifications may be intentional (e.g., by site-directed mutagenesis) or may be accidental (e.g., by mutations in the host producing the protein or due to errors in PCR amplification).

[0048] In certain embodiments, antibodies with reduced effector function are provided herein. As used herein, the phrase "effector function" is intended to include the functional capabilities conferred by Fc-containing proteins upon binding to FcγRs. Without being bound by any one theory, the formation of the Fc / FcγR complex recruits a variety of effector cells to the site of antigen binding, typically resulting in different intracellular signaling events and important subsequent immune responses. Effector function refers to both antibody-dependent cell cytotoxicity and complement-dependent cell cytotoxicity. In vitro and / or in vivo cytotoxicity assays can be performed to confirm a reduction / abrogation of CDC and / or ADCC activity. For example, an Fc receptor (FcR) binding assay can be performed to ensure that the antibody lacks FcγR binding (and thus may lack ADCC activity), but retains FcRn binding ability. Non-limiting examples of in vitro assays for evaluating the ADCC activity of a test molecule are described in U.S. Patent Nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive assay methods (e.g., ACTI TM and CytoTox non-radioactive cytotoxicity assays) can be employed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

[0049] The percent sequence identity (%) with respect to a reference polypeptide sequence is the percentage of amino acid residues in the candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence after aligning the sequences to achieve the maximum percent sequence identity and introducing gaps (if necessary) and not considering any conservative substitutions as part of the sequence identity. The alignment for the purpose of determining the percent amino acid sequence identity can be achieved in a variety of known ways, such as using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning the sequences can be determined, including the algorithms required to achieve the maximum alignment over the full length of the sequences being compared. However, for the purposes herein, the amino acid sequence identity % values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was written by Genentech, Inc., and the source code has been submitted in the user documentation to the U.S. Copyright Office, Washington, 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or can be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system (including Digital UNIX V4.0D). All sequence comparison parameters are set by the ALIGN-2 program and are not changed.

[0050] In the case of amino acid sequence comparison using ALIGN-2, the percent amino acid sequence identity of a given amino acid sequence A with (to, with or against) a given amino acid sequence B (which may alternatively be phrased as a given amino acid sequence A having or containing a certain percent amino acid sequence identity with (to, with or against) a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches in the alignment of A and B by the sequence alignment program ALIGN-2, and where Y is the total number of amino acid residues in B. It will be understood that in the case where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A with B will not be equal to the percent amino acid sequence identity of B with A. Unless otherwise expressly stated, all percent amino acid sequence identity values used herein are obtained using the ALIGN-2 computer program as described in the immediately preceding paragraph.

[0051] In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. Variants generally differ from the polypeptides expressly disclosed herein by one or more substitutions, deletions, additions, and / or insertions. Such variants can be naturally occurring or can be synthetically produced, e.g., by modifying one or more of the polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptides as described herein, and / or using any of a number of known techniques. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct has the desired characteristics (e.g., antigen binding).

[0052] In some embodiments, antibody variants having one or more amino acid substitutions are provided. Target sites for mutagenesis by substitution include CDRs and FRs. Amino acid substitutions can be introduced into the antibody of interest, and the desired activities of the product can be screened, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0053] In some embodiments, substitutions, insertions, or deletions can occur within one or more CDRs, where the substitution, insertion, or deletion does not substantially reduce the binding of the antibody to the antigen. For example, conservative substitutions that do not substantially reduce the binding affinity can be made in the CDRs. Such alterations can be outside of CDR "hot spots". In some embodiments of the variant V H and V L sequences, each CDR is unaltered.

[0054] Changes (e.g., substitutions) can be made in the CDRs, e.g., to improve antibody affinity. Such changes can occur at a higher mutation rate in the CDR-encoding codons during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and the binding affinities of the resulting variants can be tested. Affinity maturation (e.g., using error-prone PCR, chain shuffling, randomization of CDRs, or oligonucleotide-directed mutagenesis) can be used to improve antibody affinity (see, e.g., Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (2001)). CDR residues involved in antigen binding can be specifically identified, e.g., using alanine-scanning mutagenesis or modeling (see, e.g., Cunningham and Wells Science, 244:1081-1085 (1989)). In particular, CDR-H3 and CDR-L3 are typically targeted. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact points between the antibody and the antigen. Such contact residues and neighboring residues can be targeted or eliminated as substitution candidates. Variants can be screened to determine whether they possess the desired properties.

[0055] Amino acid sequence insertions and deletions include amino- and / or carboxyl-terminal fusions of polypeptides ranging in length from one residue to polypeptides containing one hundred or more residues, as well as in-sequence insertions and deletions of single or multiple amino acid residues. Examples of terminal insertions include antibodies having an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N-terminal or C-terminal of the antibody with an enzyme (e.g., for ADEPT) or a polypeptide that increases the serum half-life of the antibody. Examples of in-sequence insertion variants of antibody molecules include the insertion of three amino acids in the light chain. Examples of terminal deletions include antibodies having a deletion of seven or fewer amino acids at the light chain terminus.

[0056] In some embodiments, the antibody is altered to increase or decrease its glycosylation (e.g., by altering the amino acid sequence such that one or more glycosylation sites are created or removed). The carbohydrates attached to the Fc region of the antibody can be altered. Native antibodies from mammalian cells typically contain branched biantennary oligosaccharides that are attached by an N-linkage to Asn in the CH2 domain of the Fc region 297(See, e.g., Wright et al., TIBTECH 15:26-32 (1997)). Oligosaccharides can be various carbohydrates such as mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, fucose attached to GlcNAc in the stem of a biantennary oligosaccharide structure. For example, the oligosaccharides in an antibody can be modified to produce antibody variants with certain improved properties. Antibody glycosylation variants can have improved ADCC and / or CDC function. In some embodiments, antibody variants are provided that have a carbohydrate structure lacking (directly or indirectly) fucose attached to the Fc region. For example, the amount of fucose in such an antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn 297 relative to the sum of all sugar structures attached to Asn297 (see, e.g., WO 08 / 077546). Asn 297 refers to the asparagine residue located at approximately position 297 in the Fc region (EU numbering of Fc region residues; see, e.g., Edelman et al., Proc Natl Acad Sci U S A. May 1969; 63(1):78–85). However, due to minor sequence variations in the antibody, Asn 297 can also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated variants can have improved ADCC function (see, e.g., Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); and Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004)). Cell lines (e.g., knockout cell lines) and methods of using them can be used to produce defucosylated antibodies, such as Lec13 CHO cells lacking protein fucosylation and α-1,6-fucosyltransferase gene (FUT8) knockout CHO cells (see, e.g., Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006)). Also included are other antibody glycosylation variants (see, e.g., U.S. Patent No. 6,602,684).

[0057] In some embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody provided herein to generate an Fc region variant. The Fc region herein is the C-terminal region of an immunoglobulin heavy chain containing at least a portion of the constant region. The Fc region includes the native sequence Fc region and variant Fc regions. Fc region variants can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains amino acid modifications (e.g., substitutions) at one or more amino acid positions.

[0058] Antibodies can have an increased half-life and improved binding to the neonatal Fc receptor (FcRn) (see, e.g., US2005 / 0014934). Such antibodies can comprise an Fc region having one or more substitutions that improve binding of the Fc region to FcRn and include those having substitutions at one or more of the following Fc region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424, or 434 according to the EU numbering system (see, e.g., U.S. Patent No. 7,371,826). Other examples of Fc region variants are also contemplated (see, e.g., Duncan and Winter, Nature 322:738-40 (1988); U.S. Patent Nos. 5,648,260 and 5,624,821; and WO 94 / 29351).

[0059] In some embodiments, it may be desirable to generate cysteine-engineered antibodies, such as “thioMAb,” in which one or more residues of the antibody are replaced with cysteine residues. In some embodiments, the residues to be replaced are at accessible sites of the antibody. The reactive thiol groups can be positioned at sites for conjugation with other moieties, such as a drug moiety or a linker-drug moiety, to generate an immunoconjugate. In some embodiments, any one or more of the following residues can be replaced with cysteine: V205 (Kabat numbering) of the light chain; A118 (EU numbering) of the heavy chain; and S400 (EU numbering) of the heavy chain Fc region.

[0060] In some embodiments, the antibodies provided herein can be further modified to contain additional non-protein moieties that are known and available. Moieties suitable for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol / propanediol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, poly(propylene oxide / ethylene oxide) copolymer, poly(oxyethylated polyols) (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer can have any molecular weight and can be branched or unbranched. The number of polymers attached to the antibody can vary, and if two or more polymers are attached, they can be the same or different molecules.

[0061] The antibodies described herein can be encoded by nucleic acids. A nucleic acid is a type of polynucleotide that contains two or more nucleotide bases. In certain embodiments, the nucleic acid is a component of a vector that can be used to transfer a polypeptide-encoding polynucleotide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a genomic integration vector or "integrating vector", which can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, such as a nucleic acid capable of extrachromosomal replication. A vector capable of directing the expression of a gene to which it is operably linked is referred to herein as an "expression vector". Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, etc. In an expression vector, regulatory elements for controlling transcription such as promoters, enhancers, polyadenylation signals can be derived from mammalian, microbial, viral, or insect genes. The ability to replicate in a host (usually conferred by an origin of replication) and a selectable gene for facilitating the identification of transformants can also be incorporated additionally. Vectors derived from viruses such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, etc. can be used. A plasmid vector can be linearized for integration into a chromosomal location. The vector can contain sequences that direct site-specific integration into a defined location or a restricted set of sites in the genome (e.g., AttP-AttB recombination). Additionally, the vector can contain sequences derived from transposable elements.

[0062] As used herein, the terms “homologous,” “homology,” or “percent homology” when used herein to describe an amino acid sequence or nucleic acid sequence relative to a reference sequence can be determined using the formula described below: Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, as modified in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). This formula is incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). The percent homology of a sequence can be determined using the most recent version of BLAST as of the filing date of this application.

[0063] The nucleic acid encoding the antibodies described herein can be used to infect, transfect, transform suitable cells or otherwise render suitable cells nucleic acid transgenic, thereby enabling the production of antibodies for commercial or therapeutic use. Standard cell lines and methods for producing antibodies from large-scale cell culture are known in the art. See, e.g., Li et al., “Cell culture processes for monoclonal antibody production.” Mabs. September–October 2010;2(5):466–477. In certain embodiments, the cells are eukaryotic cells. In certain embodiments, the eukaryotic cells are mammalian cells. In certain embodiments, the mammalian cells are Chinese hamster ovary (CHO) cells, NS0 murine myeloma cells or cells. In certain embodiments, the nucleic acid encoding the antibody is integrated into a genomic locus of a cell that can be used to produce the antibody. In certain embodiments, methods for preparing antibodies are described herein that include culturing in vitro a cell comprising a nucleic acid encoding the antibody under conditions sufficient to permit production and secretion of the antibody.

[0064] In certain embodiments, described herein is a master cell bank comprising: (a) a mammalian cell line comprising one or more nucleic acids encoding an antibody as described herein integrated at a genomic locus; and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol, DMSO, or a combination thereof. In certain embodiments, the master cell bank comprises: (a) a CHO cell line comprising a nucleic acid encoding an antibody integrated at a genomic locus, the antibody having (i) a heavy chain amino acid sequence that is at least 90% identical to the heavy chain amino acid sequence shown in SEQ ID NO: 13; (ii) a light chain amino acid sequence that is at least 90% identical to the light chain amino acid sequence shown in SEQ ID NO: 14; and (b) a cryoprotectant. In certain embodiments, the cryoprotectant comprises glycerol, DMSO, or a combination thereof. In certain embodiments, the master cell bank is contained in a suitable vial or container capable of withstanding liquid nitrogen freezing.

[0065] Also described herein are methods of making the antibodies described herein. Such methods include incubating a cell or cell line comprising a nucleic acid encoding an antibody in cell culture medium under conditions sufficient to permit antibody expression and secretion, and further harvesting the antibody from the cell culture medium. The harvesting may also include one or more purification steps to remove live cells, cell debris, non-antibody proteins or polypeptides, unwanted salts, buffers, and culture components. In certain embodiments, one or more additional purification steps include centrifugation, ultracentrifugation, dialysis, desalting, protein A, protein G, protein A / G, or protein L purification, and / or ion exchange chromatography.

[0066] Anti-periostin antibody

[0067] This document describes antibodies that inhibit the function of periostin (POSTN). Such antibodies can be used to treat cancer. The antibodies described herein reduce the collagen content of tumors, decrease the infiltration of granulocytes and tumor-associated macrophages while increasing the polarization of macrophages towards the M1 phenotype, and increase the accumulation and anti-tumor properties of tumor-infiltrating T cells. In certain embodiments, the anti-periostin antibody reduces the tumor collagen content by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% compared to untreated or control treatment. In certain embodiments, the anti-periostin antibody reduces the infiltration of granulocytes and tumor-associated macrophages by at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to untreated or control treatment. In certain embodiments, the anti-periostin antibody reduces the infiltration of CD11b+ cells by at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to untreated or control treatment. In certain embodiments, the anti-periostin antibody increases the polarization of tumor-associated macrophages towards the M1 type (CD11b+, MHC class II+, CD206-) by at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to untreated or control treatment. In certain embodiments, the anti-periostin antibody increases the accumulation of CD4+ and / or CD8+ T cells in the tumor by at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to untreated or control treatment. In certain embodiments, the anti-periostin antibody increases the production of interferon γ by tumor-infiltrating CD8+ T cells by at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to untreated or control treatment.

[0068] The present invention describes a recombinant antibody that binds periostin or an antigen-binding fragment of said recombinant antibody, wherein the antibody or the antigen-binding fragment of the antibody comprises: (a) an immunoglobulin heavy chain CDR1 (CDR-H1) that comprises the amino acid sequence shown in SEQ ID NO: 1 (GYTFTSYG); (b) an immunoglobulin heavy chain CDR2 (CDR-H2) that comprises the amino acid sequence shown in SEQ ID NO: 16 (ISAYXGNT); (c) an immunoglobulin heavy chain CDR3 (CDR-H3) that comprises the amino acid sequence shown in SEQ ID NO: 17 (DXLVVPFDY); (d) an immunoglobulin light chain CDR1 (CDR-L1) that comprises the amino acid sequence shown in SEQ ID NO: 10 (SSDIGSNR); (e) an immunoglobulin light chain CDR2 (CDR-L2) that comprises the amino acid sequence shown in SEQ ID NO: 11 (SND); or (f) an immunoglobulin light chain CDR3 (CDR-L3) that comprises the amino acid sequence shown in SEQ ID NO: 12 (AAWDDSLSTYV); wherein X is any amino acid.

[0069] The present invention describes a recombinant antibody that binds periostin or an antigen-binding fragment of said recombinant antibody, wherein the antibody or the antigen-binding fragment of the antibody comprises any one, two, three, four, or five of the following complementary determining regions: (a) an immunoglobulin heavy chain CDR1 (CDR-H1) that comprises the amino acid sequence shown in SEQ ID NO: 1 (GYTFTSYG); (b) an immunoglobulin heavy chain CDR2 (CDR-H2) that comprises the amino acid sequence shown in SEQ ID NO: 16 (ISAYXGNT); (c) an immunoglobulin heavy chain CDR3 (CDR-H3) that comprises the amino acid sequence shown in SEQ ID NO: 17 (DXLVVPFDY); (d) an immunoglobulin light chain CDR1 (CDR-L1) that comprises the amino acid sequence shown in SEQ ID NO: 10 (SSDIGSNR); (e) an immunoglobulin light chain CDR2 (CDR-L2) that comprises the amino acid sequence shown in SEQ ID NO: 11 (SND); and (f) an immunoglobulin light chain CDR3 (CDR-L3) that comprises the amino acid sequence shown in SEQ ID NO: 12 (AAWDDSLSTYV); wherein X is any amino acid.

[0070] The present disclosure describes a recombinant antibody or an antigen-binding fragment of the recombinant antibody that binds periostin, wherein the antibody or the antigen-binding fragment of the antibody comprises: (a) an immunoglobulin heavy chain CDR1 (CDR-H1) that comprises the amino acid sequence shown in SEQ ID NO:1 (GYTFTSYG); (b) an immunoglobulin heavy chain CDR2 (CDR-H2) that comprises the amino acid sequence shown in any one of SEQ ID NO:2 (ISAYNGNT), 3 (ISAYSGNT), 4 (ISAYQGNT), 5 (ISAYTGNT), or 6 (ISAYDGNT); (c) an immunoglobulin heavy chain CDR3 (CDR-H3) that comprises the amino acid sequence shown in any one of SEQ ID NO:7 (DILVVPFDY), 8 (DVLVVPFDY), or 9 (DMLVVPFDY); (d) an immunoglobulin light chain CDR1 (CDR-L1) that comprises the amino acid sequence shown in SEQ ID NO:10 (SSDIGSNR); (e) an immunoglobulin light chain CDR2 (CDR-L2) that comprises the amino acid sequence shown in SEQ ID NO:11 (SND); and (f) an immunoglobulin light chain CDR3 (CDR-L3) that comprises the amino acid sequence shown in SEQ ID NO:12 (AAWDDSLSTYV). In certain embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In certain embodiments, the antibody is an IgG antibody. In certain embodiments, the antibody described herein may comprise an Fc portion having reduced or lacking effector function. In certain embodiments, the antibody has an IC50 of less than about 50 nanomolar in a cell adhesion assay performed with human lung fibroblasts and / or mouse fibroblasts. In certain embodiments, the antibody has an IC50 of less than about 40 nanomolar in a cell adhesion assay performed with human lung fibroblasts and / or mouse fibroblasts. In certain embodiments, the antibody has an IC50 of less than about 30 nanomolar in a cell adhesion assay performed with human lung fibroblasts and / or mouse fibroblasts.

[0071] The present disclosure also describes recombinant antibodies or antigen-binding fragments, wherein the antibody or the antigen-binding fragment of the antibody comprises: (a) an immunoglobulin heavy chain CDR1 (CDR-H1) that comprises the amino acid sequence set forth in SEQ ID NO:1 (GYTFTSYG); (b) an immunoglobulin heavy chain CDR2 (CDR-H2) that comprises the amino acid sequence set forth in SEQ ID NO:2 (ISAYNGNT); (c) an immunoglobulin heavy chain CDR3 (CDR-H3) that comprises the amino acid sequence set forth in SEQ ID NO:9 (DMLVVPFDY); (d) an immunoglobulin light chain CDR1 (CDR-L1) that comprises the amino acid sequence set forth in SEQ ID NO:10 (SSDIGSNR); (e) an immunoglobulin light chain CDR2 (CDR-L2) that comprises the amino acid sequence set forth in SEQ ID NO:11 (SND); and (f) an immunoglobulin light chain CDR3 (CDR-L3) that comprises the amino acid sequence set forth in SEQ ID NO:12 (AAWDDSLSTYV). In certain embodiments, the antibody is a human antibody, a humanized antibody, or a chimeric antibody. In certain embodiments, the antibody is an IgG antibody. In certain embodiments, the antibodies described herein may comprise an Fc portion having reduced or lacking effector function. In certain embodiments, the antibody has an IC50 of less than about 50 nanomolar in a cell adhesion assay performed with human lung fibroblasts and / or mouse fibroblasts. In certain embodiments, the antibody has an IC50 of less than about 40 nanomolar in a cell adhesion assay performed with human lung fibroblasts and / or mouse fibroblasts. In certain embodiments, the antibody has an IC50 of less than about 30 nanomolar in a cell adhesion assay performed with human lung fibroblasts and / or mouse fibroblasts.

[0072] The present disclosure also describes a recombinant antibody or an antigen-binding fragment of the recombinant antibody that binds periostin, the recombinant antibody or the antigen-binding fragment of the recombinant antibody comprising an immunoglobulin heavy chain and an immunoglobulin light chain: (a) wherein the immunoglobulin heavy chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 13; and (b) wherein the immunoglobulin light chain comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99%, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 14, wherein amino acid residue number 55 of SEQ ID NO: 13 is asparagine, serine, glutamine, threonine, or aspartic acid, and wherein amino acid residue number 100 of SEQ ID NO: 13 is methionine, isoleucine, or valine. In certain embodiments, the antibody is an IgG antibody. In certain embodiments, the antibodies described herein may comprise an Fc portion having reduced or lacking effector function. In certain embodiments, the antibody has an IC50 of less than about 50 nanomolar in a cell adhesion assay performed with human lung fibroblasts and / or mouse fibroblasts. In certain embodiments, the antibody has an IC50 of less than about 40 nanomolar in a cell adhesion assay performed with human lung fibroblasts and / or mouse fibroblasts. In certain embodiments, the antibody has an IC50 of less than about 30 nanomolar in a cell adhesion assay performed with human lung fibroblasts and / or mouse fibroblasts.

[0073] The antibody binding regions described herein (including variable regions and CDR regions) can be appropriately formatted as part of an antibody with reduced effector function. In certain embodiments, the antibody can be an F(ab')2 lacking the Fc region. In certain embodiments, the antibody can comprise one or more mutations in the constant region of the antibody heavy chain that reduce effector function, such as antibody-dependent cytotoxicity or complement-dependent cytotoxicity. In certain embodiments, the antibody can comprise an IgG4 constant region. In certain embodiments, the one or more mutations that reduce one or more effector functions of the recombinant antibody or the antigen-binding fragment of the recombinant antibody include mutations or groups of mutations selected from: N434A, N434H, T307A / E380A / N434A, M252Y / S254T / T256E, 433K / 434F / 436H, T250Q, T250F, M428L, M428F, T250Q / M428L, N434S, V308W, V308Y, V308F, M252Y / M428L, D259I / V308F, M428L / V308F, Q311V / N434S, T307Q / N434A, E258F / V427T, S228P, L235E, S228P / L235E / R409K, S228P / L235E, K370Q, K370E, deletion of G446, deletion of K447, and any combination thereof according to the EU numbering system for IgG4. In certain embodiments, the antibody can comprise an IgG4 constant region having a mutation corresponding to S228P of the heavy chain according to the EU numbering system. In certain embodiments, the antibody can comprise an IgG4PAA constant region having mutations corresponding to S228P, F234A, and L235A of the heavy chain according to the EU numbering system. See Parekh et al., “Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay.” MAbs May 1, 2012; 4(3):310-318.

[0074] In some embodiments, the antibodies described herein exhibit a reduced affinity for Clq relative to the corresponding wild-type antibody. In some embodiments, the antibody exhibits an affinity for the Clq receptor that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold lower than that of the corresponding wild-type antibody.

[0075] In some embodiments, the antibodies described herein exhibit at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, at least 40%, at least 30%, at least 20%, at least 10%, or at least 5% lower affinity for Clq than the corresponding wild-type antibodies. In some embodiments, the antibodies described herein exhibit an affinity for Clq between about 100 nM and about 100 μM, or about 100 nM and about 10 μM, or about 100 nM and about 1 μM, or about 1 nM and about 100 μM, or about 10 nM and about 100 μM, or about 1 μM and about 100 μM, or about 10 μM and about 100 μM. In some embodiments, the antibodies described herein exhibit an affinity for Clq greater than 1 μM, greater than 5 μM, greater than 10 μM, greater than 25 μM, greater than 50 μM, or greater than 100 μM.

[0076] In some embodiments, the antibodies described herein exhibit reduced CDC activity compared to the corresponding wild-type Fc antibodies. In some embodiments, the antibodies described herein exhibit at least 2-fold, or at least 3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least 50-fold, or at least 100-fold lower CDC activity than the CDC activity of the corresponding wild-type antibodies. In some embodiments, relative to the corresponding wild-type antibodies, the antibodies described herein exhibit at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 200%, or at least 300%, or at least 400%, or at least 500% reduction in CDC activity. In some embodiments, the antibodies described herein do not exhibit detectable CDC activity. In some embodiments, the reduction and / or elimination of CDC activity can be attributed to a reduced affinity of the antibodies described herein for Fc ligands and / or receptors.

[0077] It will be understood in the art that biotherapies can have adverse toxicity issues related to the complex nature of guiding the immune system to recognize and attack unwanted cells and / or targets. Consequences such as adverse toxicity can occur when recognition and / or targeted attack do not occur where treatment is needed. For example, antibody staining of non-target tissues can indicate potential toxicity issues. In some embodiments, the antibodies described herein exhibit reduced non-target tissue staining compared to the corresponding wild-type antibodies. In some embodiments, the antibodies described herein exhibit reduced non-target tissue staining that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold lower than the non-target tissue staining of the corresponding wild-type antibody. In some embodiments, the antibodies described herein exhibit reduced non-target tissue staining that is at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 200%, or at least 300%, or at least 400%, or at least 500% less than the non-target tissue staining of the corresponding wild-type antibody.

[0078] In some embodiments, the antibodies described herein exhibit reduced antibody-related toxicity compared to the corresponding wild-type antibodies. In some embodiments, the antibodies described herein exhibit toxicity that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold lower than the toxicity of the corresponding wild-type antibody. In some embodiments, the antibodies described herein exhibit at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 200%, or at least 300%, or at least 400%, or at least 500% reduced toxicity compared to the corresponding wild-type antibody.

[0079] It will be understood in the art that biotherapeutics may have adverse effects on the aggregation of coagulation cells. In vitro and in vivo assays can be used to measure the aggregation of coagulation cells. In some embodiments, the antibodies described herein exhibit reduced coagulation cell aggregation in in vitro assays compared to the corresponding wild-type antibodies. In some embodiments, the antibodies described herein exhibit reduced coagulation cell aggregation in in vitro assays that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold lower than the coagulation cell aggregation of the corresponding wild-type antibodies. In some embodiments, the antibodies described herein exhibit reduced coagulation cell aggregation in in vitro assays that is at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 200%, or at least 300%, or at least 400%, or at least 500% less than the corresponding wild-type antibodies.

[0080] In some embodiments, the antibodies described herein exhibit reduced in vivo coagulation cell aggregation compared to the corresponding wild-type antibodies. In some embodiments, the antibodies described herein exhibit reduced coagulation cell aggregation in in vivo assays that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold lower than the coagulation cell aggregation of the corresponding wild-type antibodies. In some embodiments, the antibodies described herein exhibit reduced coagulation cell aggregation in in vivo assays that is at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 200%, or at least 300%, or at least 400%, or at least 500% less than the corresponding wild-type antibodies.

[0081] In some embodiments, the antibodies described herein exhibit reduced platelet activation and / or platelet aggregation compared to the corresponding wild-type antibodies.

[0082] Epitopes bound by therapeutically useful periostin antibodies

[0083] This document describes unique epitopes or regions of human periostin that, upon binding, inhibit the biological activity of periostin (e.g., integrin-mediated cell attachment) and alter the tumor microenvironment (collagen remodeling and changes in immune cells). This binding is a combination of weak (van der Waals attraction), moderate (hydrogen bonding), and strong (salt bridge) interactions between antibody CDR amino acid residues and amino acid residues in periostin (e.g., contact residues). In certain embodiments, the contact residues are residues on periostin that form hydrogen bonds with residues on the anti-periostin antibody. In certain embodiments, the contact residues are residues on periostin that form salt bridges with residues on the anti-periostin antibody. In certain embodiments, the contact residues are residues on periostin that produce van der Waals attraction with residues on the anti-periostin antibody and are within at least 5, 4, or 3 angstroms of the residues on the anti-periostin antibody.

[0084] In certain embodiments, the anti-periostin antibodies described herein do not bind to tenascin C or type I collagen.

[0085] In certain embodiments, this document describes isolated antibodies that bind any one, two, three, four, five, or six of the following residues of periostin (SEQ ID NO:15): N276, R284, E288, L287, V295, or K302. In certain embodiments, this document describes isolated antibodies that bind all of the following residues of SEQ ID NO:15: N276, R284, E288, L287, V295, or K302. In certain embodiments, the antibody binds only to residues involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues involved in strong interactions with the antibody. In some embodiments, the antibody increases interferon γ expression and / or release by CD8+ T cells in the tumor site. In some embodiments, the antibody reduces the accumulation of immunosuppressive granulocytic myeloid cells and / or TAM (tumor-associated macrophages) in infiltrating tumors. In some embodiments, the antibody increases the pro-inflammatory M1 macrophage phenotype and / or reduces the M2 macrophage phenotype in infiltrating tumors. In some embodiments, the antibody increases CD8+ T cells to the tumor site, reduces TAM in infiltrating tumors, and increases the pro-inflammatory M1 macrophage phenotype.

[0086] In certain embodiments, antibodies are described herein that comprise CDRs having the amino acid sequences shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, which bind any one, two, three, four, five, or six of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, antibodies are described herein that comprise CDRs having the amino acid sequences shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, which bind all of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, the antibody binds only to residues that participate in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that participate in strong interactions with the antibody.

[0087] In certain embodiments, antibodies are described herein that comprise CDRs having amino acid sequences different from those shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, which bind any one, two, three, four, five, or six of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, antibodies are described herein that comprise CDRs having amino acid sequences different from those shown in any one of SEQ ID NOs: 11, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, which bind any one, two, three, four, five, or six of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, the antibody binds only to residues that participate in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that participate in strong interactions with the antibody.

[0088] In certain embodiments, antibodies are described herein that comprise CDRs that differ by 1, 2, 3, 4, or 5 amino acid residues from the amino acid sequences shown in any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and that bind any one, two, three, four, five, or six of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, antibodies are described herein that comprise CDRs that are different from the amino acid sequences shown in any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and that bind any one, two, three, four, five, or six of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, the antibody binds only to residues that participate in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that participate in strong interactions with the antibody.

[0089] In certain embodiments, antibodies are described herein that specifically bind periostin and that comprise a human or humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 13; and a human or humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 14, and that bind one, two, three, four, five, or six of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, antibodies are described herein that specifically bind periostin and that comprise a human or humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 13; and a human or humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 14, and that bind all of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, the antibody binds only to residues that participate in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that participate in strong interactions with the antibody.

[0090] In certain embodiments, antibodies are described herein that comprise CDRs having the amino acid sequences shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and comprise a human or humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 13; and a human or humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 14, and that binds to any one, two, three, four, five, or six of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, antibodies are described herein that comprise CDRs having the amino acid sequences shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and that bind to all of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, the antibody binds only to residues that participate in a strong or moderate interaction with the antibody. In certain embodiments, the antibody binds only to residues that participate in a strong interaction with the antibody.

[0091] In certain embodiments, antibodies that specifically bind periostin are described herein, the antibodies comprising a human or humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98% or about 99% identical to the amino acid sequence shown in SEQ ID NO:13, wherein amino acid residue number 55 of SEQ ID NO:13 is asparagine, serine, glutamine, threonine or aspartic acid, and wherein amino acid residue number 100 of SEQ ID NO:13 is methionine, isoleucine or valine; and a human or humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98% or about 99% identical to the amino acid sequence shown in SEQ ID NO:14, and that binds to one, two, three, four, five or six of the following residues of SEQ ID NO:15: N276, R284, E288, L287, V295 or K302. In certain embodiments, antibodies that specifically bind periostin are described herein, the antibodies comprising a human or humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98% or about 99% identical to the amino acid sequence shown in SEQ ID NO:13, wherein amino acid residue number 55 of SEQ ID NO:13 is asparagine, serine, glutamine, threonine or aspartic acid, and wherein amino acid residue number 100 of SEQ ID NO:13 is methionine, isoleucine or valine; and a human or humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98% or about 99% identical to the amino acid sequence shown in SEQ ID NO:14, and that binds to all of the following residues of SEQ ID NO:15: N276, R284, E288, L287, V295 or K302. In certain embodiments, the antibody binds only to residues that participate in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that participate in strong interactions with the antibody.

[0092] In certain embodiments, antibodies that specifically bind periostin are described herein, the antibodies comprising CDRs having the amino acid sequences shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and a human or humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 13, wherein amino acid residue number 55 of SEQ ID NO: 13 is asparagine, serine, glutamine, threonine, or aspartic acid, and wherein amino acid residue number 100 of SEQ ID NO: 13 is methionine, isoleucine, or valine; and a human or humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 14, and that binds to one, two, three, four, five, or six of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, antibodies that specifically bind periostin are described herein, the antibodies comprising CDRs having the amino acid sequences shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and a human or humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 13, wherein amino acid residue number 55 of SEQ ID NO: 13 is asparagine, serine, glutamine, threonine, or aspartic acid, and wherein amino acid residue number 100 of SEQ ID NO: 13 is methionine, isoleucine, or valine; and a human or humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 14, and that binds to all of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, the antibody binds only to residues involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues involved in strong interactions with the antibody.

[0093] In certain embodiments, antibodies are described herein that compete for binding with an antibody that comprises a CDR having an amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 and that binds any one, two, three, four, five, or six of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, antibodies are described herein that compete for binding with an antibody that comprises a CDR having an amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 and that binds all of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, the antibody binds only to residues that participate in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that participate in strong interactions with the antibody.

[0094] In certain embodiments, antibodies are described herein that comprise a binding region that at least partially overlaps with the binding region of an antibody that comprises a CDR having an amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 and that binds any one, two, three, four, five, or six of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, antibodies are described herein that comprise a binding region that at least partially overlaps with the binding region of an antibody that comprises a CDR having an amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 and that binds all of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, the antibody binds only to residues that participate in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that participate in strong interactions with the antibody.

[0095] In certain embodiments, antibodies are described herein that compete for binding with an antibody comprising a human or humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 13; and a human or humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 14.

[0096] In certain embodiments, antibodies are described herein that comprise a binding region that at least partially overlaps with the binding region of an antibody comprising a human or humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 13; and a human or humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 14.

[0097] In certain embodiments, antibodies are described herein that compete for binding with an antibody comprising CDRs having the amino acid sequences shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and comprising a human or humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 13; and a human or humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 14, and that binds to any one, two, three, four, five, or six of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, antibodies are described herein that comprise CDRs having the amino acid sequences shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and that bind to all of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, the antibody binds only to residues involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues involved in strong interactions with the antibody.

[0098] In certain embodiments, antibodies are described herein that comprise a binding region that at least partially overlaps with the binding region of an antibody that comprises CDRs having the amino acid sequences shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and that comprises a human or humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 13; and a human or humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 14, and that binds to any one, two, three, four, five, or six of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, antibodies are described herein that comprise CDRs having the amino acid sequences shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and that bind to all of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, the antibody binds only to residues that participate in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that participate in strong interactions with the antibody.

[0099] In certain embodiments, antibodies are described herein that compete for binding with an antibody comprising a CDR having the amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and comprising a human or humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 13, wherein amino acid residue number 55 of SEQ ID NO: 13 is asparagine, serine, glutamine, threonine, or aspartic acid, and wherein amino acid residue number 100 of SEQ ID NO: 13 is methionine, isoleucine, or valine; and a human or humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 14, and binds to any one, two, three, four, five, or six of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, antibodies are described herein that comprise a CDR having the amino acid sequence shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 and that bind to all of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, the antibody binds only to residues involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues involved in strong interactions with the antibody.

[0100] In certain embodiments, antibodies are described herein that comprise a binding region that at least partially overlaps with the binding region of an antibody that comprises CDRs having the amino acid sequences shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and that comprises a human or humanized heavy chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 13, wherein the amino acid at residue 55 of SEQ ID NO: 13 is asparagine, serine, glutamine, threonine, or aspartic acid, and wherein the amino acid at residue 100 of SEQ ID NO: 13 is methionine, isoleucine, or valine; and a human or humanized light chain variable region amino acid sequence that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence shown in SEQ ID NO: 14, and that binds to any one, two, three, four, five, or six of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, antibodies are described herein that comprise CDRs having the amino acid sequences shown in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and that bind to all of the following residues of SEQ ID NO: 15: N276, R284, E288, L287, V295, or K302. In certain embodiments, the antibody binds only to residues that participate in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that participate in strong interactions with the antibody.

[0101] Therapeutic methods

[0102] The antibodies disclosed herein are antibodies that can be used to treat cancer or tumors. Treatment refers to a method that seeks to improve or alleviate the treated condition. With respect to cancer, treatment includes, but is not limited to, a reduction in tumor volume, a decrease in tumor volume growth, an increase in progression-free survival or overall life expectancy. In certain embodiments, treatment will achieve remission of the treated cancer. In certain embodiments, treatment encompasses use as a prophylactic or maintenance dose that is intended to prevent recurrence or progression of a previously treated cancer or tumor. Those skilled in the art will understand that while the antibody may be safe and effective, not all individuals will respond equivalently to the administered treatment, yet such individuals are considered to have been treated.

[0103] Tumors that can be treated by the antibodies described herein include those that express periostin. Periostin is a component of the extracellular matrix that is secreted by cancer-associated fibroblasts and, thus, most tumors will express or contain periostin. In certain embodiments, the tumors being treated are periostin-positive tumors that have detectable periostin or tumors known to have detectable periostin based on population analysis of similar tumors. In certain embodiments, periostin-high tumors are tumors that have an IHC score of about 50 or higher.

[0104] In certain embodiments, the cancer or tumor is a solid cancer or tumor. In certain embodiments, the cancer or tumor is a hematological cancer or tumor. In certain embodiments, the cancer or tumor includes breast tumor, heart tumor, lung tumor, small intestine tumor, colon tumor, spleen tumor, kidney tumor, bladder tumor, head tumor, neck tumor, ovarian tumor, prostate tumor, brain tumor, pancreatic tumor, skin tumor, bone tumor, bone marrow tumor, blood tumor, thymus tumor, uterine tumor, testicular tumor or liver tumor. In certain embodiments, the tumors or cancers that can be treated with the antibodies of the present invention include adenoma, adenocarcinoma, angiosarcoma, astrocytoma, epithelial carcinoma, germ cell tumor, glioblastoma, glioma, hemangioendothelioma, angiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma and / or teratoma. In certain embodiments, the tumor / cancer is selected from acral lentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytic tumor, Bartholin gland adenocarcinoma, basal cell carcinoma, bronchioloalveolar carcinoma, capillary carcinoid, carcinoma, carcinosarcoma, cholangiocarcinoma, chondrosarcoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymosarcoma, Swing's sarcoma, focal nodular hyperplasia, gastroma, germ cell line tumor, glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, insulinoma, intraepithelial neoplastic lesion, intraepithelial squamous cell neoplastic lesion, invasive squamous cell carcinoma, large cell carcinoma, liposarcoma, lung cancer, lymphoblastic leukemia, lymphocytic leukemia, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelial tumor, neurilemmoma, medulloblastoma, medulloepithelioma, mesothelioma, mucoepidermoid carcinoma, myeloid leukemia, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian cancer, papillary serous adenocarcinoma, pituitary tumor, plasmacytoma, pseudosarcoma, prostate cancer, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, squamous cell carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous cell carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, verrucous carcinoma, vaginal / vulvar carcinoma, vasoactive intestinal polypeptide-secreting tumor (VIPoma) and Wilm's tumor.In certain embodiments, the tumors / cancers to be treated with one or more antibodies of the present invention include brain cancer, head and neck cancer, head and neck squamous cell carcinoma, colorectal cancer, acute myeloid leukemia, pre-B cell acute lymphoblastic leukemia, bladder cancer, astrocytoma (preferably grade II, III or IV astrocytoma), glioblastoma, glioblastoma multiforme, small cell carcinoma and non-small cell carcinoma (preferably non-small cell lung cancer), lung adenocarcinoma, metastatic melanoma, non-androgen-dependent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, prostatic adenocarcinoma and breast cancer (preferably ductal carcinoma of the breast and / or breast cancer). In some embodiments, the cancer includes glioblastoma, pancreatic cancer, breast cancer, bladder cancer, kidney cancer, head and neck cancer, ovarian cancer, skin cancer, gastric cancer, mesothelioma, liver cancer, endometrial cancer, colon cancer, cervical cancer, prostate cancer or lung cancer. In certain embodiments, the cancer treated with the antibody of the present disclosure includes glioblastoma. In certain embodiments, the cancer treated with one or more antibodies of the present disclosure includes pancreatic cancer. In certain embodiments, the cancer treated with one or more antibodies of the present disclosure includes ovarian cancer. In certain embodiments, the cancer treated with one or more antibodies of the present disclosure includes lung cancer. In certain embodiments, the cancer treated with one or more antibodies of the present disclosure includes squamous cell lung cancer. In certain embodiments, the cancer treated with one or more antibodies of the present disclosure includes prostate cancer. In certain embodiments, the cancer treated with one or more antibodies of the present disclosure includes colon cancer. In certain embodiments, the cancer treated includes glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer or lung cancer. In one embodiment, the cancer is refractory to other treatments. In one embodiment, the cancer treated is recurrent. In one embodiment, the cancer is recurrent / refractory glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer or lung cancer.

[0105] In certain embodiments, the antibody can be administered to a subject in need thereof by any route suitable for administering an antibody-containing pharmaceutical composition, such as subcutaneous, intraperitoneal, intravenous, intramuscular, intratumoral, or intracerebral, etc. In certain embodiments, the antibody is administered intravenously. In certain embodiments, the antibody is administered subcutaneously. In certain embodiments, the antibody is administered intratumorally. In certain embodiments, the antibody is administered according to a suitable dosing schedule (e.g., weekly, twice a week, monthly, twice a month, once every two weeks, once every three weeks, or once a month, etc.). In certain embodiments, the antibody is administered once every three weeks. The antibody can be administered in any therapeutically effective amount. In certain embodiments, the therapeutically acceptable amount is between about 0.1 mg / kg and about 50 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 1 mg / kg and about 40 mg / kg. In certain embodiments, the therapeutically acceptable amount is between about 5 mg / kg and about 30 mg / kg. A therapeutically effective amount includes those amounts sufficient to alleviate one or more symptoms associated with the disease or affliction to be treated.

[0106] The anti-periostin antibodies described herein can also be used in a method for reducing the collagen content in a tumor in an individual.

[0107] The anti-periostin antibodies described herein can also be used in a method for increasing the M1 macrophage phenotype and / or reducing the M2 macrophage phenotype in a tumor in an individual.

[0108] The anti-periostin antibodies described herein can also be used in a method for reducing the accumulation of immunosuppressive granulocytic myeloid cells and / or tumor-associated macrophages in an individual.

[0109] The anti-periostin antibodies described herein can also be used in a method for increasing the frequency of CD4+ and / or CD8+ T cells in a tumor of an individual.

[0110] The anti-periostin antibodies described herein can also be used in a method for increasing the interferon-γ expression and / or release of CD8+ T cells in a tumor in an individual.

[0111] The antibodies described herein can be used to manufacture a medicament for reducing the collagen content in a tumor in an individual.

[0112] The antibodies described herein can be used to manufacture a medicament for increasing the M1 macrophage phenotype and / or reducing the M2 macrophage phenotype in a tumor in an individual.

[0113] The antibodies described herein can be used to manufacture a medicament for reducing the accumulation of immunosuppressive granulocytic myeloid cells and / or tumor-associated macrophages in an individual.

[0114] The antibodies described herein can be used to manufacture a medicament for increasing the frequency of CD4+ and / or CD8+ T cells in a tumor of an individual.

[0115] The antibodies described herein can be used to manufacture a medicament for increasing the interferon-γ expression and / or release of CD8+ T cells in a tumor in an individual.

[0116] Pharmaceutically acceptable excipients, carriers and diluents

[0117] The antibodies described herein can be provided in an isolated and purified form that is sufficiently pure for administration to a human individual.

[0118] In certain embodiments, the periostin antibodies of the present disclosure are included in a pharmaceutical composition that comprises one or more pharmaceutically acceptable excipients, carriers, and diluents. In certain embodiments, the antibodies of the present disclosure are administered by suspension in a sterile solution. In certain embodiments, the solution comprises 0.9% NaCl or 5% dextrose, glucose, or sucrose. In certain embodiments, the solution further comprises one or more of the following: buffers, such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, and tris(hydroxymethyl)aminomethane (Tris); surfactants, such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; polyols / di-saccharides / polysaccharides, such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; amino acids, such as glycine or arginine; antioxidants, such as ascorbic acid, methionine; or chelating agents, such as EDTA or EGTA.

[0119] In certain embodiments, the antibodies of the present disclosure are transported / stored in a lyophilized form and reconstituted prior to administration. In certain embodiments, the lyophilized antibody formulation comprises a bulking agent, such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or a combination thereof. The lyophilized formulation can be contained in a vial made of glass or other suitable non-reactive material. The antibody can be buffered at a certain pH (usually less than 7.0) upon formulation (whether reconstituted or not). In certain embodiments, the pH can be between 4.5 and 6.5, 4.5 and 6.0, 4.5 and 5.5, 4.5 and 5.0, or 5.0 and 6.0.

[0120] The present disclosure also describes a kit that comprises one or more of the antibodies described herein in a suitable container and one or more additional components selected from the group consisting of instructions for use, diluents, excipients, carriers, and devices for administration.

[0121] In certain embodiments, methods of preparing a cancer therapeutic agent are described herein, which include mixing one or more pharmaceutically acceptable excipients, carriers or diluents with the antibodies of the present disclosure. In certain embodiments, methods of preparing a cancer therapeutic agent for storage or transportation are described herein, which include lyophilizing one or more antibodies of the present disclosure.

[0122] Examples

[0123] The following illustrative examples represent embodiments of the compositions and methods described herein and are not meant to be limiting in any way.

[0124] Example 1 - Antibody Generation and Screening

[0125] A phage display antibody discovery campaign was conducted to isolate binders against periostin using a fully human phage library. Briefly, three rounds of panning were performed using recombinant human periostin, recombinant mouse periostin, or a combination thereof, with a focus on identifying murine cross-reactive binders. From this panning strategy, 78 sequence-unique ScFvs that cross-react with murine periostin were identified and produced in human IgG1 form for functional screening in a cell attachment assay. See Figure 1.

[0126] Recombinant human or mouse periostin was coated overnight on 96-well plates at 4°C. The next day, the plates were washed with PBS and blocked with 2% BSA for 1 hour at 37°C. After blocking, the antibodies were added to the plates and incubated for 30 min at 37°C. After incubation, 50,000 IMR90 human lung fibroblasts or 50,000 MLG mouse fibroblasts were then added to the wells and allowed to incubate for 2 h at 37°C. The plates were then washed twice with PBS, and the confluence of the wells was measured using the IncuCyte platform. From a high-concentration single-dose screen at 500 nM, 21 IgGs were identified as having >50% inhibition, as shown in Figure 1, and advanced to a binding screen to determine the relative affinities for human and mouse periostin, as shown in Table 1 below.

[0127] To determine the relative affinities for recombinant human or mouse periostin, these proteins were coated overnight on maxisorp plates at 4°C. The next day, the plates were blocked with casein blocking buffer for 1 h at 37°C. Titrations of each antibody were added to the plates and allowed to bind for 1 h at room temperature. The plates were washed 4 times with PBST, after which they were incubated with an HRP-conjugated anti-human Fc secondary antibody for 30 min at room temperature. The plates were then washed 4 times again with PBST, and then developed using a TMB substrate and 1 M HCl. From this screen, 4 clones (bold and italicized in Table 1) were selected that had binding EC50 values <1 nM for both human and mouse periostin.

[0128]

[0129] Example 2 - Generation of NB0828 and Sequence Variants

[0130] Four candidates were retested in a dose - response in the cell attachment assay to determine the IC50 values. From this screen, NB0627 was identified as a particularly suitable IgG (Table 2).

[0131]

[0132]

[0133] NB0627 was then converted to the effector - silent IgG4PAA isotype, thereby generating the lead candidate NB0828. Sequence analysis of NB0828 identified two post - translational modification - susceptible sites in the VH region. The first site, the deamidation site, is located in CDR - H2, and the second site, the oxidation site, is located in CDR - H3. Therefore, to remove these susceptible sites, several single - mutants and double - mutants were generated and their binding and activity were measured. The results of the IC50 and EC50 values of NB0828 and its variants are summarized in Table 3.

[0134]

[0135] Example 3 - In Vivo Efficacy of NB0828 in Mouse Bladder MB49 and Colon CT26 Tumor Models

[0136] The efficacy of NB0828 was tested in two independent tumor models, the bladder MB49 and colon CT26 tumor models. Briefly, 250,000 MB49 cells were injected intradermally into the flanks of female C57BL / 6 mice, or 50,000 CT26 cells were injected intradermally into the flanks of female Balb / c mice. Three days after tumor implantation, the mice were treated intraperitoneally with NB0828 (50 mg / kg, 3QW) or vehicle control (PBS). Tumor volume was assessed twice weekly by caliper measurement and calculated as (length x width 2 ) / 2. Mice were euthanized when the tumor size exceeded 15 mm in any single dimension or due to tumor ulceration as a humane endpoint.

[0137] As shown in Figures 2 (MB49) and 5 (CT26), NB0828 has an effect of reducing tumor growth in both models. In the MB49 model, this reduction in tumor growth is associated with a lower frequency of intratumoral granulocytic myeloid cells as shown in Figure 3 and a lower collagen content as shown in Figure 4. Similar to the MB49 model, the CT26 model shows a reduction in granulocytic myeloid cells. In addition, NB0828 reduces the frequency of tumor-infiltrating macrophages, and due to the treatment with NB0828, the existing macrophages skew towards the M1 phenotype, as shown in Figure 6. In the CT26 mouse model, NB0828 treatment is also associated with a higher frequency of tumor-infiltrating CD8+ and CD4+ T cells and an increase in CD8+ T cell function as measured by the higher expression of interferon γ, as shown in Figure 7.

[0138] Immunophenotyping

[0139] As described, starting from day 3, mice bearing MB49 or CT26 tumors were treated with NB0828 or vehicle control. For the data shown, immunophenotyping was performed on day 20 after tumor implantation in MB49 and day 18 after tumor implantation in CT26, respectively. Tumors were excised using a surgical blade, the skin was removed and mechanically disrupted, and then enzymatic digestion was performed using the Miltenyi mouse tumor dissociation enzyme mix. The digested samples were passed through a 40 μm filter, washed in RPMI, and then washed a second time in RPMI + 10% FBS. The cells were then resuspended for counting, and up to 2x10 6 white blood cells / sample were plated and stained for analysis by flow cytometry. To evaluate CD8+ tumor-infiltrating lymphocyte function in the CT26 model, the digested single-cell suspension from the tumor was incubated at 37 °C in the presence of anti-CD28 and brefeldin A with the AH1 peptide [H2-L dThe restricted gp70(423 - 431) MuLV epitope, i.e., the immunodominant CD8+ T cell epitope expressed by CT26 cells, was used to stimulate for 5 hours. After stimulation, cells were stained by standard surface / intracellular staining methods to detect IFN-γ production by CD8+ T cells using flow cytometry. The flow staining panel used to evaluate the indicated cell populations is included in Table 4 below. A viability stain (Thermo Fisher, Live / Dead Fixable Violet Stain) was used to enable interrogation of only live cell events, and the pan-leukocyte marker CD45 was included to enable normalization of populations within the immune compartment. The following phenotypic / functional definitions were made for the immune populations of interest: total myeloid cells (CD45+CD11b+), granulocytes (CD45+CD11b+Gr-1high or CD45+CD11b+Ly6G+Ly6C low), macrophages (CD45+CD11b+Ly6G-Ly6C low / negative F4 / 80+), M1 macrophages (MHCII+CD206-), M2 macrophages (MHC II-CD206+), CD8+ TILs (CD45+CD11b-CD3+CD90.2+CD8+), CD4+ TILs (CD45+CD11b-CD3+CD90.2+CD4+), IFN-γ+CD8+ TILs (CD45+CD11b-CD3+CD8+IFN-γ+). The median fluorescence intensity (MFI) was used to determine the IFN-γ staining intensity from IFN-γ+CD8+ TILs.

[0140]

[0141] Collagen content

[0142] The total collagen content of tumors was evaluated by quantifying hydroxyproline using the Quickzyme Total Collagen Assay. To prepare samples, when tumors reached endpoint, MB49 tumors were excised from tumor-bearing mice, snap-frozen in liquid nitrogen and stored at -80 °C until analysis. The tumor material was weighed and resuspended in 6 M HCl at 200 mg tumor / ml HCl, vortexed and incubated at 95 °C for 20 h. The tubes were cooled, centrifuged at 13,000 RPM for 10 minutes, and the supernatant was collected. The supernatant was diluted in Milli Q water and then in 4 M HCl according to the manufacturer's recommended protocol and plated in technical replicates to detect hydroxyproline using the provided buffer and detection reagent. The OD570 nm value was measured and compared to a standard curve generated using the provided collagen to calculate the amount of collagen in each sample. The calculated total collagen (μg) for each sample was divided by the total mass (mg) of the tumor input to normalize the data across tumor samples.

[0143] Example 4 - In Vivo Efficacy of NB0828 in a Murine Colon MC38 Tumor Model

[0144] The efficacy of NB0828 in reducing tumor growth in a murine colon MC38 tumor model was tested, and the results are shown in Figure 8. Briefly, 200,000 MC38 cells were injected intradermally into the flanks of female C57BL / 6 mice. Three days after tumor implantation, mice were treated intraperitoneally with NB0828 (50 mg / kg, 3QW) or vehicle control (PBS). For depletion of CD8+ T cells, anti - murine CD8a (clone 2.43) or IgG isotype control (clone LTF - 2) was delivered together with NB0828 for the first 6 doses (10 mg / kg, 3QW). Depletion of T cells in the blood was confirmed by flow cytometry using the flow staining panel listed in Table 5. Tumor volume was measured using the same method described for the MB49 and CT26 models. NB0828 effectively reduced tumor growth in the MC38 model (Figure 8). NB0828 also effectively altered the tumor microenvironment to increase CD8+ T cells, decrease the frequency of tumor - associated macrophages (TAMs), and increase the ratio of pro - inflammatory macrophages, as shown in Figures 9A and 9C. The efficacy of NB0828 in reducing tumor growth in this model was dependent on CD8+ T cells, because depletion of CD8+ T cells during NB0828 treatment reversed the beneficial effects of NB0828, as shown in 9D. Overall, this data indicates that NB0828 effectively reduced tumor growth and increased CD8+ T cells to the tumor site, decreased TAMs in infiltrating tumors, and increased the pro - inflammatory M1 macrophage phenotype.

[0145] Immunophenotyping

[0146] Tumors were excised using a scalpel blade, the skin was removed and mechanically disrupted, and then enzymatic digestion was performed using the Miltenyi mouse tumor dissociation enzyme mix (incubated for 45 min at 37 °C on an orbital shaker). The digested samples were passed through a 40 μm filter, washed in RPMI, and then washed a second time in RPMI + 10% FBS. Cells were then resuspended for counting and plated and stained for up to 2x10 6 white blood cells / sample for analysis by flow cytometry. To evaluate CD8+ tumor - infiltrating lymphocyte (CD8+ TIL) function in the MC38 model, single - cell suspensions from digested tumors (up to 2x10 6 white blood cells / sample) were incubated at 37 °C in the presence of anti - CD28 and brefeldin A with p15E peptide [H2 - K expressed by the MC38 tumor bThe restricted p15E(604 - 611) MuLV epitope was stimulated for 5 h. After stimulation, cells were stained by standard surface / intracellular staining methods using the eBioscience Intracellular Fix & Perm Buffer Kit to detect IFN-γ production by CD8+ T cells using flow cytometry. The flow staining panel used to evaluate the indicated cell populations is included in the table below. A viability stain (Thermo Fisher, Live / Dead Fixable Violet Stain) was used to enable interrogation of only live cell events, and the pan-leukocyte marker CD45 was included to enable normalization of populations within the immune compartment. For studies in MC38, the following phenotypic / functional definitions were made for the immune populations reported: total myeloid cells (CD45+CD11b+), macrophages (CD45+CD11b+Ly6G-Ly6C low / negative F4 / 80+), M1 macrophages (MHC II+), M2 macrophages (MHC II-), CD8+ TIL (CD45+CD11b-CD3+SSC low CD8+), IFN-γ+ CD8+ TIL (CD45+CD11b-CD3+SSC low CD8+IFN-γ+).

[0147]

[0148] Example 5 - NB0828 binds to the FAS2 domain of periostin (POSTN)

[0149] Study the binding region of NB0828 to further characterize the antibody.

[0150] Production of BIGH3 / POSTN chimeric proteins

[0151] The closest homolog of periostin by sequence is transforming growth factor-β-induced protein (BIGH3), which has 48% sequence homology in the EMI-FAS4 region of the protein. Although the sequence homology between these two proteins is low, the overall domain structure is highly similar, containing an EMI domain and four tandem fasciclin (FAS) domains. NB0828 binds to periostin with high affinity but does not bind to BIGH3. To further characterize the binding domain of NB0828, BIGH3 / POSTN chimeric proteins were generated in which each domain of periostin was replaced by the corresponding domain of BIGH3, resulting in five BIGH3 / POSTN chimeras (Figure 10). NB0828 binding studies were performed on these proteins by ELISA. As shown in Figure 11A, NB0828 retained binding to all BIGH3 / POSTN chimeras except the FAS2 chimera. This observable loss of binding when the POSTN FAS2 domain was replaced with the BIGH3 FAS2 domain indicates that NB0828 binds to the FAS2 domain of POSTN.

[0152] Production of POSTN FAS2 variants

[0153] The FAS2 domain of periostin is a conformational structure composed of 132 amino acids. Single amino acid substitution (alanine) variants were generated at different positions across the FAS2 domain to further delimit the NB0828 epitope or binding region and identify key contact residues (Table 5). Using the published crystal structure (Liu et al., 2018; PDB#5YJG), residues for mutagenesis were identified based on surface exposure in the computational analysis tool MOE.

[0154]

[0155]

[0156] In the initial round, 23 alanine variants of residues spanning the entire FAS2 domain were generated. These variants were subjected to NB0828 binding studies, and variants with >50% loss of the maximum signal within the assay were identified as residues critical for binding. Figure 11B shows the results from this screen. Two variants representing key amino acids for NB0828 binding were identified: NB1205 (R284A) and NB1207 (E288A). Given the close proximity of these two amino acids (Figure 12), an additional 11 variants were generated in a second round, thus more specifically focusing on residues within this region. Figure 11C shows the results from the second screen. Based on the ELISA results from the second screen, four additional variants representing amino acids critical for NB0828 binding were identified: NB1243 (L287A), NB1246 (V295A), NB1248 (K302A), and NB1202 (N276A). Notably, NB1190 (D245A), which was very close to but did not fall below the >50% cut-off of the maximum signal, lies on a loop separated from the remaining residues but forms a contact point with N276 within the FAS2 domain and on the NB0828 binding loop (Figure 12). Given that N276 was identified as a key residue for NB0828 binding, the D245 residue may also be a contact residue for NB0828 or provide structural integrity important for binding to the NB0828 epitope loop.

[0157] Determination of NB0828 affinity across species

[0158] The binding affinity of NB0828 across human, mouse, rat, and cynomolgus monkey species was determined. The NB0828 affinity was determined using the octet red system. Briefly, NB0828 was captured using an anti-human Fc (AHC) biosensor, followed by the association of titrated recombinant human, mouse, rat, or cynomolgus monkey periostin (100 nM → 0 nM; 1:2 dilutions). Recombinant human, mouse, and rat periostin were purchased commercially from R&D systems, while cynomolgus monkey periostin was made in-house. The results showed that across these four species, the affinity of NB0828 for periostin was highly similar, ranging from 0.1 - 0.5 nM. This data strongly supports the epitope mapping experiments detailed above, as the sequence identity within the described NB0828 binding loop is 100% across these four species.

[0159]

[0160]

[0161] Example 6 - NB0828 does not block the binding of periostin to tenascin C and type I collagen but blocks cell attachment

[0162] To determine whether NB0828 blocks the binding of periostin to extracellular proteins, namely tenascin C and type I collagen, a competitive ELISA assay was performed. Recombinant human periostin (2 ng / mL) was coated onto maxisorp plates overnight at 4 °C. The next day, the plates were blocked with casein blocking buffer for 1 h at 37 °C. To determine the binding EC 80 of recombinant human tenascin C and human type I collagen, the proteins were biotinylated at a 10:1 ratio using EZ-Link TM NHS-PEG4-biotin (Fisher), and titrations of each biotinylated protein were added to the plates and incubated for 1 h at room temperature. The plates were washed 4 times with PBST, followed by detection with avidin-HRP for 30 min. The plates were then washed 4 times again with PBST, and developed using TMB substrate and 1 M HCl. The EC 80 of each protein was determined using Graphpad Prism 7 software. For the competitive ELISA, titrations of NB0828, control IgG, or PBST were added to the plates, and the plates were incubated with shaking for 30 min at room temperature. The plates were then washed 4 times with PBST, and the binding EC 80 concentration of biotinylated tenascin C (0.9 nM, 13A, left) or collagen (100 nM, 13B, left) or a pre-mixed blocking control was added to the plates. The pre-mixed blocking control was prepared by mixing the EC 80 concentration of biotinylated protein with titrations of recombinant human periostin, which was incubated with shaking for 30 min at room temperature and then added to the plates. The plates were then incubated with shaking for 1 h at room temperature, and washed, detected, and developed as described above.

[0163] Tenascin C and type I collagen bind to periostin, with their E C80 bindings being 0.9 nM and 100 nM, respectively (Figure 13A and Figure 13B). As shown in Figure 13A and Figure 13B, NB0828 does not inhibit the binding of periostin to tenascin C and type I collagen compared to the pre-mixed positive control. Taken together, these data indicate that NB0828 does not block the interaction of periostin with extracellular matrix proteins, namely tenascin C and type I collagen.

[0164] Example 7 - Immunohistochemical (IHC) Evaluation of Periostin Expression across Human Tumor Indications

[0165] To assess periostin expression levels across different human cancers, an immunohistochemistry (IHC) prevalence study was conducted. Tissue microarrays (TMAs) containing 18 tumor indications and approximately 750 individual samples were evaluated for periostin expression. Samples were stained with anti-periostin antibody EPR20806 (Abcam catalog id: ab215199, lot id: GR3192974-3) diluted 1:50. Staining was quantified using digital pathology methods (HALO, Indica labs) to calculate the IHC score (IHC score = [% low-intensity staining area * 1] + [% medium-intensity staining area * 2] + [% high-intensity staining area * 3]). Based on the approximate mean periostin IHC score across all samples, the cut-off point for low / high periostin staining was calculated as a periostin IHC score of 50. The prevalence study demonstrated the range of periostin staining across the test indications and within the test indications, with pancreatic cancer, breast cancer, and squamous lung cancer showing the highest levels of periostin staining (Figure 14A). Periostin-high tumors were present in all indications but at different frequencies (Figure 14A). Representative IHC images of periostin expression in breast cancer are shown in Figure 14B. Collectively, these data demonstrate that periostin is widely expressed across multiple tumor types.

[0166] While the preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

[0167] All publications, patent applications, issued patents, and other documents mentioned in this specification are hereby incorporated by reference as if each individual publication, patent application, issued patent, or other document were specifically and individually indicated to be incorporated by reference in its entirety. To the extent that the definitions contained in the text incorporated by reference conflict with the definitions in the present disclosure, they are excluded.

[0168]

[0169]

Claims

1. A recombinant antibody that binds periostin or an antigen-binding fragment of said recombinant antibody, wherein said antibody or antigen-binding fragment of said antibody comprises: a) Immunoglobulin heavy chain CDR1 (CDR-H1) having the amino acid sequence as set forth in SEQ ID NO:1, GYTFTSYG; b) Immunoglobulin heavy chain CDR2 (CDR-H2) having the amino acid sequence as set forth in SEQ ID NO:2, ISAYNGNT; c) Immunoglobulin heavy chain CDR3 (CDR-H3) having the amino acid sequence as set forth in SEQ ID NO:9, DMLVVPFDY; d) Immunoglobulin light chain CDR1 (CDR-L1) having the amino acid sequence as set forth in SEQ ID NO:10, SSDIGSNR; e) Immunoglobulin light chain CDR2 (CDR-L2) having the amino acid sequence as set forth in SEQ ID NO:11, SND; and f) Immunoglobulin light chain CDR3 (CDR-L3) having the amino acid sequence as set forth in SEQ ID NO:12, AAWDDSLSTYV.

2. The recombinant antibody or antigen-binding fragment of said recombinant antibody according to claim 1, wherein said recombinant antibody or antigen-binding fragment of said recombinant antibody is human, chimeric or humanized.

3. The recombinant antibody or antigen-binding fragment of said recombinant antibody according to claim 1 or 2, wherein said recombinant antibody or antigen-binding fragment of said recombinant antibody is an IgG antibody.

4. The recombinant antibody or antigen-binding fragment of said recombinant antibody according to claim 1 or 2, wherein said recombinant antibody or antigen-binding fragment of said recombinant antibody comprises one or more mutations to reduce one or more effector functions of said recombinant antibody or antigen-binding fragment of said recombinant antibody.

5. The recombinant antibody or antigen-binding fragment of said recombinant antibody according to claim 4, wherein said one or more mutations that reduce one or more effector functions of said recombinant antibody or antigen-binding fragment of said recombinant antibody comprise one or more mutations or groups of mutations selected from: N434A, N434H, T307A / E380A / N434A, M252Y / S254T / T256E, 433K / 434F / 436H, T250Q, T250F, M428L, M428F, T250Q / M428L, N434S, V308W, V308Y, V308F, M252Y / M428L, D259I / V308F, M428L / V308F, Q311V / N434S, T307Q / N434A, E258F / V427T, S228P, L235E, S228P / L235E / R409K, S228P / L235E, K370Q, K370E, G446 deletion, K447 deletion and combinations thereof according to the EU numbering system of IgG4.

6. The recombinant antibody or antigen-binding fragment of the recombinant antibody according to claim 4, wherein the plurality of mutations that reduce one or more effector functions of the recombinant antibody or the antigen-binding fragment of the recombinant antibody comprise: S228P, F234A, and L235A mutations of IgG4 according to the EU numbering system.

7. The recombinant antibody or antigen-binding fragment of the recombinant antibody according to claim 1 or 2, wherein the recombinant antibody or antigen-binding fragment of the recombinant antibody is a Fab, F(ab)2, single-domain antibody, or single-chain variable fragment (scFv).

8. The recombinant antibody or antigen-binding fragment of the recombinant antibody according to claim 1 or 2, the recombinant antibody or antigen-binding fragment of the recombinant antibody comprising an immunoglobulin heavy-chain variable region and an immunoglobulin light-chain variable region: a) wherein the immunoglobulin heavy-chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 13; and b) wherein the immunoglobulin light-chain variable region comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO:

14.

9. The recombinant antibody or antigen-binding fragment of the recombinant antibody according to claim 1 or 2, the recombinant antibody or antigen-binding fragment of the recombinant antibody comprising an immunoglobulin heavy-chain variable region and an immunoglobulin light-chain variable region: a) wherein the immunoglobulin heavy-chain variable region comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 13; and b) wherein the immunoglobulin light-chain variable region comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO:

14.

10. The recombinant antibody or antigen-binding fragment of the recombinant antibody according to claim 1 or 2, the recombinant antibody or antigen-binding fragment of the recombinant antibody comprising an immunoglobulin heavy-chain variable region and an immunoglobulin light-chain variable region: a) wherein the immunoglobulin heavy-chain variable region comprises an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO: 13; and b) wherein the immunoglobulin light-chain variable region comprises an amino acid sequence that is at least 97% identical to the amino acid sequence shown in SEQ ID NO:

14.

11. The recombinant antibody or antigen-binding fragment of the recombinant antibody according to claim 1 or 2, the recombinant antibody or antigen-binding fragment of the recombinant antibody comprising an immunoglobulin heavy-chain variable region and an immunoglobulin light-chain variable region: a) wherein the immunoglobulin heavy-chain variable region comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO: 13; and b) wherein the immunoglobulin light-chain variable region comprises an amino acid sequence that is at least 99% identical to the amino acid sequence shown in SEQ ID NO:

14.

12. The recombinant antibody or antigen-binding fragment of the recombinant antibody according to claim 1 or 2, the recombinant antibody or antigen-binding fragment of the recombinant antibody comprising an immunoglobulin heavy-chain variable region and an immunoglobulin light-chain variable region: a) wherein the immunoglobulin heavy-chain variable region comprises the amino acid sequence shown in SEQ ID NO: 13; and b) wherein the immunoglobulin light chain variable region comprises the amino acid sequence as shown in SEQ ID NO:

14.

13. A recombinant antibody that binds periostin or an antigen-binding fragment of the recombinant antibody, the recombinant antibody or the antigen-binding fragment of the recombinant antibody comprising: a) an immunoglobulin heavy chain variable region having the amino acid sequence as shown in SEQ ID NO: 13; and b) an immunoglobulin light chain variable region having the amino acid sequence as shown in SEQ ID NO:

14.

14. A nucleic acid encoding the recombinant antibody or the antigen-binding fragment of the recombinant antibody according to any one of claims 1 to 13.

15. A cell line comprising the nucleic acid according to claim 14.

16. The cell line according to claim 15, wherein the cell line is a Chinese hamster ovary cell line.

17. A pharmaceutical composition comprising the recombinant antibody or the antigen-binding fragment of the recombinant antibody according to any one of claims 1 to 13 and a pharmaceutically acceptable excipient.

18. A pharmaceutical composition comprising the recombinant antibody or the antigen-binding fragment of the recombinant antibody according to any one of claims 1 to 13 and a pharmaceutically acceptable carrier.

19. A pharmaceutical composition comprising the recombinant antibody or the antigen-binding fragment of the recombinant antibody according to any one of claims 1 to 13 and a pharmaceutically acceptable diluent.

20. The pharmaceutical composition according to any one of claims 17-19, which is formulated for intravenous administration.

21. The pharmaceutical composition according to any one of claims 17-19, which is formulated for subcutaneous administration.

22. The pharmaceutical composition according to any one of claims 17-19, which is formulated for intratumoral administration.

23. Use of the recombinant antibody or the antigen-binding fragment of the recombinant antibody according to any one of claims 1 to 13 or a pharmaceutical composition comprising the recombinant antibody or the antigen-binding fragment of the recombinant antibody according to any one of claims 1 to 13 for the preparation of a medicament for reducing the collagen content in a tumor of bladder cancer in an individual.

24. Use of the recombinant antibody or the antigen-binding fragment of the recombinant antibody according to any one of claims 1 to 13 or a pharmaceutical composition comprising the recombinant antibody or the antigen-binding fragment of the recombinant antibody according to any one of claims 1 to 13 for the preparation of a medicament for treating bladder cancer or colon cancer in an individual.

25. A method for preparing a composition for reducing the collagen content in a tumor, which comprises mixing the recombinant antibody or the antigen-binding fragment of the recombinant antibody according to any one of claims 1 to 13 with a pharmaceutically acceptable excipient.

26. A method for preparing a composition for reducing the collagen content in a tumor, which comprises mixing the recombinant antibody or the antigen-binding fragment of the recombinant antibody according to any one of claims 1 to 13 with a pharmaceutically acceptable carrier.

27. A method of preparing a composition for reducing the collagen content in a tumor, which comprises mixing the recombinant antibody or an antigen-binding fragment of the recombinant antibody according to any one of claims 1 to 13 with a pharmaceutically acceptable diluent.

28. A method of producing the recombinant antibody or an antigen-binding fragment of the recombinant antibody according to any one of claims 1 to 13, which comprises incubating the cell line according to claim 15 or 16 in a cell culture medium under conditions sufficient to permit the expression and secretion of the recombinant antibody or an antigen-binding fragment of the recombinant antibody according to any one of claims 1 to 13.

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