Anti-periostin antibodies and uses thereof

Abstract

To provide antibodies that block the function of periostin hypothesized to suppress anti-tumor immunity and drive tumor growth and progression.SOLUTION: The invention provides a recombinant antibody or antigen binding fragment thereof that binds periostin, where the antibody or antigen binding fragment thereof comprises an immunoglobulin heavy chain CDR1 (CDR-H1), an immunoglobulin heavy chain CDR2 (CDR-H2), an immunoglobulin heavy chain CDR3 (CDR-H3), an immunoglobulin light chain CDR1 (CDR-L1), an immunoglobulin light chain CDR2 (CDR-L2) and an immunoglobulin light chain CDR3 (CDR-L3) each of which comprises a specific amino acid sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 779,996, filed December 14, 2018, and U.S. Provisional Application No. 62 / 899,075, filed September 11, 2019, all of which are incorporated by reference in their entireties.

[0002] background Periostin (POSTN) is a multicellular protein involved in many pathological processes, including epithelial-mesenchymal transition (EMT), cell-matrix interactions, and inflammation. POSTN is overexpressed in several pathological conditions, including inflammation, fibrosis, and cancer, where it correlates with poor prognosis. In cancer, POSTN is typically expressed by stromal cells, such as cancer-associated fibroblasts (CAFs), but POSTN expression has also been reported in cancer-initiating cells (CICs) and bone marrow-derived immunosuppressive cells. POSTN regulates extracellular matrix 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, epithelial-mesenchymal transition, angiogenesis, and immune cell recruitment. POSTN is hypothesized to drive tumor growth and progression by acting on multiple aspects of cancer biology, including suppressing antitumor immunity by promoting immune clearance and by increasing immune suppression by tumor-infiltrating myeloid cells.

[0003] summary Described herein are antibodies that inhibit periostin functions, such as integrin-mediated cell adhesion. Such antibodies are useful for the treatment of cancer. The anti-periostin antibodies described herein reduce the collagen content of tumors, increase the polarization of macrophages to the M1 phenotype, decrease the infiltration of suppressive myeloid cell populations, such as granulocytes and tumor-associated macrophages, and increase the accumulation and anti-tumor properties of tumor-infiltrating T cells.

[0004] This specification describes a recombinant antibody or an antigen-binding fragment thereof that binds to periostin, wherein the antibody or its antigen-binding fragment comprises an immunoglobulin heavy chain CDR1 (CDR-H1) containing the amino acid sequence shown in SEQ ID NO: 1 (GYTFTSYG); an immunoglobulin heavy chain CDR2 (CDR-H2) containing 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) containing 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) containing the amino acid sequence shown in SEQ ID NO: 10 (SSDIGSNR); an immunoglobulin light chain CDR2 (CDR-L2) containing the amino acid sequence shown in SEQ ID NO: 11 (SND); and an immunoglobulin light chain CDR3 (CDR-L3) containing the amino acid sequence shown in SEQ ID NO: 12 (AAWDDSLSTYV). In some embodiments, the antibody or its antigen-binding fragment comprises an immunoglobulin heavy chain CDR1 (CDR-H1) containing the amino acid sequence shown in SEQ ID NO: 1 (GYTFTSYG); an immunoglobulin heavy chain CDR2 (CDR-H2) containing the amino acid sequence shown in SEQ ID NO: 2 (ISAYNGNT); an immunoglobulin heavy chain CDR3 (CDR-H3) containing the amino acid sequence shown in SEQ ID NO: 9 (DMLVVPFDY); an immunoglobulin light chain CDR1 (CDR-L1) containing the amino acid sequence shown in SEQ ID NO: 10 (SSDIGSNR); an immunoglobulin light chain CDR2 (CDR-L2) containing the amino acid sequence shown in SEQ ID NO: 11 (SND); and an immunoglobulin light chain CDR3 (CDR-L3) containing the amino acid sequence shown in SEQ ID NO: 12 (AAWDDSLSTYV). In some embodiments, the antibody or its antigen-binding fragment is human, chimeric, or humanized. In some embodiments, the recombinant antibody or its antigen-binding fragment is an IgG antibody. In certain embodiments, the recombinant antibody or its antigen-binding fragment comprises one or more mutations that reduce one or more effector functions of the recombinant antibody or its antigen-binding fragment.In certain embodiments, the one or more mutations that reduce one or more effector functions of the recombinant antibody or antigen-binding fragment thereof are selected from the group consisting of N434A, N434H, T307A / E380A / N434A, M252Y / S254T / T256E, 433K / 434F / 436H, T250Q, T250F, M428L, M428F, T250Q / M428L, N434S, V308W, V308W, V308H ... The recombinant antibody or antigen-binding fragment thereof may comprise one or more mutations or a set of mutations selected from: 08Y, 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 combinations thereof. In certain embodiments, the one or more mutations that reduce one or more effector functions of the recombinant antibody or antigen-binding fragment thereof comprise 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, a single domain antibody, or a 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% identical, or 100% identical to the amino acid sequence set forth 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% identical, or 100% identical to the amino acid sequence set forth in SEQ ID NO: 14, wherein amino acid at amino acid residue number 55 of SEQ ID NO: 13 is asparagine, serine, glutamine, threonine, or aspartic acid, and wherein 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 (N276, R284, E288, L287, V295, or K302) for specific binding to periostin. 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 (N276, R284, E288, L287, V295, or K302) for specific binding to periostin. In some embodiments, the recombinant antibody or antigen-binding fragment thereof requires at least all of the following residues of SEQ ID NO: 15 (N276, R284, E288, L287, V295, or K302) for specific binding to periostin. Described herein are pharmaceutical compositions comprising the recombinant antibody or antigen-binding fragment thereof and a pharma- ceutically 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 thereof or pharmaceutical composition for use in reducing collagen content in a tumor.In some embodiments, the recombinant antibody or antigen-binding fragment thereof or pharmaceutical composition is for use in treating cancer. 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. Also described herein is a method of decreasing collagen content in a tumor of an individual, comprising administering the recombinant antibody or antigen-binding fragment thereof or pharmaceutical composition to the individual. Also described herein is a method of increasing M1 macrophage phenotype and / or decreasing M2 macrophage phenotype in a tumor of an individual, comprising administering the recombinant antibody or antigen-binding fragment thereof or pharmaceutical composition to the individual. Also described herein is a method of decreasing accumulation of suppressive granulocytic myeloid cells and / or tumor-associated macrophages in an individual, comprising administering the recombinant antibody or antigen-binding fragment thereof or pharmaceutical composition to the individual. Also described herein is a method for increasing the frequency of CD4+ and / or CD8+ T cells in a tumor of an individual, comprising administering the recombinant antibody or antigen-binding fragment thereof or pharmaceutical composition to the individual. Also described herein is a method for enhancing the function of CD8+ T cells in a tumor, as measured by the expression and / or release of interferon-gamma by CD8+ T cells in the individual, comprising administering the recombinant antibody or antigen-binding fragment thereof or pharmaceutical composition to the individual. Also described herein is a method for treating cancer in an individual, comprising administering a therapeutically effective amount of the recombinant antibody or antigen-binding fragment thereof or pharmaceutical composition to the individual. 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. Also described herein is a method for making a composition for reducing collagen content in a tumor, comprising the step of mixing the recombinant antibody or antigen-binding fragment thereof with a pharma- ceutically acceptable excipient, carrier, or diluent.Also described herein is a method for making a composition for increasing M1 macrophage phenotype and / or decreasing M2 macrophage phenotype in a tumor, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharma- ceutically acceptable excipient, carrier, or diluent. Also described herein is a method for making a composition for decreasing accumulation of suppressive granulocytic myeloid cells and / or tumor-associated macrophages in an individual, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharma- ceutically acceptable excipient, carrier, or diluent. Also described herein is a method for making a composition for increasing frequency of CD4- and / or CD8-positive T cells in a tumor in an individual, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharma- ceutically acceptable excipient, carrier, or diluent. Also described herein is a method for making a composition for enhancing the function of CD8 positive T cells in a tumor, as measured by expression and / or release of interferon gamma by CD8 positive T cells in an individual, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharma- ceutically acceptable excipient, carrier, or diluent. Also described herein is a method for making a composition for treating cancer, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharma- ceutically 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.

[0005] Also described herein is a recombinant antibody or antigen-binding fragment thereof that binds to periostin, 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% identical, or 100% identical to the amino acid sequence set forth 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% identical, 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 some embodiments, the recombinant antibody or antigen-binding fragment thereof is a human antibody. In some embodiments, the recombinant antibody or antigen-binding fragment thereof is an IgG antibody. In certain embodiments, the recombinant antibody or antigen-binding fragment thereof comprises one or more mutations that reduce one or more effector functions of the recombinant antibody or antigen-binding fragment thereof. 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 N434A, N434H, T307A / E380A / N434A, M252Y / S254T / T256E, 433K / 434F / 436H, T250Q, T250F, M428L, M428F, T250Q / M428L, N434S, V308W ... In a particular embodiment, the one or more mutations that reduce one or more effector functions of the recombinant antibody or antigen-binding fragment thereof comprise one or more mutations or a set of mutations selected from 08Y, 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 combinations thereof. In a particular embodiment, the one or more mutations that reduce one or more effector functions of the recombinant antibody or antigen-binding fragment thereof comprise IgG4 S228P, F234A, and L235A mutations 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). Also described herein is a pharmaceutical composition comprising the recombinant antibody or antigen-binding fragment thereof and a pharma- ceutically 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 thereof or pharmaceutical composition for use in reducing collagen content in a tumor. In some embodiments, the recombinant antibody or antigen-binding fragment thereof or pharmaceutical composition is for use in treating cancer. 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. Also described herein is a method for decreasing collagen content in a tumor of an individual, comprising administering the recombinant antibody or antigen-binding fragment thereof, or pharmaceutical composition to the individual. Also described herein is a method for increasing M1 macrophage phenotype and / or decreasing M2 macrophage phenotype in a tumor of an individual, comprising administering the recombinant antibody or antigen-binding fragment thereof, or pharmaceutical composition to the individual. Also described herein is a method for decreasing accumulation of suppressive granulocytic myeloid cells and / or tumor-associated macrophages in an individual, comprising administering the recombinant antibody or antigen-binding fragment thereof, or pharmaceutical composition to the individual. Also described herein is a method for increasing frequency of CD4-positive and / or CD8-positive T cells in a tumor of an individual, comprising administering the recombinant antibody or antigen-binding fragment thereof, or pharmaceutical composition to the individual. Also described herein is a method for enhancing function of CD8-positive T cells in a tumor, as measured by expression and / or release of interferon gamma by CD8-positive T cells in the individual, comprising administering the recombinant antibody or antigen-binding fragment thereof, or pharmaceutical composition to the individual.Also described herein is a method for treating cancer in an individual, comprising administering to the individual a therapeutically effective amount of the recombinant antibody or antigen-binding fragment thereof or 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. Also described herein is a method for making a composition for reducing collagen content in a tumor, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharmaceutically acceptable excipient, carrier, or diluent. Also described herein is a method for making a composition for increasing M1 macrophage phenotype and / or decreasing M2 macrophage phenotype in a tumor, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharmaceutically acceptable excipient, carrier, or diluent. Also described herein is a method for making a composition for reducing accumulation of suppressive granulocytic myeloid cells and / or tumor associated macrophages in an individual, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharma- ceutically acceptable excipient, carrier, or diluent. Also described herein is a method for making a composition for increasing the frequency of CD4- and / or CD8-positive T cells in a tumor of an individual, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharma- ceutically acceptable excipient, carrier, or diluent. Also described herein is a method for making a composition for enhancing the function of CD8-positive T cells in a tumor, as measured by expression and / or release of interferon-gamma by CD8-positive T cells in an individual, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharma- ceutically acceptable excipient, carrier, or diluent. Also described herein is a method for making a composition for treating cancer, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharma- ceutically 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] Also described herein are recombinant antibodies or antigen-binding fragments thereof that bind to periostin, where when bound to periostin, the recombinant antibodies or antigen-binding fragments thereof bind to the fasciclin 2 (FAS2) domain of periostin. In some embodiments, the recombinant antibodies or antigen-binding fragments thereof bind to any residue between amino acid residues 276 and 302 (inclusive) of periostin (SEQ ID NO: 15). In some embodiments, the recombinant antibodies or antigen-binding fragments thereof bind to at least one of the following residues of periostin (SEQ ID NO: 15): N276, R284, E288, L287, V295, or K302. In some embodiments, the recombinant antibodies or antigen-binding fragments thereof bind 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, the recombinant antibody or antigen-binding fragment thereof, when bound to periostin, binds to all of the following residues of periostin (SEQ ID NO: 15): N276, R284, E288, L287, V295, or K302. In some embodiments, the recombinant 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% identical, or 100% identical to the amino acid sequence set forth 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% identical, 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 some embodiments, the antibody or antigen-binding fragment thereof comprises an immunoglobulin heavy chain CDR1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO: 1 (GYTFTSYG); an immunoglobulin heavy chain CDR2 (CDR-H2) comprising the amino acid sequence set forth in any one of SEQ ID NOs: 2 (ISAYNGNT), 3 (ISAYSGNT), 4 (ISAYQGNT), 5 (ISAYTGNT), or 6 (ISAYDGNT); an immunoglobulin heavy chain CDR3 (CDR-H4) comprising the amino acid sequence set forth in SEQ ID NOs: 7 (DILVVPFDY), 8 (DVLVVPFDY), or 9 (DMLVVPFDY); immunoglobulin light chain CDR1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO: 10 (SSDIGSNR); immunoglobulin light chain CDR2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO: 11 (SND); and immunoglobulin light chain CDR3 (CDR-L3) comprising the amino acid sequence set forth 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 comprising the recombinant antibody or antigen-binding fragment thereof and a pharma- ceutical 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 thereof or pharmaceutical composition for use in reducing collagen content in a tumor. In some embodiments, the recombinant antibody or antigen-binding fragment thereof or pharmaceutical composition is for use in treating cancer. 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. Also described herein is a method for reducing collagen content in a tumor of an individual, comprising administering to the individual the recombinant antibody or antigen-binding fragment thereof or pharmaceutical composition.Also described herein is a method for increasing M1 macrophage phenotype and / or decreasing M2 macrophage phenotype in a tumor of an individual, comprising administering the recombinant antibody or antigen-binding fragment thereof, or pharmaceutical composition to the individual. Also described herein is a method for decreasing accumulation of suppressive granulocytic myeloid cells and / or tumor-associated macrophages in an individual, comprising administering the recombinant antibody or antigen-binding fragment thereof, or pharmaceutical composition to the individual. Also described herein is a method for increasing the frequency of CD4-positive and / or CD8-positive T cells in a tumor of an individual, comprising administering the recombinant antibody or antigen-binding fragment thereof, or pharmaceutical composition to the individual. Also described herein is a method for enhancing the function of CD8-positive T cells in a tumor, as measured by expression and / or release of interferon-gamma by CD8-positive T cells in the individual, comprising administering the recombinant antibody or antigen-binding fragment thereof, or pharmaceutical composition to the individual. Also described herein is a method for treating cancer in an individual, comprising administering a therapeutically effective amount of the recombinant antibody or antigen-binding fragment thereof, or pharmaceutical composition to the individual. 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. Also described herein is a method of making a composition for reducing collagen content in a tumor, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharmaceutically acceptable excipient, carrier, or diluent. Also described herein is a method of making a composition for increasing M1 macrophage phenotype and / or decreasing M2 macrophage phenotype in a tumor, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharmaceutically acceptable excipient, carrier, or diluent. Also described herein is a method of making a composition for reducing accumulation of suppressive granulocytic myeloid cells and / or tumor-associated macrophages in an individual, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharmaceutically acceptable excipient, carrier, or diluent.Also described herein is a method for making a composition for increasing the frequency of CD4-positive and / or CD8-positive T cells in a tumor of an individual, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharma- ceutically acceptable excipient, carrier, or diluent. Also described herein is a method for making a composition for increasing the function of CD8-positive T cells in a tumor, as measured by expression and / or release of interferon-gamma by CD8-positive T cells in an individual, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharma- ceutically acceptable excipient, carrier, or diluent. Also described herein is a method for making a composition for treating cancer, comprising mixing the recombinant antibody or antigen-binding fragment thereof with a pharma- ceutically 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] Also described herein are nucleic acids encoding any one of the above recombinant antibodies or antigen-binding fragments thereof.

[0008] Also described herein is a cell line comprising the above nucleic acid. In some embodiments, the cell line is a Chinese Hamster Ovary cell line. Also described herein is a method for producing the recombinant antibody or antigen-binding fragment thereof, comprising incubating the cell line in a cell culture medium under conditions sufficient to allow expression and secretion of the recombinant antibody or any one of the antigen-binding fragments thereof. [Brief description of the drawings]

[0009] The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features and advantages thereof will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the features described herein are utilized, and the accompanying drawings, in which: [Figure 1]FIG. 1 shows inhibition of periostin (POSTN)-mediated cell adhesion by 78 sequence unique IgGs tested at a single concentration of 500 nM. [Diagram 2] FIG. 2 shows tumor growth in a mouse MB49 bladder cancer model following treatment with NB0828 or vehicle control. [Diagram 3] Figure 3 shows the effect of treatment with NB0828 on intratumoral myeloid cell accumulation. MB49 tumor-bearing mice were treated with NB0828 or vehicle as described in Figure 2. Data are presented as a percentage of total CD45-positive immune infiltrate. [Figure 4] Figure 4 shows the change in total tumor collagen content following treatment with NB0828. MB49 tumor-bearing mice were treated as described in Figure 2, and total intratumoral collagen content of MB49 tumors at endpoint was assessed as described in Methods. [Diagram 5] FIG. 5 shows tumor growth in a mouse CT26 colon cancer model following treatment with NB0828 or vehicle control. [Figure 6] FIG. 6 shows decreased intratumoral accumulation of granulocytic cells / TAMs (tumor associated macrophages) and bias of macrophages towards the M1 phenotype in CT26 tumor-bearing mice treated with NB0828. [Figure 7] FIG. 7 shows increased accumulation of CD8- and CD4-positive infiltrating lymphocytes (TILs) and enhanced CD8-positive tumor-infiltrating lymphocyte function in CT26 tumor-bearing mice treated with NB0828. [Figure 8] FIG. 8 shows tumor growth in a mouse MC38 colon cancer model following treatment with NB0828 or vehicle control. [Figure 9A] Figures 9A-9D show that in the MC38 colon cancer model, NB0828 reduces the total amount of tumor-associated macrophages (9A) while increasing the frequency of proinflammatory type I macrophages (9B) and CD8+ T cells (9C), and that the efficacy of NB0828 against tumors is dependent on CD8+ T cells (9D). [Figure 9B] Figures 9A-9D show that in the MC38 colon cancer model, NB0828 reduces the total amount of tumor-associated macrophages (9A) while increasing the frequency of proinflammatory type I macrophages (9B) and CD8+ T cells (9C), and that the efficacy of NB0828 against tumors is dependent on CD8+ T cells (9D). [Figure 9C] Figures 9A-9D show that in the MC38 colon cancer model, NB0828 reduces the total amount of tumor-associated macrophages (9A) while increasing the frequency of proinflammatory type I macrophages (9B) and CD8+ T cells (9C), and that the efficacy of NB0828 against tumors is dependent on CD8+ T cells (9D). [Figure 9D] Figures 9A-9D show that in the MC38 colon cancer model, NB0828 reduces the total amount of tumor-associated macrophages (9A) while increasing the frequency of proinflammatory type I macrophages (9B) and CD8+ T cells (9C), and that the efficacy of NB0828 against tumors is dependent on CD8+ T cells (9D). [Figure 10] FIG. 10 shows a diagram for generating transforming growth factor beta-induced protein (BIGH3) / periostin chimeras for epitope mapping studies. [Figure 11A] 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 mutations in the FAS2 domain of POSTN EMI-FAS4. [Figure 11B] 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 mutations in the FAS2 domain of POSTN EMI-FAS4. [Figure 11C]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 mutations in the FAS2 domain of POSTN EMI-FAS4. [Figure 12] FIG. 12 shows the crystal structure of dimeric POSTN EMI-FAS4, with the location of the NB0828 epitope boxed and magnified in the lower half. [Figure 13A] 13A and 13B show the binding EC80 of human tenascin-C to periostin and the function blocking activity of NB0828 (13A), and the binding EC80 of human type I collagen to periostin and the function blocking activity of NB0828 (13B). [Figure 13B] 13A and 13B show the binding EC80 of human tenascin-C to periostin and the function blocking activity of NB0828 (13A), and the binding EC80 of human type I collagen to periostin and the function blocking activity of NB0828 (13B). [Figure 14A] FIG. 14A shows the prevalence of periostin expression in various tumor types as determined by immunohistochemistry. [Figure 14B] FIG. 14B shows representative images of immunohistochemical staining on breast cancer samples expressing low, moderate, and high periostin.

[0010] Detailed Description Described herein is a recombinant antibody or antigen-binding fragment thereof that binds to periostin, wherein the antibody or antigen-binding fragment thereof comprises: (a) an immunoglobulin heavy chain CDR1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO:1 (GYTFTSYG); (b) an immunoglobulin heavy chain CDR2 (CDR-H2) comprising the amino acid sequence set forth in any one of SEQ ID NOs:2 (ISAYNGNT), 3 (ISAYSGNT), 4 (ISAYQGNT), 5 (ISAYTGNT), or 6 (ISAYDGNT); (c) an immunoglobulin heavy chain CDR3 (CDR-H3) comprising the amino acid sequence set forth in SEQ ID NO:7 (DILVVPFD (d) an immunoglobulin heavy chain CDR1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO: 10 (SSDIGSNR); (e) an immunoglobulin light chain CDR2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO: 11 (SND); and (f) an immunoglobulin light chain CDR3 (CDR-L3) comprising the amino acid sequence set forth in SEQ ID NO: 12 (AAWDDSLSTYV).

[0011] Described herein is a recombinant antibody or antigen-binding fragment thereof that binds to periostin, wherein the antibody or antigen-binding fragment thereof comprises: (a) an immunoglobulin heavy chain CDR1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO: 1 (GYTFTSYG); (b) an immunoglobulin heavy chain CDR2 (CDR-H2) comprising the amino acid sequence set forth in SEQ ID NO: 16 (ISAYXGNT); (c) an immunoglobulin heavy chain CDR3 (CDR4) comprising the amino acid sequence set forth in SEQ ID NO: 17 (DXLVVPFDY). (d) an immunoglobulin light chain CDR1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO: 10 (SSDIGSNR); (e) an immunoglobulin light chain CDR2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO: 11 (SND); and (f) an immunoglobulin light chain CDR3 (CDR-L3) comprising the amino acid sequence set forth in SEQ ID NO: 12 (AAWDDSLSTYV), where X is any amino acid residue.

[0012] Described herein is a recombinant antibody or antigen-binding fragment thereof that binds periostin, comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, (a) where the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% identical, or 100% identical to the amino acid sequence set forth in SEQ ID NO:13; and (b) where the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% identical, or 100% identical to the amino acid sequence set forth in SEQ ID NO:14, where amino acid residue number 55 of SEQ ID NO:13 is asparagine, serine, glutamine, threonine, or aspartic acid, or where amino acid residue number 100 of SEQ ID NO:13 is methionine, isoleucine, or valine.

[0013] Also described herein is a recombinant antibody or antigen-binding fragment thereof that binds periostin, comprising an immunoglobulin heavy chain variable region and an immunoglobulin light chain variable region, wherein (a) the immunoglobulin heavy chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% identical, or 100% identical to the amino acid sequence set forth in SEQ ID NO:13; and (b) the immunoglobulin light chain variable region comprises an amino acid sequence that is at least about 90%, 95%, 97%, 99% identical, or 100% identical to the amino acid sequence set forth in SEQ ID NO:14.

[0014] Described herein is a recombinant antibody or antigen-binding fragment thereof that binds periostin, 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 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 identical to the amino acid sequence set forth in SEQ ID NO:14.

[0015] Described herein is a recombinant antibody or antigen-binding fragment thereof that binds to the fasciclin 2 (FAS2) domain of periostin. In certain embodiments, the recombinant antibody or antigen-binding fragment thereof, when bound to periostin, contacts an amino acid residue selected from amino acids 276-302 of SEQ ID NO: 15. In certain embodiments, the recombinant antibody or antigen-binding fragment thereof, when bound to periostin, contacts one of the following amino acid residues of SEQ ID NO: 15 (N276, R284, E288, L287, V295, or K302).

[0016] In the following description, certain specific details are presented to provide a thorough understanding of various embodiments. However, one of ordinary skill in the art will understand that the provided examples may be practiced without these details. Unless otherwise required by the context, throughout the specification and the following claims, the word "comprise" and its variations, such as "comprises" and "comprising," should be taken in an open and inclusive sense, i.e., "including but not limited to." As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally used in its sense including "and / or" unless the content clearly dictates otherwise. Moreover, the headings provided herein are merely for convenience and do not interpret the scope or meaning of the claimed embodiments.

[0017] As used herein, the term "about" refers to an amount closer to the stated amount by no more than 10%.

[0018] As used herein, the term "individual", "patient" or "subject" refers to an individual who has been diagnosed with, is suspected of having, or is at risk of developing at least one disease that the described compositions and methods are useful for treating. 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.

[0019] The term "treat" or "treating" as used herein refers to an intervention in an individual's physiological or disease state designed or intended to ameliorate at least one sign or symptom associated with the individual's physiological or disease state. Those skilled in the art will recognize that given a heterogeneous population of individuals suffering from a disease, not all individuals will respond equally or at all to a given treatment. An individual is considered to be treated regardless of any objective response criteria.

[0020] The antibodies provided include monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific and polyreactive antibodies), and antibody fragments. The antibodies include antibody conjugates, and molecules comprising antibodies, such as chimeric molecules. Thus, antibodies include full-length and naturally occurring antibodies, as well as fragments and portions thereof that retain their binding specificity, including any specific binding portion thereof, including those having any number of immunoglobulin classes and / or isotypes (e.g., IgG1, IgG2, IgG3, IgG4, IgA, IgD, IgE, and IgM); and Fab, F(ab') 2The term "monoclonal antibody" includes, but is not limited to, biologically relevant (antigen-binding) fragments or specific binding portions thereof, including, but not limited to, Fv, Fv, and scFv (single chain or related entities). Monoclonal antibodies are generally antibodies within a composition of substantially homogeneous antibodies; thus, any individual antibodies contained within the monoclonal antibody composition are identical except for possible naturally occurring mutations that may be present in small numbers. Polyclonal antibodies are preparations containing different antibodies of various sequences, generally directed against two or more different determinants (epitopes). The monoclonal antibody may comprise a human IgG1 constant region. The monoclonal antibody may comprise a human IgG4 constant region.

[0021] The term "antibody" is used herein in the broadest sense and includes polyclonal and monoclonal antibodies, such as intact antibodies and functional (antigen-binding) antibody fragments thereof, such as antigen-binding fragments (Fab), F(ab'), and the like. 2 The term includes fragments, Fab' fragments, Fv fragments, recombinant IgG (rIgG) fragments, single chain antibody fragments, such as single chain variable fragments (sFv or scFv), and single domain antibody (e.g. sdAb, sdFv, nanobody) fragments. The term encompasses engineered and / or otherwise modified forms of immunoglobulins, such as intrabodies, peptibodies, chimeric antibodies, fully human antibodies, humanized antibodies, and heteroconjugate antibodies, multispecific antibodies, such as bispecific antibodies, diabodies, triabodies, and tetrabodies, tandem di-scFv, tandem tri-scFv. Unless otherwise specified, the term "antibody" should be understood to encompass functional antibody fragments thereof. The term also encompasses intact or full-length antibodies, such as antibodies of any class or subclass, such as IgG and its subclasses, IgM, IgE, IgA, and IgD. The antibody may comprise a human IgG1 constant region. The antibody may comprise a human IgG4 constant region.

[0022] The terms "complementarity determining region" and "CDR" are synonymous with "hypervariable region" or "HVR", which are known in the art to refer to non-contiguous amino acid sequences in an antibody variable region that confer antigen specificity and / or binding affinity. Generally, there are three CDRs in each heavy chain variable region (CDR-H1, CDR-H2, CDR-H3) and there are three CDRs in each light chain variable region (CDR-L1, CDR-L2, CDR-L3). The terms "framework region" and "FR" are known in the art to refer to the non-CDR portions of the heavy and light chain variable regions. Generally, there are four FRs in each full-length heavy chain variable region (FR-H1, FR-H2, FR-H3, and FR-H4) and there are four FRs in each full-length light chain variable region (FR-L1, FR-L2, FR-L3, and FR-L4).The precise amino acid sequence boundaries of a given CDR or FR can be determined by Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering system); Al-Lazikani et al., (1997) JMB 273,927-948 ("Chothia" numbering system); 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 system); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains," Dev Comp Immunol, 2003. Jan;27(1):55-77 (the "IMGT" numbering system); Honegger A and Pluckthun A, "Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool," J Mol Biol, 2001 Jun 8;309(3):657-70, (the "Aho" numbering system); and Whitelegg NR and Rees AR, "WAM: an improved algorithm for modelling antibodies on the WEB," Protein Eng. 2000 Dec;13(12):819-24 (the "AbM" numbering system).

[0023] The boundaries of a given CDR or FR may vary depending on 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 in both the Kabat and Chothia schemes is based on the sequence length of the most common antibody regions, with insertions accommodated by insertion letters, e.g., "30a", and deletions occurring in some antibodies. The two schemes place certain insertions and deletions ("insertions") at different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is in many ways similar to the Chothia numbering system.

[0024] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The heavy and light chain variable domains of a native antibody (V H and V L ) generally have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs (see, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). H Domain or V L The domain may be sufficient to confer antigen-binding specificity. Moreover, an antibody that binds to a particular antigen may have a V domain derived from the antibody that binds the antigen. H Domain or V L Each of the complementary V domains is L Domain or V H They can be isolated by screening libraries of domains (see, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628(1991)).

[0025] Among the antibodies provided are antibody fragments. An "antibody fragment" refers to a molecule that is not an intact antibody and contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds. Examples of antibody fragments include Fv, Fab, Fab', Fab'-SH, F(ab') 2 These include, but are not limited to, diabodies, linear antibodies, single chain antibody molecules (e.g., scFv or scFv), and multispecific antibodies formed from antibody fragments. In certain embodiments, the antibody is a single chain antibody fragment comprising a heavy chain variable region and / or a light chain variable region, such as an scFv.

[0026] The term "specific binding" or "binding" as used herein refers to binding mediated by one or more amino acid residues of the CDR of the referenced antibody or fragment, or one or more variable region amino acid residues of the referenced antibody or fragment. The term "contact" or "contacting" as used herein in relation to the binding or binding of an antibody to a specific target refers to the amino acid residues of the variable region or CDR coming within 5 Å, 4 Å, 3 Å, or less of the listed contacting residues. Contacting includes hydrogen bonds, van der Waals interactions, and salt bridge formation between amino acid residues of the variable region or CDR of the antibody and the listed residues.

[0027] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, as well as recombinant host cell production. In some embodiments, the antibody is a recombinantly produced fragment, e.g., a fragment that contains an arrangement that does not occur in nature, e.g., a fragment having two or more antibody regions or antibody chains connected by a synthetic linker, e.g., a polypeptide linker, and / or a fragment that is not produced by enzymatic digestion of a naturally occurring intact antibody. In some aspects, the antibody fragment is an scFv.

[0028] A "humanized" antibody is an antibody in which all or substantially all CDR amino acid residues are derived from a non-human CDR and all or substantially all FR amino acid residues are derived from a human FR. A humanized antibody may optionally contain at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of a non-human antibody typically refers to a variant of a non-human antibody that has undergone humanization to reduce immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are replaced with the corresponding residues from a non-human antibody (e.g., the antibody from which the CDR residues are derived), e.g., to restore or improve the specificity or affinity of the antibody.

[0029] Among the antibodies provided are human antibodies. A "human antibody" is an antibody having an amino acid sequence that corresponds to that of an antibody produced by a human, or a human cell, or a non-human source utilizing a human antibody repertoire or other human antibody coding sequence, such as a human antibody library. The term excludes humanized forms of non-human antibodies that contain non-human antigen-binding regions, such as antibodies in which all or substantially all CDRs are non-human.

[0030] 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 challenge. Such animals typically contain all or part of a human immunoglobulin locus that replaces an endogenous immunoglobulin locus or is present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic animals, the endogenous immunoglobulin locus is generally inactivated. Human antibodies can also be derived from or selected from human antibody libraries, including phage display libraries and cell-free libraries, that contain antibody coding sequences derived from the human repertoire. In certain embodiments, human antibodies can have sequence defects removed or their affinity enhanced by successive rounds of selection by methods such as phage display.

[0031] The terms "polypeptide" and "protein" are used synonymously and refer to a polymer of amino acid residues and are not limited to a minimum length. Polypeptides (including antibodies and antibody chains provided and other peptides, such as linkers and binding peptides) can contain amino acid residues, including natural and / or non-natural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some embodiments, the polypeptide may contain modifications relative to the native or native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, such as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts producing the protein or errors due to PCR amplification.

[0032] In certain embodiments, antibodies with reduced effector functions are provided herein. The phrase "effector function" as used herein is meant to include the functional capabilities conferred by Fc-containing proteins when bound to FcγR. Without wishing to be bound to any one theory, the formation of Fc / FcγR complexes recruits various effector cells to the bound antigen site, typically resulting in a variety of signaling events and important subsequent immune responses within the cell. Effector function refers to both antibody-dependent cellular cytotoxicity and complement-dependent cellular cytotoxicity. In vitro and / or in vivo cytotoxicity assays can be performed to confirm the reduction / elimination of complement-dependent cellular cytotoxicity and / or antibody-dependent cellular cytotoxicity. For example, Fc receptor (FcR) binding assays can be performed to confirm that the antibody lacks binding to FcγR (and thus may lack antibody-dependent cellular cytotoxicity), but retains the ability to bind to FcRn. Non-limiting examples of in vitro assays for evaluating antibody-dependent cellular cytotoxicity of a molecule of interest are described in U.S. Patent Nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive assay methods may be used (e.g., ACTI™ and CytoTox96™ non-radioactive cytotoxicity assays). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs), monocytes, macrophages, and natural killer (NK) cells.

[0033] Percent sequence identity (%) to a reference polypeptide sequence is the proportion of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence, without considering any conservative substitutions as part of the sequence identity, after aligning the sequences and, if necessary, introducing gaps to achieve maximum sequence identity. Alignment for purposes of determining amino acid sequence identity can be accomplished in a variety of ways known in the art, for example, using publicly available computer software, such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment over the full length of the sequences being compared. However, for purposes herein, percent amino acid sequence identity values ​​are calculated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and the source code, together with user documentation, has been submitted to the United States Copyright Office, Washington, DC 20559, where it is registered under United States Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc. (South San Francisco, Calif.) or may be compiled from the source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0034] In the context where ALIGN-2 is used for comparing amino acid sequences, the % amino acid sequence identity of a given amino acid sequence A to a given amino acid sequence B (alternatively, it may be expressed as a given amino acid sequence A having or containing a particular % amino acid sequence identity to a given amino acid sequence B) is calculated as follows: fraction X / Y x 100, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in the program's alignment of A and B, and Y is the total number of amino acid residues in B. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not be equal to the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0035] In some embodiments, amino acid sequence variants of the antibodies provided herein are contemplated. Variants typically differ from the polypeptides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be synthetically produced, for example, by modifying one or more of the above polypeptide sequences of the invention and evaluating one or more biological activities of the polypeptide 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 antibodies may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, provided that the final construct has the desired characteristics, e.g., binding to the antigen.

[0036] In some embodiments, antibody variants are provided that have one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include CDRs and FRs. Amino acid substitutions can be introduced into an antibody of interest and the products screened for a desired activity, such as retained / improved antigen binding, reduced immunogenicity, or improved ADCC (antibody-dependent cellular cytotoxicity) or CDC (complement-dependent cytotoxicity).

[0037] In some embodiments, substitutions, insertions, or deletions may occur within one or more CDRs, where the substitutions, insertions, or deletions do not substantially reduce the binding of the antibody to the antigen. For example, conservative substitutions that do not substantially reduce binding affinity may be made within a CDR. Such modifications may be outside the "hot spots" of a CDR. H Sequence and V L In some embodiments of the sequences, each CDR is unaltered.

[0038] Modifications (e.g., substitutions) can be made within the CDRs, for example, to improve the affinity of the antibody. Such modifications can be made in the codons encoding the CDRs, which have a high mutation rate during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196(2008)), and the resulting mutants can be tested for binding affinity. Affinity maturation (e.g., using error-prone PCR, chain shuffling, randomization of CDRs, or oligonucleotide-directed mutagenesis) can be used to improve the affinity of the antibody (see, e.g., Hoogenboom et al. in Methods in Molecular Biology 178:1-37(2001)). CDR residues involved in binding to the antigen can be specifically identified, for example, using alanine scanning mutagenesis or modeling (see, e.g., Cunningham and Wells Science, 244:1081-1085(1989)). CDR-H3 and CDR-L3 are often specifically targeted. Alternatively or additionally, a crystal structure of an antigen-antibody complex to identify contact points between the antibody and antigen. Such contact and adjacent residues can be targeted or eliminated as candidates for substitution. Mutants can be screened to determine whether they contain the desired properties.

[0039] Amino acid sequence insertions and deletions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intersequence insertions and deletions of one or more amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody to an enzyme (e.g., for antibody-directed enzyme prodrug therapy) or a polypeptide which increases the serum half-life of the antibody. An example of an intrasequence insertional variant of an antibody molecule includes an insertion of three amino acids into the light chain. An example of a terminal deletion is an antibody with up to seven amino acids deleted at the end of the light chain.

[0040] In some embodiments, the antibody is modified to increase or decrease its glycosylation (e.g., by modifying the amino acid sequence so that one or more glycosylation sites are created or removed). The carbohydrate attached to the Fc region of the antibody may be modified. Natural antibodies derived from mammalian cells typically contain branched, biantennary oligosaccharides attached by N-linkage to Asn297 in the CH2 domain of the Fc region (see, e.g., Wright et al. TIBTECH 15:26-32 (1997)). The oligosaccharides can be various carbohydrates, e.g., mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, fucose attached to the GlcNAc at the "base" of the biantennary oligosaccharide structure. Modification of the oligosaccharides in the antibody can be performed, for example, to generate antibody variants with specific improved properties. Antibody glycosylation variants can have improved ADCC and / or CDC function. In some embodiments, antibody variants are provided that have a glycostructure that lacks fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such antibodies can be between 1% and 80%, between 1% and 65%, between 5% and 65%, or between 20% and 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycostructure at Asn297 compared to the sum of all glycostructures attached to Asn297 (see, e.g., WO 08 / 077546). Asn297 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 US A. 1969 May; 63(1):78-85). However, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., at positions 294 to 300, due to small sequence variations within the antibody. Such fucosylation variants may 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, such as knockout cell lines, e.g., Lec13 CHO cells deficient in protein fucosylation, and α-1,6-fucosyltransferase gene (FUT8) knockout CHO cells, and methods of use thereof, can be used to produce defucosylated antibodies (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)). Other antibody glycosylation mutants are also included (see, e.g., U.S. Pat. No. 6,602,684).

[0041] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of an antibody provided herein, thereby generating an Fc region variant. An Fc region herein is a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. Fc regions include native sequence Fc regions and variant Fc regions. An Fc region variant may comprise the sequence of a human Fc region (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) that contains an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0042] The antibodies may have extended half-life and improved binding to fetal Fc receptor (FcRn) (see, e.g., U.S. Patent Application Publication No. 2005 / 0014934). Such antibodies may comprise an Fc region having one or more substitutions in the Fc region that improve binding of the Fc region to FcRn, including those having substitutions at one or more of the 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 contemplated (see, e.g., Duncan & Winter, Nature 322:738-40 (1988); U.S. Patent Nos. 5,648,260 and 5,624,821; and WO 94 / 29351).

[0043] In some embodiments, it may be desirable to generate cysteine ​​engineered antibodies, e.g., "thiomonoclonal antibodies," in which one or more residues of an antibody are substituted with a cysteine ​​residue. In some embodiments, the substituted residue occurs at a site accessible to the antibody. The reactive thiol group may be located at a site for conjugation to another moiety, e.g., a drug moiety or a linker drug moiety, thereby generating an immunoconjugate. In some embodiments, any one or more of the following residues may be substituted with a 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.

[0044] In some embodiments, the antibodies provided herein may be further modified to contain additional non-proteinaceous moieties that are known and available. Moieties suitable for derivatization of 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), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone) polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated 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 may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and when more than one polymer is attached, they can be the same molecule or different molecules.

[0045] The antibodies described herein may be encoded by a nucleic acid. A nucleic acid is a type of polynucleotide that contains two or more nucleotide bases. In certain embodiments, a nucleic acid is a component of a vector that can be used to introduce a polypeptide that encodes a polynucleotide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule that can transport another nucleic acid to which it is linked. One type of vector is a vector that is integrated into a genome, i.e., an "integrative vector," which is capable of becoming integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid capable of extrachromosomal replication. A vector that can direct 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, and the like. Regulatory sequences, such as promoters, enhancers, and polyadenylation signals, used to control transcription in an expression vector 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 selection gene that facilitates recognition of transformants may additionally be incorporated. Vectors derived from viruses, such as lentiviruses, retroviruses, adenoviruses, adeno-associated viruses, etc., may be used. Plasmid vectors may be linearized for integration into chromosomal locations. Vectors may contain sequences that direct site-specific integration into a predefined location or a limited set of sites within the genome (e.g., AttP-AttB recombination). Additionally, vectors may contain sequences derived from transposable elements.

[0046] The terms "homologous", "homology" or "percent identity" as used herein to describe an amino acid or nucleic acid sequence compared to a reference sequence may be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87: 2264-2268, 1990, modified as in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such formula is incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215: 403-410, 1990). Percent identity of sequences may be determined using the most recent version of BLAST as of the filing date of this application.

[0047] Nucleic acids encoding the antibodies described herein can be used to infect, transfect, transform, or otherwise transfect suitable cells, thereby allowing the production of antibodies for commercial or therapeutic use. Standard cell line and large scale cell culture production methods are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production." Mabs. 2010 Sep-Oct; 2(5): 466-477. In certain embodiments, the cell is a eukaryotic cell. In certain embodiments, the eukaryotic cell is a mammalian cell. In certain embodiments, the mammalian cell is a Chinese Hamster Ovary (CHO) cell, an NS0 mouse myeloma cell, or a PER.C6® cell. In certain embodiments, the nucleic acid encoding the antibody is integrated into a genomic locus of a cell useful for producing the antibody. In certain embodiments, described herein are methods for making an antibody comprising culturing a cell containing nucleic acid encoding the antibody under in vitro conditions sufficient to allow production and secretion of the antibody.

[0048] 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 described herein integrated at a genomic location; 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, integrated at a genomic location, a nucleic acid encoding an antibody having (i) a heavy chain amino acid sequence that is at least 90% identical to the amino acid sequence set forth in SEQ ID NO: 13; and (ii) a light chain amino acid sequence that is at least 90% identical to the amino acid sequence set forth 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 that can withstand freezing with liquid nitrogen.

[0049] Also described herein are methods of making the antibodies described herein. Such methods include incubating a cell or cell line containing nucleic acid encoding the antibody in cell culture medium under conditions sufficient to allow expression and secretion of the antibody, and further harvesting the antibody from the cell culture medium. Harvesting may further include one or more purification steps to remove viable cells, cell debris, non-antibody proteins or polypeptides, undesired salts, buffers, and medium components. In certain embodiments, the additional purification step(s) include centrifugation, ultracentrifugation, dialysis, desalting, purification with Protein A, Protein G, Protein A / G, or Protein L, and / or ion exchange chromatography.

[0050] Anti-periostin antibody Described herein are antibodies that inhibit periostin (POSTN) function. Such antibodies are useful for the treatment of cancer. The antibodies described herein reduce tumor collagen content, increase macrophage polarization to the M1 phenotype, reduce infiltration of granulocytes and tumor-associated macrophages, and increase accumulation and anti-tumor properties of tumor-infiltrating T cells. In certain embodiments, anti-periostin antibodies reduce tumor collagen content by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, or 40% compared to no treatment or control treatment. In certain embodiments, anti-periostin antibodies reduce infiltration of granulocytes and tumor-associated macrophages by at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to no treatment or control treatment. In certain embodiments, anti-periostin antibodies reduce infiltration of CD11b-positive cells by at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to no treatment or control treatment. In certain embodiments, anti-periostin antibodies increase polarization of tumor-associated macrophages to M1 type (CD11b-positive, MHC class II-positive, CD206-negative) by at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to no treatment or control treatment. In certain embodiments, anti-periostin antibodies increase accumulation of CD4-positive and / or CD8-positive T cells in tumors by at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to no treatment or control treatment. In certain embodiments, the anti-periostin antibody increases interferon-gamma production by tumor-infiltrating CD8-positive T cells by at least about 20%, 25%, 30%, 35%, 40%, 45%, or 50% compared to no treatment or a control treatment.

[0051] Described herein is a recombinant antibody or antigen-binding fragment thereof that binds to periostin, wherein the antibody or antigen-binding fragment thereof comprises (a) an immunoglobulin heavy chain CDR1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO:1 (GYTFTSYG); (b) an immunoglobulin heavy chain CDR2 (CDR-H2) comprising the amino acid sequence set forth in SEQ ID NO:16 (ISAYXGNT); (c) an immunoglobulin heavy chain CDR3 (CDR-H3) comprising the amino acid sequence set forth in SEQ ID NO:17 (DXLVVPFDY); (d) an immunoglobulin light chain CDR1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO:10 (SSDIGSNR); (e) an immunoglobulin light chain CDR2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO:11 (SND); or (f) an immunoglobulin light chain CDR3 (CDR-L3) comprising the amino acid sequence set forth in SEQ ID NO:12 (AAWDDSLSTYV), where X is any amino acid.

[0052] Described herein is a recombinant antibody or antigen-binding fragment thereof that binds to periostin, wherein the antibody or antigen-binding fragment thereof comprises: (a) an immunoglobulin heavy chain CDR1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO:1 (GYTFTSYG); (b) an immunoglobulin heavy chain CDR2 (CDR-H2) comprising the amino acid sequence set forth in SEQ ID NO:16 (ISAYXGNT); (c) an immunoglobulin heavy chain CDR3 (CDR-H3) comprising the amino acid sequence set forth in SEQ ID NO:17 (DXLVVPFDY). (d) an immunoglobulin light chain CDR1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO: 10 (SSDIGSNR); (e) an immunoglobulin light chain CDR2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO: 11 (SND); and (f) an immunoglobulin light chain CDR3 (CDR-L3) comprising the amino acid sequence set forth in SEQ ID NO: 12 (AAWDDSLSTYV), wherein X is any amino acid.

[0053] Described herein is a recombinant antibody or antigen-binding fragment thereof that binds to periostin, wherein the antibody or antigen-binding fragment thereof comprises: (a) an immunoglobulin heavy chain CDR1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO:1 (GYTFTSYG); (b) an immunoglobulin heavy chain CDR2 (CDR-H2) comprising the amino acid sequence set forth in any one of SEQ ID NOs:2 (ISAYNGNT), 3 (ISAYSGNT), 4 (ISAYQGNT), 5 (ISAYTGNT), or 6 (ISAYDGNT); (c) an immunoglobulin heavy chain CDR3 (CDR-H3) comprising the amino acid sequence set forth in SEQ ID NO:7 (DILVVPFD (d) an immunoglobulin heavy chain CDR1 (CDR-L1) comprising the amino acid sequence set forth in any one of SEQ ID NO: 10 (SSDIGSNR); (e) an immunoglobulin light chain CDR2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO: 11 (SND); (f) and an immunoglobulin light chain CDR3 (CDR-L3) comprising 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 with deleted or reduced 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.

[0054] Also described herein is a recombinant antibody or antigen-binding fragment thereof, wherein the antibody or antigen-binding fragment thereof comprises: (a) an immunoglobulin heavy chain CDR1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO:1 (GYTFTSYG); (b) an immunoglobulin heavy chain CDR2 (CDR-H2) comprising the amino acid sequence set forth in SEQ ID NO:2 (ISAYNGNT); (c) an immunoglobulin heavy chain CDR3 (CDR-H3) comprising the amino acid sequence set forth in SEQ ID NO:9 (DMLVVPFDY); (d) an immunoglobulin light chain CDR1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO:10 (SSDIGSNR); (e) an immunoglobulin light chain CDR2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO:11 (SND); and (f) an immunoglobulin light chain CDR3 (CDR-L3) comprising 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 antibody described herein may comprise an Fc portion with deleted or reduced 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.

[0055] Also described herein is a recombinant antibody or antigen-binding fragment thereof that binds to periostin, 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% identical, 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% identical, 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 with deleted or reduced 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.

[0056] The antibody binding region, including the variable and CDR regions described herein, may be suitably formatted as part of an antibody with reduced effector function. In certain embodiments, the antibody is an F(ab') antibody that lacks the Fc region. 2In certain embodiments, the antibody may comprise one or more mutations to the constant region of the antibody heavy chain that reduce effector functions, such as antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. In certain embodiments, the antibody may 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 antigen-binding fragment thereof may be N434A, N434H, T307A / E380A / N434A, M252Y / S254T / T256E, 433K / 434F / 436H, T250Q, T250F, M428L, M428F, T250Q / M428L, N434S, V308W, The antibody comprises a mutation or set of mutations selected from 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 combinations thereof. In a particular embodiment, the antibody comprises an IgG4 constant region with a mutation corresponding to S228P in the heavy chain according to the EU numbering system. In certain embodiments, the antibody may comprise an IgG4 PAA constant region with mutations corresponding to S228P, F234A, and L235A in 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 2012 May 1; 4(3): 310-318.

[0057] In some embodiments, the antibodies described herein exhibit reduced affinity for C1q compared to the corresponding wild-type antibody, hi some embodiments, the antibodies exhibit affinity for the C1q 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 less than the corresponding wild-type antibody.

[0058] In some embodiments, the antibodies described herein exhibit an affinity for C1q that is 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 than the corresponding wild-type antibody. In some embodiments, the antibodies described herein exhibit an affinity for C1q of about 100 nM to about 100 μM, or about 100 nM to about 1 μM, or about 1 nM to about 100 μM, or about 10 nM to about 100 μM, or about 1 μM to about 100 μM, or about 10 μM to about 100 μM. In some embodiments, the antibodies described herein exhibit an affinity for C1q 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.

[0059] In some embodiments, the antibodies described herein exhibit reduced complement dependent cytotoxicity 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 less complement dependent cytotoxicity than the corresponding wild-type antibodies. 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% less complement dependent cytotoxicity compared to the corresponding wild-type antibodies. In some embodiments, the antibodies described herein do not exhibit any detectable complement dependent cytotoxicity. In some embodiments, the reduction and / or elimination of complement dependent cytotoxicity activity may result from a decreased affinity of the antibodies described herein for Fc ligands and / or receptors.

[0060] It is understood in the art that biological therapies can have adverse toxicity issues with the complex nature of directing the immune system to recognize and attack unwanted cells and / or targets. If the recognition and / or targeting for attack does not occur where treatment is needed, consequences such as adverse toxicity can occur. For example, staining of non-targeted tissues with an antibody can indicate a potential toxicity issue. In some embodiments, the antibodies described herein exhibit reduced staining of non-targeted tissues compared to the corresponding wild-type antibody. In some embodiments, the antibodies described herein exhibit reduced staining of non-targeted tissues 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 corresponding wild-type antibody. In some embodiments, the antibodies described herein exhibit reduced staining of non-targeted tissues 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% reduced compared to the corresponding wild-type antibody.

[0061] In some embodiments, the antibodies described herein exhibit reduced antibody-associated toxicity compared to the corresponding wild-type antibodies. In some embodiments, the antibodies described herein exhibit 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 less toxicity than the corresponding wild-type antibodies. 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 antibodies.

[0062] It is understood in the art that biological therapies may have adverse effects on platelet aggregation. In vitro and in vivo assays can be used to measure platelet aggregation. In some embodiments, the antibodies described herein exhibit reduced platelet aggregation in in vitro assays compared to the corresponding wild-type antibodies. In some embodiments, the antibodies described herein exhibit reduced platelet 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 corresponding wild-type antibodies. In some embodiments, the antibodies described herein exhibit reduced platelet aggregation in an in vitro assay 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% reduced compared to the corresponding wild-type antibody.

[0063] In some embodiments, the antibodies described herein exhibit reduced platelet aggregation in vivo compared to the corresponding wild-type antibody. In some embodiments, the antibodies described herein exhibit reduced platelet aggregation in an in vivo assay 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 corresponding wild-type antibody. In some embodiments, the antibodies described herein exhibit reduced platelet aggregation in an in vivo assay 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% reduced compared to the corresponding wild-type antibody.

[0064] In some embodiments, the antibodies described herein exhibit reduced platelet activation and / or platelet aggregation compared to the corresponding wild-type antibody.

[0065] Epitopes Bound by Therapeutically Useful Periostin Antibodies Described herein are unique epitopes or regions of human periostin that, when bound, inhibit periostin biological activities (e.g., integrin-mediated cell adhesion) and alter the tumor microenvironment (collagen remodeling and immune cell alterations). This binding is a combination of weak (van der Waals attractions), moderate (hydrogen bonds), and strong (salt bridges) interactions between the CDR amino acid residues of the antibody and amino acid residues in periostin (e.g., contact residues). In certain embodiments, contact residues are residues on periostin that form hydrogen bonds with residues on the anti-periostin antibody. In certain embodiments, contact residues are residues on periostin that form salt bridges with residues on the anti-periostin antibody. In certain embodiments, contact residues are residues on periostin that form van der Waals attractions with residues on the anti-periostin antibody and that are within at least 5 Å, 4 Å, or 3 Å of the residues on the anti-periostin antibody.

[0066] In certain embodiments, the anti-periostin antibodies described herein do not bind to tenascin-C or type I collagen.

[0067] In certain embodiments, described herein is an isolated antibody that binds to 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, described herein is an isolated antibody 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 are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in strong interactions with the antibody. In some embodiments, the antibody increases interferon gamma expression and / or release by CD8 positive T cells at the tumor site. In some embodiments, the antibody reduces accumulation of suppressive granulocytic myeloid cells and / or tumor associated macrophages in invasive tumors. In some embodiments, the antibody increases the proinflammatory M1 macrophage phenotype and / or decreases the M2 macrophage phenotype in infiltrating tumors, hi some embodiments, the antibody increases CD8 positive T cells to the tumor site, decreases tumor associated macrophages, and increases the proinflammatory M1 macrophage phenotype in infiltrating tumors.

[0068] In certain embodiments, described herein is an antibody comprising a CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, which binds to any one, two, three, four, five, or six of the following residues (N276, R284, E288, L287, V295, or K302) of SEQ ID NO: 15. In certain embodiments, described herein is an antibody comprising a CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, which binds to all of the following residues (N276, R284, E288, L287, V295, or K302) of SEQ ID NO: 15. In certain embodiments, the antibody binds only to residues that are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in strong interactions with the antibody.

[0069] In certain embodiments, described herein are antibodies that comprise a CDR different from the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 that binds to any one, two, three, four, five, or six of the following residues (N276, R284, E288, L287, V295, or K302) of SEQ ID NO: 15. In certain embodiments, described herein are antibodies that comprise a CDR different from the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 that binds to any one, two, three, four, five, or six of the following residues (N276, R284, E288, L287, V295, or K302) of SEQ ID NO: 15. In certain embodiments, the antibody binds only to residues that are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in strong interactions with the antibody.

[0070] In certain embodiments, described herein are antibodies that differ by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 from the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 by 1, 2, 3, 4, 5, or 5 amino acid residues, and that bind to any one, 2, 3, 4, 5, or 6 of the following residues (N276, R284, E288, L287, V295, or K302) of SEQ ID NO: 15. In certain embodiments, described herein are antibodies that differ by CDRs from the amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12 that bind to any one, 2, 3, 4, 5, or 6 of the following residues (N276, R284, E288, L287, V295, or K302) of SEQ ID NO: 15. 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.

[0071] In certain embodiments, described herein are antibodies that specifically bind periostin, 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 set forth 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 set forth in SEQ ID NO:14, and which bind 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, described herein is an antibody that specifically binds periostin, 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 set forth 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 set forth in SEQ ID NO: 14, and 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 are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in strong interactions with the antibody.

[0072] In certain embodiments, described herein is an antibody comprising a CDR having an amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, 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 set forth 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 set forth in SEQ ID NO: 14, and which 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, described herein is an antibody comprising a CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and binding 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.

[0073] In certain embodiments, described herein are antibodies that specifically bind periostin 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 set forth in SEQ ID NO:13, where amino acid residue number 55 of SEQ ID NO:13 is asparagine, serine, glutamine, threonine, or aspartic acid and 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 set forth 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, described herein are antibodies that specifically bind periostin 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 set forth in SEQ ID NO:13, where amino acid residue number 55 of SEQ ID NO:13 is asparagine, serine, glutamine, threonine, or aspartic acid and 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 set forth in SEQ ID NO:14, 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 are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in strong interactions with the antibody.

[0074] In certain embodiments, provided herein are CDRs having an amino acid sequence as set forth 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 as set forth 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 SEQ ID NO: and amino acid residue number 100 of SEQ ID NO:13 is a 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 set forth in SEQ ID NO:14, and which 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, provided herein are CDRs having an amino acid sequence as set forth 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 as set forth 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 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 set forth in SEQ ID NO: 14, and 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 are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in strong interactions with the antibody.

[0075] In certain embodiments, described herein are antibodies that compete for binding with an antibody comprising a CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and bind 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, described herein are antibodies that compete for binding with an antibody comprising a CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and bind to all of the following residues of SEQ ID NO: 15 (N276, R284, E288, L287, V295, or K302). In certain embodiments, the antibodies bind only to residues that are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in strong interactions with the antibody.

[0076] In certain embodiments, described herein are antibodies comprising a binding region that at least partially overlaps with a binding region of an antibody comprising a CDR having an amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, 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, described herein are antibodies comprising a binding region that at least partially overlaps with a binding region of an antibody comprising a CDR having an amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and 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 are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in strong interactions with the antibody.

[0077] In certain embodiments, described herein are antibodies 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 set forth 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 set forth in SEQ ID NO:14.

[0078] In certain embodiments, described herein are antibodies comprising 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 set forth 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 set forth in SEQ ID NO:14.

[0079] In certain embodiments, described herein are antibodies that compete for binding with an antibody comprising a CDR having an amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, 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 set forth 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 set forth in SEQ ID NO: 14, and that bind 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, described herein is an antibody comprising a CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and binding 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.

[0080] In certain embodiments, described herein are antibodies comprising a binding region that at least partially overlaps with a binding region of an antibody comprising a CDR having an amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth 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 set forth 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, described herein is an antibody comprising a CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and binding 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.

[0081] In certain embodiments, provided herein is a human or humanized heavy chain variable region amino acid sequence that competes for binding with an antibody comprising a CDR having an amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and that is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence set forth in SEQ ID NO: 13, where amino acid residue number 55 of SEQ ID NO: 13 is an asparagine, serine, glutamine, threonine, or asparagine. 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 set forth in SEQ ID NO: 14, and binds to any one, two, three, four, five, or six of the following residues (N276, R284, E288, L287, V295, or K302) of SEQ ID NO: 15. In certain embodiments, described herein are antibodies comprising a CDR having an amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and binds to all of the following residues (N276, R284, E288, L287, V295, or K302) of SEQ ID NO: 15. In certain embodiments, the antibody binds only to residues that are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in strong interactions with the antibody.

[0082] In certain embodiments, provided herein is a human or humanized heavy chain variable region amino acid sequence that at least partially overlaps with a binding region of an antibody comprising a CDR having an amino acid sequence as set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12, and is at least about 80%, about 90%, about 95%, about 97%, about 98%, or about 99% identical to the amino acid sequence as set forth in SEQ ID NO: 13 (wherein the amino acid at residue number 55 of SEQ ID NO: 13 is an asparagine, serine, glutamine, threonine, or asparagine). 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 set forth in SEQ ID NO: 14, and comprising a binding region that binds to any one, two, three, four, five, or six of the following residues (N276, R284, E288, L287, V295, or K302) of SEQ ID NO: 15. In certain embodiments, described herein are antibodies that comprise a CDR having an amino acid sequence set forth in any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, and 12. In certain embodiments, the antibody binds only to residues that are involved in strong or moderate interactions with the antibody. In certain embodiments, the antibody binds only to residues that are involved in strong interactions with the antibody.

[0083] treatment method The antibodies disclosed herein are useful for the treatment of cancer or tumor. Treatment refers to a method that seeks to improve or ameliorate the condition being treated. Treatments for cancer include, but are not limited to, reducing tumor volume, reducing tumor volume growth, progression-free survival or lifespan extension. In certain embodiments, treatments will affect the remission of the cancer being treated. In certain embodiments, treatments include use as a prophylactic or maintenance dose to prevent the recurrence or progression of a previously treated cancer or tumor. It will be understood by those skilled in the art that although the antibodies may be safe and effective, not all individuals will respond equally to the treatment administered, and these individuals will nonetheless be considered to be treated.

[0084] Tumors treatable with the antibodies described herein include those that express periostin. Periostin is a component of the extracellular matrix secreted by cancer-associated fibroblasts, and thus most tumors will express or contain periostin. In certain embodiments, the tumors to be treated are periostin positive, have detectable periostin, or are known to have detectable periostin based on population analysis of high probability tumors. In certain embodiments, periostin-high tumors are those with an IHC score of about 50 or greater.

[0085] 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 tumors of the breast, heart, lung, small intestine, large intestine, spleen, kidney, bladder, head, neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testis, and liver. In certain embodiments, tumors or cancers that may be treated with the antibodies of the present invention include adenoma, adenocarcinoma, hemangiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, hemangiosarcoma, hematoma, hepatoblastoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and / or teratoma. In certain embodiments, the tumor / cancer is selected from the group consisting of acral lentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytoma, Bartholin's adenocarcinoma, basal cell carcinoma, bronchial adenocarcinoma, capillary carcinoid, cellular carcinoma, carcinosarcoma, cholangiocarcinoma, chondrosarcoma, cystadenoma, yolk sac tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrioid adenocarcinoma, ependymoma, Ewing's sarcoma, focal nodular hyperplasia, gastrinoma, germ line tumor, glioma, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, hepatic adenoma, hepatic adenomatosis, hepatocellular carcinoma, insulinite, intraepithelial neoplasia, intraepithelial squamous cell neoplasm, invasive squamous cell carcinoma, large cell carcinoma, The cancer is selected from the group consisting of liposarcoma, lung cancer, lymphoblastic leukemia, lymphocytic leukemia, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelioma, schwannoma, medulloblastoma, medulloepithelioma, mesothelioma, mucoepidermoid carcinoma, myeloid leukemia, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian cancer, serous papillary 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 cancer, VIP (vasoactive intestinal peptide)-secreting tumor, and Wilms' tumor.In certain embodiments, the tumors / cancers to be treated with one or more antibodies of the invention include brain cancer, head and neck cancer, head and neck squamous cell carcinoma, colorectal cancer, acute myeloid leukemia, precursor 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, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, prostate adenocarcinoma, and breast cancer, preferably ductal carcinoma, 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 this disclosure comprises glioblastoma. In certain embodiments, the cancer treated with one or more antibodies of this disclosure comprises pancreatic cancer. In certain embodiments, the cancer treated with one or more antibodies of this disclosure comprises ovarian cancer. In certain embodiments, the cancer treated with one or more antibodies of this disclosure comprises lung cancer. In certain embodiments, the cancer treated with one or more antibodies of this disclosure comprises squamous cell lung cancer. In certain embodiments, the cancer treated with one or more antibodies of this disclosure comprises prostate cancer. In certain embodiments, the cancer treated with one or more antibodies of this disclosure comprises colon cancer. In certain embodiments, the cancer treated comprises glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer. In certain embodiments, the cancer is refractory to other treatments. In certain embodiments, the cancer treated has recurred. In certain embodiments, the cancer is relapsed / refractory glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer.

[0086] In certain embodiments, the antibody may be administered to a subject in need thereof by any route suitable for administration of a pharmaceutical composition containing the antibody, such as subcutaneous, intraperitoneal, intravenous, intramuscular, intratumoral, or intracerebral. 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 in an appropriate dosing regimen, such as once weekly, twice weekly, once monthly, twice monthly, once every two weeks, once every three weeks, or once monthly. In certain embodiments, the antibody is administered once every three weeks. The antibody may be administered in any therapeutically effective amount. In certain embodiments, a therapeutically acceptable amount is from about 0.1 mg / kg to about 50 mg / kg. In certain embodiments, a therapeutically acceptable amount is from about 1 mg / kg to about 40 mg / kg. In certain embodiments, a therapeutically acceptable amount is from about 5 mg / kg to about 30 mg / kg. A therapeutically effective amount includes an amount sufficient to ameliorate one or more symptoms associated with the disease or affliction being treated.

[0087] The anti-periostin antibodies described herein are also useful in methods for reducing collagen content in a tumor of an individual.

[0088] The anti-periostin antibodies described herein are also useful in methods for increasing the M1 macrophage phenotype and / or decreasing the M2 macrophage phenotype in a tumor of an individual.

[0089] The anti-periostin antibodies described herein are also useful in methods for reducing the accumulation of suppressive granulocytic myeloid cells and / or tumor-associated macrophages in an individual.

[0090] The anti-periostin antibodies described herein are also useful in methods for increasing the frequency of CD4+ and / or CD8+ T cells in a tumor of an individual.

[0091] The anti-periostin antibodies described herein are also useful in methods for increasing interferon-gamma expression and / or release by CD8 positive T cells within a tumor in an individual.

[0092] The antibodies described herein are useful in the manufacture of a medicament for reducing collagen content in a tumor in an individual.

[0093] The antibodies described herein are useful in the manufacture of a medicament for increasing the M1 macrophage phenotype and / or decreasing the M2 macrophage phenotype in a tumor of an individual.

[0094] The antibodies described herein are useful in the manufacture of a medicament for reducing the accumulation of suppressive granulocytic myeloid cells and / or tumor associated macrophages in an individual.

[0095] The antibodies described herein are useful in the manufacture of a medicament for increasing the frequency of CD4+ and / or CD8+ T cells in a tumor of an individual.

[0096] The antibodies described herein are useful in the manufacture of a medicament for increasing interferon-gamma expression and / or release by CD8 positive T cells within a tumor in an individual.

[0097] Pharmaceutically acceptable excipients, carriers, and diluents The antibodies described herein may be provided in an isolated and purified form that is sufficiently pure for administration to a human individual.

[0098] In certain embodiments, the anti-periostin antibodies of the present disclosure are included in a pharmaceutical composition comprising one or more pharma- ceutically acceptable excipients, carriers, and diluents. In certain embodiments, the antibodies of the present disclosure are administered suspended in a sterile solution. In certain embodiments, the solution comprises about 0.9% NaCl or about 5% dextrose, glucose, or sucrose. In certain embodiments, the solution further comprises one or more of a buffer, such as acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant, such as polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; a polyol / disaccharide / polysaccharide, such as glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, such as glycine or arginine; an antioxidant, such as ascorbic acid, methionine; or a chelating agent, such as EDTA or EGTA.

[0099] In certain embodiments, the antibodies of the present disclosure are shipped / stored lyophilized and reconstituted prior to administration. In certain embodiments, the lyophilized antibody formulation includes a bulking agent, such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or a combination thereof. The lyophilized formulation may be contained in a vial of glass or other suitable non-reactive material. When formulated, whether reconstituted or not, the antibody may be buffered at a particular pH, generally below 7.0. In certain embodiments, the pH may be 4.5-6.5, 4.5-6.0, 4.5-5.5, 4.5-5.0, or 5.0-6.0.

[0100] Also described herein are kits that include one or more antibodies described herein in a suitable container and one or more additional components selected from instructions for use, diluents, excipients, carriers, and devices for administration.

[0101] In certain embodiments, described herein are methods of preparing a cancer treatment comprising mixing one or more pharma- ceutically acceptable excipients, carriers, or diluents with an antibody of the present disclosure. In certain embodiments, described herein are methods of preparing a cancer treatment for storage or shipment comprising lyophilizing one or more antibodies of the present disclosure.

[0102] Working Example The following illustrative examples are representative of embodiments of the compositions and methods described herein, and are not meant to be limiting in any way.

[0103] Example 1 - Antibody generation and screening A phage display antibody discovery campaign was performed using a fully human phage library to isolate binders to periostin. Briefly, three rounds of panning were performed using either recombinant human periostin, recombinant mouse periostin, or a combination, with emphasis on identifying mouse cross-reactive binders. From this panning strategy, 78 sequence-unique ScFvs that cross-reacted with mouse periostin were identified and generated in human IgG1 format for functional screening in cell adhesion assays. See Figure 1.

[0104] Recombinant human or mouse periostin was coated onto 96-well plates overnight at 4°C. The next day, the plates were washed with PBS and blocked with 2% bovine serum albumin for 1 hour at 37°C. After blocking, antibodies were added to the plates and incubated at 37°C for 30 minutes. After incubation, 50,000 IMR90 human lung fibroblasts or 50,000 MLG mouse fibroblasts were then added to the wells and allowed to incubate at 37°C for 2 hours. The plates were then washed twice with PBS and the confluency of the wells was measured using the IncuCyte platform. From the high concentration single dose screen at 500 nM, 21 IgGs were identified as having greater than 50% inhibition, as shown in Figure 1, and were advanced into binding screening to determine their relative affinity for human and mouse periostin, as shown in Table 1 below.

[0105] To determine the relative affinity for recombinant human or mouse periostin, these proteins were coated on maxisorp plates overnight at 4°C. The next day, the plates were blocked with casein blocking buffer for 1 hour at 37°C. A titration of each antibody was added to the plate and allowed to bind for 1 hour at room temperature. The plate was washed 4 times with PBST and then incubated with anti-human Fc secondary antibody conjugated to horseradish peroxidase for 30 minutes at room temperature. The plate was then washed again with 4x PBST and then developed using TMB substrate and 1M HCl. From this screen, four clones were selected (marked by bold and italics in Table 1) that had binding EC50 values ​​of less than 1 nM for both human and mouse periostin.

[0106] [Table 1]

[0107] Example 2 - Generation of NB0828 and sequence variants The four candidates were retested in a dose-response cell adhesion assay to determine IC50 values. From this screen, NB0627 was identified as a particularly suitable IgG (Table 2).

[0108] [Table 2]

[0109] NB0627 was then converted to an effector-silent IgG4 PAA isotype to generate the lead candidate NB0828. Sequence analysis of NB0828 identified two post-translational modification defects in the VH region. The first deamidation site is located in CDR-H2, and the second oxidation site is located in CDR-H3. Therefore, in an attempt to remove these defects, several single and double mutants were generated and their binding and activity were measured. A summary of the results of IC50 and EC50 values ​​for NB0828 and its mutants is listed in Table 3.

[0110] [Table 3]

[0111] Example 3 - In vivo efficacy of NB0828 in mouse bladder MB49 and colon CT26 tumor models The efficacy of NB0828 was tested in two separate tumor models, bladder MB49 and colon CT26. 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, mice were treated intraperitoneally with either NB0828 (50 mg / kg, three times a week) or vehicle control (PBS). Tumor volume was assessed twice a week by caliper measurement and was calculated as (length × width). 2 The tumor size was calculated as: ) / 2. Mice were euthanized when tumor size exceeded 15 mm in any single direction or due to tumor ulceration as the humane endpoint.

[0112] As shown in Figure 2 (MB49) and Figure 5 (CT26), NB0828 was effective in reducing tumor growth in both models. This reduction in tumor growth in the MB49 model was accompanied by a lower frequency of intratumoral granulocytic myeloid cells, as shown in Figure 3, and lower collagen content, as shown in Figure 4. Similar to the MB49 model, the CT26 model showed a reduction in granulocytic myeloid cells. Furthermore, NB0828 reduced the frequency of tumor-infiltrating macrophages, and those macrophages that were present were biased toward an M1 phenotype as a result of treatment with NB0828, as shown in Figure 6. In the CT26 mouse model, treatment with NB0828 was also accompanied by a higher frequency of tumor-infiltrating CD8+ and CD4+ T cells, as well as enhanced CD8+ T cell function as measured by higher expression of interferon-gamma, as shown in Figure 7.

[0113] Immunophenotyping MB49 or CT26 tumor-bearing mice were treated with NB0828 or vehicle control starting on day 3 as indicated. For data shown, immunophenotyping was performed on days 20 and 18 after tumor implantation for MB49 and CT26, respectively. Tumors were excised, skin removed, mechanically disrupted using scalpel blades, and then enzymatically digested using Miltenyi Mouse Tumor Dissociation Enzyme Cocktail. Digested samples were passed through a 40 μm strainer, washed with RPMI, followed by a second wash with RPMI + 10% fetal bovine serum. Cells were then resuspended for counting and counted up to 2 × 10 per sample. 6 White blood cells were plated and stained for analysis by flow cytometry. For the evaluation of the function of CD8-positive tumor-infiltrating lymphocytes in the CT26 model, single cell suspensions of digested tumors were incubated with the AH1 peptide [H2-L] in the presence of anti-CD28 antibody and brefeldin A. dCells were stimulated for 5 hours at 37°C with the restricted gp70(423-431) MuLV epitope, an immunodominant CD8+ T cell epitope expressed by CT26 cells. After stimulation, cells were stained by standard surface / intracellular staining methods and flow cytometry was used to detect IFN-γ production by CD8+ T cells. 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 allow for examination of only live cell events and the pan-leukocyte marker CD45 was included to allow for normalization of populations within the immune compartment. Immune populations of interest were phenotypically / functionally defined as follows: total myeloid cells (CD45+CD11b+), granulocytes (CD45+CD11b+Gr-1 hi or CD45+CD11b+Ly6G+Ly6C lo), macrophages (CD45+CD11b+Ly6G+Ly6C lo / negative F4 / 80+), M1 macrophages (MHC II+CD206+), M2 macrophages (MHC II+CD206+), CD8+ tumor-infiltrating lymphocytes (CD45+CD11b+CD3+CD90.2+CD8+), CD4+ tumor-infiltrating lymphocytes (CD45+CD11b+CD3+CD90.2+CD4+), IFN-γ+CD8+ tumor-infiltrating lymphocytes (CD45+CD11b+CD3+CD8+IFN-γ+). Median fluorescence intensity (MFI) was used for determination of IFN-γ staining intensity derived from IFN-γ positive CD8 positive tumor infiltrating lymphocytes.

[0114] [Table 4]

[0115] Collagen Content Total collagen content of tumors was assessed by quantification of hydroxyproline using the QuickZyme total collagen assay. For sample preparation, MB49 tumors were excised from tumor-bearing mice when tumors reached the endpoint, flash frozen in liquid nitrogen, stored at -80°C, and subsequently analyzed. Tumor material was weighed, resuspended in 6M HCl at a ratio of 200 mg tumor per ml HCl, vortexed, and incubated at 95°C for 20 hours. The tubes were cooled, centrifuged at 13,000 RPM for 10 minutes, and the supernatant was collected. The supernatant was diluted with Milli-Q water and then 4M HCl according to the manufacturer's recommended protocol and plated for detection of hydroxyproline using the supplied buffer and detection reagents, with technical replicates. Absorbance readings at 570 nm were measured and the amount of collagen in each sample was calculated by comparison to a standard curve generated using the supplied collagen. The calculated total collagen amount (μg) for each sample was divided by the total tumor input mass (mg) to normalize the data between tumor samples.

[0116] Example 4 - In vivo efficacy of NB0828 in the mouse colon MC38 tumor model NB0828 was tested for its efficacy in reducing tumor growth in a mouse colon MC38 tumor model, and the results are shown in Figure 8. Briefly, 200,000 MC38 cells were injected intradermally into the flank of female C57BL / 6 mice. Three days after tumor implantation, mice were treated intraperitoneally with either NB0828 (50 mg / kg, 3 times per week) or vehicle control (PBS). For depletion of CD8-positive T cells, anti-mouse CD8a antibody (clone 2.43) or IgG isotype control (clone LTF-2) was delivered along with NB0828 for the first six doses (10 mg / kg, 3 times per week). Depletion of T cells in the blood was confirmed by flow cytometry using the flow staining panel listed in Table 5. Tumor volumes were measured using the same methods as described for the MB49 and CT26 models. NB0828 was effective in reducing tumor growth in the MC38 model (Figure 8). NB0828 was also effective in increasing CD8 positive T cells, decreasing the frequency of tumor-associated macrophages (TAMs), and increasing the ratio of proinflammatory macrophages by altering the tumor microenvironment, as shown in Figures 9A and 9C. The efficacy of NB0828 in reducing tumor growth in this model was dependent on CD8 positive T cells, because removal of CD8 positive T cells during treatment with NB0828 reversed the beneficial effects of NB0828, as shown in Figure 9D. In summary, this data indicates that NB0828 effectively reduces tumor growth, increases CD8 positive T cells to the tumor site, decreases tumor-associated macrophages, and increases the proinflammatory M1 macrophage phenotype in infiltrating tumors.

[0117] Immunophenotyping Tumors were excised, skin removed, mechanically disrupted using a scalpel, and then enzymatically digested using Miltenyi Mouse Tumor Dissociation Enzyme Cocktail (45 min incubation at 37°C on a shaking platform). Digested samples were passed through a 40 μm strainer, washed with RPMI, followed by a second wash with RPMI + 10% fetal bovine serum. Cells were then resuspended for counting, up to 2 × 10 per sample. 6 White blood cells were plated and stained for analysis by flow cytometry. For the assessment of CD8-positive tumor infiltrating lymphocyte (CD8-positive TIL) function in the MC38 model, single cell suspensions of digested tumors (up to 2 × 10 per sample) were plated and stained for analysis by flow cytometry. 6 leukocytes) in the presence of anti-CD28 antibody and brefeldin A, the p15E peptide [expressed by MC38 tumors, H2-K b Restrictive p15E(604-611) MuLV epitope] for 5 hours at 37°C. After stimulation, cells were stained to detect IFN-γ production by CD8+ T cells using flow cytometry with standard surface / intracellular staining methods using eBioscience Intracellular Fixation and Permeabilization Buffer Set. 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 allow for examination of only live cell events, and the pan-leukocyte marker CD45 was included to allow for normalization of populations within the immune compartment. For studies in MC38, reported immune populations of interest were phenotypically / functionally defined as follows: total myeloid cells (CD45+CD11b+), macrophages (CD45+CD11b+Ly6G+Ly6C lo / negative F4 / 80+), M1 macrophages (MHC II+), M2 macrophages (MHC II+), CD8+ tumor infiltrating lymphocytes (CD45+CD11b+CD3+SSC lo CD8+), IFN-γ+CD8+ tumor infiltrating lymphocytes (CD45+CD11b+CD3+SSC lo CD8+IFN-γ+).

[0118] [Table 5]

[0119] Example 5 - NB0828 binds to the FAS2 domain of periostin (POSTN) The binding region of NB0828 was examined to further characterize the antibody.

[0120] Construction of BIGH3 / POSTN chimeric protein The closest homologous protein to periostin in sequence is transforming growth factor beta-induced protein (BIGH3), which has 48% sequence homology within the EMI-FAS4 region of the protein. Although the sequence homology between the two proteins is low, the overall domain structure is very similar, containing one EMI domain and four tandem fasciclin (FAS) domains. NB0828 binds with high affinity to periostin but not to BIGH3. To further characterize the binding domain of NB0828, a BIGH3 / POSTN chimeric protein was generated in which each domain of periostin was replaced with the corresponding domain of BIGH3, generating five BIGH / POSTN chimeras (Figure 10). NB0828 binding studies were performed on these proteins by ELISA. As shown in Figure 11A, NB0828 retains binding to all BIGH3 / POSTN chimeras except for the FAS2 chimera. This observable decrease in binding when the POSTN FAS2 domain was replaced with the BIGH3 FAS2 domain indicates that NB0828 binds to the FAS2 domain of POSTN.

[0121] Generation of POSTN FAS2 mutants The FAS2 domain of periostin is a conformational structure composed of 132 amino acids. Single amino acid substitution (alanine) mutants at various positions between the FAS2 domains were generated to further define the NB0828 epitope or binding region and to identify important contact residues (Table 5). The published crystal structure (Liu et al., 2018; PDB # 5YJG) was used to identify residues for mutagenesis based on surface exposure in MOE, a computational analysis tool.

[0122] [Table 6]

[0123] 23 alanine mutants spanning residues across the entire FAS2 domain were generated in the first round. NB0828 binding studies were performed on these mutants, and mutants with greater than 50% reduction from the maximum signal in the assay were identified as residues important for binding. Figure 11B shows the results of that screen. Two mutants were identified that displayed amino acids important for NB0828 binding: NB1205 (R284A) and NB1207 (E288A). Due to the proximity of these two amino acids (Figure 12), 11 additional mutants were generated in the second round, focusing more specifically on residues in this region. Figure 11C shows the results of the second round of screening. Based on the ELISA results of the second round of screening, four additional mutants were identified that displayed amino acids important for NB0828 binding: NB1243 (L287A), NB1246 (V295A), NB1248 (K302A), and NB1202 (N276A). Notably, NB1190 (D245A), which was very close to, but did not fall below, a >50% reduction from the maximum signal cutoff, is located in a separate loop from the remaining residues, but is within the FAS domain and on the NB0828-binding loop, forming a contact point with N276 (Figure 12). Since N276 was identified as a residue important for binding to NB0828, the D245 residue may also be either a contact residue for NB0828 or provide structural integrity for the NB0828 epitope loop that is important for binding.

[0124] Determining affinity of NB0828 between species The binding affinity of NB0828 between human, mouse, rat, and cynomolgus monkey species was determined. The affinity of NB0828 was determined using the octet red system. Briefly, an anti-human Fc antibody (AHC) biosensor was used to capture NB0828 and then associated with titrated amounts of recombinant human, mouse, rat, or cynomolgus monkey periostin (100 nM → 0 nM; 1:2 dilution). Recombinant human, mouse, and rat periostin were purchased commercially from R&D Systems, Inc., and cynomolgus monkey periostin was produced in-house. The results show that the affinity of NB0828 for periostin between the four species is very similar, in the range of 0.1-0.5 nM. This data strongly supports the epitope mapping experiments detailed above, since the sequence identity in the binding loop of NB0828 described was 100% between the four species.

[0125] [Table 7]

[0126] Example 6 - NB0828 does not block periostin binding to tenascin-C and type I collagen but does block cell adhesion To determine whether NB0828 blocks periostin binding to the extracellular proteins 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, plates were blocked with casein blocking buffer for 1 hour at 37°C. Binding EC for recombinant human tenascin-C and human type I collagen 80To determine the EC of each protein, proteins were biotinylated 10:1 using EZ-Link™ NHS-PEG4-biotin (Fisher) and titrations of each biotinylated protein were added to the plate and incubated at room temperature for 1 hour. Plates were washed 4 times with PBST and then detected using avidin-HRP (horseradish peroxidase) for 30 minutes. Plates were then washed again 4x PBST and developed using TMB substrate and 1M HCl. The EC of each protein was 80 was determined using GraphPad Prism 7 software. For competitive ELISA, titrations of NB0828, control IgG, or PBST were added to the plate and incubated at room temperature for 30 min with shaking. Plates were then washed 4 times with PBST and assayed for EC values ​​of either biotinylated tenascin-C (0.9 nM, 13A, left) or collagen (100 nM, 13B, left). 80 Binding concentrations or premixed blocking controls were added to the plate. The premixed blocking controls were EC 80 Biotinylated proteins were prepared by mixing titrations of recombinant human periostin at concentrations of 0.1% and incubated at room temperature with shaking for 30 min before being added to the plates, which were then incubated at room temperature with shaking for 1 h, washed, detected, and developed as described above.

[0127] Tenascin-C and type I collagen had binding EC values ​​of 0.9 nM and 100 nM, respectively. 80 NB0828 bound to periostin at 100 ng / mL (Figures 13A and 13B). As shown in Figures 13A and 13B, in contrast to the premixed positive control, NB0828 did not inhibit periostin binding to tenascin-C and type I collagen. In summary, these data indicate that NB0828 does not block the interaction of periostin with the extracellular matrix proteins tenascin-C and type I collagen.

[0128] Example 7 - Immunohistochemistry (IHC) assessment of periostin expression among human tumor indications To assess periostin expression levels among various human cancers, an immunohistochemical (IHC) prevalence study was performed. 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 product identification number: ab215199, lot identification number: GR3192974-3) diluted 1:50. Staining was quantified using digital pathology (HALO, Indica labs) and an IHC score was calculated (IHC score = [% area of ​​low intensity staining]). * 1]+[Medium intensity staining area% * 2]+[High intensity staining area% * 3]). Cut points for low / high periostin staining were calculated based on the approximate mean periostin IHC score across all samples, with a periostin IHC score of 50. Prevalence studies showed a range of periostin staining across and within the indications tested, with pancreatic, breast, and squamous cell lung cancers showing the highest levels of periostin staining (Fig. 14A). Periostin-high tumors were present in all indications, but with different frequencies (Fig. 14A). Representative immunohistochemistry images of periostin expression in breast cancer are shown in Fig. 14B. Altogether, these data demonstrate that periostin is widely expressed across multiple tumor types.

[0129] While 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. Numerous variations, changes, and substitutions will readily 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 used in practicing the invention.

[0130] All publications, patent applications, issued patents, and other documents mentioned herein are incorporated by reference herein as if each individual publication, patent application, issued patent, and other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in the documents incorporated by reference are excluded to the extent they conflict with definitions in this disclosure.

[0131] [Table 8]

Claims

1. A recombinant antibody or antigen-binding fragment thereof that binds to periostin, wherein the antibody or antigen-binding fragment thereof comprises: a) an immunoglobulin heavy chain CDR1 (CDR-H1) comprising the amino acid sequence set forth in SEQ ID NO:1 (GYTFTSYG); b) an immunoglobulin heavy chain CDR2 (CDR-H2) comprising the amino acid sequence set forth in SEQ ID NO:2 (ISAYNGNT); c) an immunoglobulin heavy chain CDR3 (CDR-H3) comprising the amino acid sequence set forth in SEQ ID NO:9 (DMLVVPFDY); d) an immunoglobulin light chain CDR1 (CDR-L1) comprising the amino acid sequence set forth in SEQ ID NO: 10 (SSDIGSNR); e) an immunoglobulin light chain CDR2 (CDR-L2) comprising the amino acid sequence set forth in SEQ ID NO:11 (SND); and f) an immunoglobulin light chain CDR3 (CDR-L3) comprising the amino acid sequence set forth in SEQ ID NO: 12 (AAWDDSLSTYV); Including, The recombinant antibody or antigen-binding fragment thereof may be Fab, F(ab) ２ or a single chain variable fragment (scFv).

2. Immunoglobulin heavy chain variable region and immunoglobulin light chain variable region: a) the immunoglobulin heavy chain variable region herein comprises an amino acid sequence that is at least 90%, 95%, 97%, 99% identical, or 100% identical to the amino acid sequence set forth in SEQ ID NO:13; and b) the immunoglobulin light chain variable region herein comprises an amino acid sequence that is at least 90%, 95%, 97%, 99% identical, or 100% identical to the amino acid sequence set forth in SEQ ID NO:14; The recombinant antibody or antigen-binding fragment thereof of claim 1 .

3. A nucleic acid encoding the recombinant antibody or antigen-binding fragment thereof according to claim 1 or 2.

4. A cell line comprising the nucleic acid of claim 3.

5. A pharmaceutical composition comprising the recombinant antibody or antigen-binding fragment thereof described in claim 1 or 2 and a pharma- ceutically acceptable excipient, carrier, or diluent.

6. A recombinant antibody or antigen-binding fragment thereof according to claim 1 or 2, or a pharmaceutical composition according to claim 5, for use in the treatment of cancer.

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