Anti-GARP / TGFβ antibody and its usage

Abstract

The present disclosure provides antibodies and antibody derivatives that bind to GARP (also known as LRRC32 and CPPRDD) and / or the GARP / TGFβ complex and methods of use thereof. In some embodiments, the anti-GARP / TGFβ antibodies or antibody derivatives provided herein can inhibit the TGFβ signaling pathway in target cells.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to International Patent Application PCT / CN2020 / 133398, filed on 2 December 2020, the contents of which are incorporated herein by reference in their entirety and claim that priority.

[0002] Technical field This disclosure relates to antibodies and antibody derivatives that bind to the GARP / TGFβ complex, and to methods of using the same. [Background technology]

[0003] The glycoprotein A-led repeat sequence (GARP, also known as LRRC32 and CPPRDD) is a transmembrane cell surface docking protein for latent transforming growth factor β (TGFβ). GARP comprises three domains, a large N-terminal extracellular domain, a transmembrane domain, and a short C-terminal cytoplasmic tail region, accounting for approximately 70% of the protein. GARP plays a crucial role in multiple stringent regulatory steps of TGFβ production, accumulation, and activation. The GARP / TGFβ complex is expressed on regulatory T lymphocytes (Tregs), platelets, and several human cancer cells, where it has been reported to support cancer cell proliferation and migration by providing an excess source of TGFβ, allowing TGFβ to play a role in the tumor microenvironment and promote tumor immune evasion. Given the critical role of GARP and TGFβ signaling in immunomodulation and cancer biology, the development of therapeutic molecules and methods targeting GARP / TGFβ signaling for use in immunotherapy and cancer treatment is a need in this field. [Overview of the project]

[0004] This disclosure provides isolated monoclonal antibodies and antibody derivatives that bind specifically to the GARP / TGFβ complex with high affinity, including monospecific anti-GARP / TGFβ antibodies and multispecific antibodies that bind to the GARP / TGFβ complex and one or more other targets. In some examples, the antibodies or antibody derivatives disclosed herein include full-length antibodies that bind to the GARP / TGFβ complex. In some examples, the antibodies or antibody derivatives disclosed herein include scFv that bind to the GARP / TGFβ complex. This disclosure further provides the antibodies and antibody derivatives disclosed herein, as well as methods for preparing and using drug compositions comprising these antibodies and antibody derivatives, for example, in the treatment of diseases and disorders such as cancer. The present invention is partly based on the discovery of novel antibodies that bind to the GARP / TGFβ complex, which can target tumor cells and / or increase the immune response against tumor cells.

[0005] This disclosure provides an antibody that binds to the GARP / TGFβ complex, the antibody comprising a) heavy chain variable region CDR-H1 comprising (1) one amino acid sequence among SEQ ID NO: 1, 11, 21, 31, 41, 51, 61 and 105, or a variant of said amino acid sequence comprising at most about three amino acid substitutions; (2) heavy chain variable region CDR-H2 comprising one amino acid sequence among SEQ ID NO: 2, 12, 22, 32, 42, 52, 62 and 106, or a variant of said amino acid sequence comprising at most about three amino acid substitutions; and (3) heavy chain variable region CDR-H3 comprising one amino acid sequence among SEQ ID NO: 3, 13, 23, 33, 43, 53, 63 and 107, or a variant of said amino acid sequence comprising at most about three amino acid substitutions; and b) (1) SEQ ID NO: The light chain variable region comprises: (2) a light chain variable region CDR-L1 containing one amino acid sequence among 4, 14, 24, 34, 44, 54, 64, and 108, or a variant of said amino acid sequence containing at least approximately three amino acid substitutions; (3) a light chain variable region CDR-L3 containing one amino acid sequence among SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, and 109, or a variant of said amino acid sequence containing at least approximately three amino acid substitutions; and (4) a light chain variable region CDR-L3 containing one amino acid sequence among SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, and 110, or a variant of said amino acid sequence containing at least approximately three amino acid substitutions.

[0006] In some examples, the antibody was 1 x 10 -7 The antibody binds to the GARP / TGFβ complex with a KD of M or less. In some examples, the antibody was 1 x 10⁶ -8 The antibody binds to the GARP / TGFβ complex with a KD of M or less. In some examples, the antibody was approximately 1 x 10⁻¹⁶ -11 From M to approximately 1x10 -7 The antibody binds to the GARP / TGFβ complex at KD of M. In some examples, the antibody was approximately 1 x 10⁶ -10 M to approximately 5x10 -8 M binds to the GARP / TGFβ complex via KD.

[0007] In some examples, the antibody cross-competed with the reference anti-GARP / TGFβ antibody, which included a) a heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 6, and b) CDR-H1 containing the amino acid sequence shown in (1) SEQ ID NO: 11, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 12, and (3) SEQ ID NO: c) A heavy chain variable domain (VH) sequence including CDR-H3 containing the amino acid sequence shown in 13, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 14, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 15, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 16, c) A heavy chain variable domain (VH) sequence including CDR-H1 containing the amino acid sequence shown in (1) SEQ ID NO: 21, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 22, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 23, and (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 24, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 25, and (3) SEQ ID NO: CDR-L3 containing the amino acid sequence shown in 26 and a light chain variable domain (VL) sequence, d) (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 31, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 32, (3) SEQ ID NO:e) A heavy chain variable domain (VH) sequence including CDR-H3 containing the amino acid sequence shown in 33, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 34, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 35, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 36, e) A heavy chain variable domain (VH) sequence including CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 41, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 42, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 43, and (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 44, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 45, and (3) SEQ ID NO: f) A light chain variable domain (VL) sequence including CDR-L3 containing the amino acid sequence shown in 46, (1) a heavy chain variable domain (VH) sequence including CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 51, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 52, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 53, and (1) a light chain variable domain (VL) sequence including CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 54, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 55, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 56, g) a light chain variable domain (VL) sequence including CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 61, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 62, and (3) SEQ ID NO: A heavy chain variable domain (VH) sequence including CDR-H3 containing the amino acid sequence shown in 63, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 64, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 65, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 66, or h)The heavy chain variable domain (VH) sequence includes (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 105, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 106, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 107, and the light chain variable domain (VL) sequence includes (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 108, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 109, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 110.

[0008] In some examples, the antibody is a) a heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain and a CDR-H3 domain, wherein the CDR-H1 domain, the CDR-H2 domain and the CDR-H3 domain each comprise a CDR-H1 domain, a CDR-H2 domain and a CDR-H3 domain included in a reference heavy chain variable region, and the reference heavy chain variable region comprises a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 85, 89, 93, 97, 101 and 111, and b) A light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain each include a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain included in a reference light chain variable region, and the reference light chain variable region comprises a light chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 83, 84, 86, 90, 94, 98, 102, and 112.

[0009] In some examples, the antibody includes a heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 6. In some examples, the antibody includes a heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 11, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 12, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 14, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 15, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 16. In some examples, the antibody includes a heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 21, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 22, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 24, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 25, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 26.In some examples, the antibody includes a heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 31, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 32, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 33, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 34, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 35, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 36. In some examples, the antibody includes a heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 41, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 42, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 43, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 44, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 45, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 46. In some examples, the antibody includes a heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 51, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 52, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 53, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 54, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 55, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 56.In some examples, the antibody includes a heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 61, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 62, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 64, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 65, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 66. In some examples, the antibody includes a heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 105, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 106, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 107, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 108, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 109, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 110.

[0010] In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 28. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 37, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 38. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 37, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 83. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 47, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 48. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 47, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 84. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 57, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 58. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 67, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 68.In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 85, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 86. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 89, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 90. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 93, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 94. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 97, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 98. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 101, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 102. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 111, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 112.

[0011] In some examples, the antibody comprises a human framework. In some examples, the antibody is a human antibody. In some examples, the antibody is a humanized antibody. In some examples, the antibody comprises full-length immunoglobulin, single-chain Fv(scFv) fragments, Fab fragments, Fab' fragments, F(ab')2, Fv fragments, disulfide-bonded stable Fv fragments (dsFv), (dsFv)2, Fv-Fc fusions, scFv-Fc fusions, scFv-Fv fusions, diabodies, triabodies, tetrabodies, or any combination thereof.

[0012] In some examples, the antibody includes an Fc region. In some examples, the Fc region includes a human Fc region. In some examples, the Fc region includes an Fc region selected from the group consisting of IgG, IgA, IgD, IgE, and IgM Fc regions.

[0013] In some examples, the Fc region includes an Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions. In some examples, the Fc region includes an IgG1 Fc region. In some examples, the Fc region includes an IgG4 Fc region. In some examples, the antibody binds to the human GARP / TGFβ complex. In some examples, the antibody binds to the cynomolgus monkey GARP / TGFβ complex. In some examples, the antibody binds to the human GARP / TGFβ complex, the cynomolgus monkey GARP / TGFβ complex, and the mouse GARP / TGFβ complex. In some examples, the Fc region includes a C-terminal lysine. In some examples, the Fc region includes a C-terminal lysine deletion.

[0014] In some examples, the antibody is a multispecific antibody, for example, a bispecific antibody, where the multispecific antibody includes a second antibody moiety that specifically binds to a second antigen. In some examples, the second antigen is a tumor-associated antigen. In some examples, tumor-associated antigens include Her-2, EGFR, PDL1, MSLN, c-Met, B-cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD47, CD49f, CD56, CD74, CD123, CD133, CD138, CD276 (B7H3), epithelial glycosphing Protein (EGP2), trophoblast cell surface antigen 2 (TROP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine kinase erb-B2, 3, 4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human telomerase reverse transcriptase (hTERT), kinase insertion domain receptor (KDR), Lewis The antigen is selected from the group consisting of A (CA 1.9.9), Lewis Y (LeY), phosphatidylinositol proteoglycan-3 (GPC3), L1 cell adhesion molecule (L1CAM), mucin 16 (Muc-16), mucin 1 (Muc-1), NG2D ligand, tumor embryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), claudin 18.2 (CLDN18.2), vascular endothelial growth factor R2 (VEGF-R2), nephroblastoma protein (WT-1), type 1 tyrosine kinase transmembrane receptor (ROR1), PVR, PVRL2, and any combination thereof. In some examples, the second antigen is an immune checkpoint modulator. In some examples, the immune checkpoint modulator is selected from the group consisting of TIGIT, PD1, CTLA4, LAG-3, 2B4, BTLA, and any combination thereof. In some examples, the second antigen is an immune costimulatory molecule or a subunit of the T cell receptor / CD3 complex.In some examples, the immunocostimulatory molecule is selected from the group consisting of CD28, ICOS, CD27, 4-1BB, OX40, CD40, and any combination thereof. In some examples, the T cell receptor / CD3 complex subunit is selected from the group consisting of CD3γ, CD3δ, CD3ε, and any combination thereof.

[0015] This disclosure provides an immunoconjugate comprising any antibody disclosed herein, which is conjugated to a therapeutic agent or label. In some examples, the therapeutic agent is a cytotoxin or a radioisotope. In some examples, the label is selected from the group consisting of radioisotopes, fluorescent dyes, and enzymes.

[0016] This disclosure provides an antigen-recognizing receptor comprising an extracellular antigen-binding domain containing an antibody disclosed herein. In some examples, the antigen-recognizing receptor is a chimeric antigen receptor (CAR) or a recombinant T cell receptor. In some examples, the antigen-recognizing receptor is a CAR. In some examples, the antibody is scFv or Fab.

[0017] This disclosure provides immune-responsive cells comprising antigen-recognition receptors disclosed herein. In some examples, the immune-responsive cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, and myeloid cells. In some examples, the immune-responsive cells are T cells.

[0018] This disclosure further provides drug compositions. In some examples, the drug composition comprises a) an antibody, immunoconjugate, or immune-responsive cell as disclosed herein, and b) a pharmaceutically acceptable carrier.

[0019] This disclosure further provides one or more nucleic acids encoding any antibody disclosed herein, one or more vectors comprising any nucleic acid disclosed herein, and host cells comprising any nucleic acid or any vector disclosed herein.

[0020] This disclosure further provides methods for preparing the antibodies disclosed herein. In some examples, the methods include expressing the antibodies in the host cells disclosed herein and isolating the antibodies from the host cells.

[0021] This disclosure further provides methods for reducing tumor burden in a subject. In some examples, the method involves administering an effective amount of an antibody, immunoconjugate, or drug composition disclosed herein to the subject.

[0022] In some embodiments, the method reduces the number of tumor cells. In some embodiments, the method reduces the size of the tumor. In some embodiments, the method eradicates the tumor in the subject. In some embodiments, the tumor exhibits high microsatellite instability (MSI). In some embodiments, the tumor is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof.

[0023] This disclosure further provides methods for treating and / or preventing cancer in a subject. In some embodiments, the method involves administering to a subject an effective amount of an antibody, immunoconjugate, or drug composition disclosed herein.

[0024] This disclosure further provides methods for extending the survival time of subjects with cancer. In some examples, the method involves administering to a subject an effective amount of an antibody, immunoconjugate, or drug composition disclosed herein.

[0025] In some embodiments, cancer exhibits high microsatellite instability (MSI). In some embodiments, cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof.

[0026] This disclosure provides any antibody used as a drug as disclosed herein. This disclosure further provides any antibody used for the treatment of cancer as disclosed herein. This disclosure further provides drug compositions used as drugs as disclosed herein. This disclosure further provides drug compositions used for the treatment of cancer as disclosed herein. In some examples, cancers exhibit high microsatellite instability (MSI). In some examples, cancers are selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, hematological cancers, and combinations thereof.

[0027] This disclosure further provides kits comprising antibodies, immunoconjugates, drug compositions, nucleic acids, vectors, or immune-responsive cells disclosed herein. In some examples, the kit includes a manual for the treatment and / or prevention of neoplasms.

[0028] This disclosure further provides a method for treating cancer in a subject, the method comprising administering an effective amount of anti-GARP / TGFβ antibody and anti-PD1 antibody to the subject. In some examples, the anti-GARP / TGFβ antibody is the anti-GARP / TGFβ antibody disclosed herein. In some examples, the cancer exhibits high microsatellite instability (MSI). In some examples, the cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof. In some examples, the anti-GARP / TGFβ antibody and anti-PD1 antibody are administered simultaneously or sequentially. In some examples, the anti-GARP / TGFβ antibody and anti-PD1 antibody are administered simultaneously. In some cases, one or more anti-PD1 antibodies are administered before administering anti-GARP / TGFβ antibodies. In some cases, subjects receive a complete course of anti-PD1 antibody therapy before administering anti-GARP / TGFβ antibodies. In some cases, anti-GARP / TGFβ antibodies are administered during a second course of anti-PD1 antibody therapy. In some cases, subjects receive at least one, at least two, at least three, or at least four anti-PD1 antibodies before administering anti-GARP / TGFβ antibodies. In some cases, at least one anti-PD1 antibody is administered concurrently with an anti-GARP / TGFβ inhibitor. In some cases, one or more anti-GARP / TGFβ antibodies are administered before administering anti-PD1 antibodies. In some cases, subjects receive at least two, at least three, at least three, or at least four anti-GARP / TGFβ antibodies before administering anti-PD1 antibodies. In some examples, at least one anti-GARP / TGFβ antibody is administered concurrently with an anti-PD1 antibody. In some examples, the anti-GARP / TGFβ antibody and the anti-PD1 antibody are administered every 1, 2, 3, 4, or 5 weeks. In some examples, the cancer is recurrent or progressive after a therapy selected from the group consisting of surgery, chemotherapy, radiotherapy, and any combination thereof. [Brief explanation of the drawing]

[0029] [Figure 1] This figure shows the GARP / latent TGFβ1 binding of the selected antibody clone. The antibody clone GA1 was selected from a natural human Fab phage library, and its binding ability was tested by flow cytometry to human GARP / latent TGFβ1-transfected CHO-S cells (1A), cynomolgus monkey GARP / latent TGFβ1-transfected CHO-S cells (1B), mouse GARP / latent TGFβ1-transfected CHO-S cells (1C), thrombin-activated human platelets (1D), and anti-CD3 / CD28 bead-activated human Treg cells (1E). The GARP reference antibody ABBV-151 analog was used as a positive control. The isotype control (bevacizumab) was used as a negative control. [Figure 2] This figure shows that GA1 inhibits the release of mature TGFβ1 from activated platelets. Platelets were stimulated with thrombin for 1 hour in the presence or absence of the specified antibody. After stimulation, the reaction supernatant was harvested and used for the quantification of mature TGFβ1. Mature TGFβ1 was detected using the TGFβ1 Duoset® ELISA kit (R&D). The GARP reference antibody ABBV-151 analog was used as a positive control. [Figure 3] This figure shows that GA1 reduces platelet-mediated T cell inhibition. CD4+ T cells were stimulated with anti-CD3 / CD28 Dynabeads (Gibco) at a bead-to-cell ratio of 1:40 and incubated with platelets and GA1 for 4 days. IFNγ was quantified in the harvested supernatant. The GARP reference antibody ABBV-151 analog was used as a positive control. The isotype control (bevacizumab) was used as a negative control. [Figure 4]This figure shows that GA1 can reverse Treg-mediated T cell inhibition. In a mixed leukocyte reaction assay, isolated Treg cells (2.5 × 10³) were added to a mixture of antibody-containing or antibody-uncontaining T cells (1 × 10⁵) and allogeneic dendritic cells (DCs) (1 × 10⁴). After incubation for 5 days, IFNγ (4A) and IL-2 (4B) secretion in the culture supernatant were quantified. The GARP reference antibody ABBV-151 analog was used as a positive control. The isotype control (bevacizumab) was used as a negative control. [Figure 5] This figure shows that GA1 can inhibit tumor growth both alone and in combination with anti-PD1 antibodies. In a syngeneic mouse model of MC38 (mouse colon cancer), MC38 cells were subcutaneously transplanted into C57BL / 6 mice (n = 6 mice / group). Four days after tumor inoculation, the first drug of each test was administered. Mice were treated intraperitoneally with the specified antibody twice a week for three weeks. RMP1-14 is a commercially available anti-mouse PD1 antibody. All data points represent the mean ± SEM. [Figure 6] This figure shows the GARP / latent TGFβ1 binding ability of GA1 precursor mutants. Flow cytometry was used to test the binding ability of top GA1 mutants selected from affinity maturation to human GARP / latent TGFβ1-transfected CHO-S cells (6A), cynomolgus monkey GARP / latent TGFβ1-transfected CHO-S cells (6B), mouse GARP / latent TGFβ1-transfected CHO-S cells (6C), thrombin-activated human platelets (6D), and anti-CD3 / CD28 bead-activated human Treg cells (6E). An isotype control (bevacizumab) was used as a negative control. [Figure 7] This figure shows the total cell binding ability between GA1 framework / constant region mutants and CHO-S cells transfected with human GARP / latent TGFβ1. The isotype control (bevacizumab) is used as a negative control. [Figure 8]This figure shows that the GA1 variant inhibits the release of mature TGFβ1 from activated platelets. Platelets were stimulated with thrombin for 1 hour in or without the specified antibody. After stimulation, the reaction supernatant was harvested and used for the quantification of mature TGFβ1. Mature TGFβ1 was detected using the TGFβ1 Duoset® ELISA kit (R&D). The GARP reference antibody ABBV-151 analog was used as a positive control. The isotype control (bevacizumab) was used as a negative control. [Figure 9] This figure shows that selected GA1 variants reduce platelet-mediated T cell inhibition. CD4+ T cells were stimulated for 4 days with or without platelets using anti-CD3 / CD28 Dynabeads (Gibco) at a bead-to-cell ratio of 1:40, either in the presence or absence of the specified antibody. IFNγ was quantified in the supernatant harvested from the reaction. The GARP reference antibody ABBV-151 analog was used as a positive control. The isotype control (bevacizumab) was used as a negative control. [Figure 10] This figure shows that the GA1 mutant can reverse Treg-mediated T cell inhibition. In a mixed leukocyte reaction assay, isolated Treg cells (2.5 × 10³) were added to a mixture of T cells (1 × 10⁵) containing or not containing the GA1 mutant and allogeneic dendritic cells (DCs) (1 × 10⁴). After incubation for 5 days, IFNγ (10A) and IL-2 (10B) secretion in the culture supernatant were quantified. The GARP reference antibody ABBV-151 analog was used as a positive control. The isotype control (bevacizumab) was used as a negative control. [Figure 11]This figure shows that GA1#8 inhibits TGFβ-mediated Smad2 phosphorylation in activated human Treg cells. Isolated Treg cells were stimulated for 24 hours with anti-CD3 / CD28 Dynabeads (Gibco) in a 1:1 bead-to-cell ratio, either in the presence or absence of the specified antibody. Cell lysates were analyzed by Western blotting using antibodies against P-Smad2 (as a read produced by activated TGFβ1) and GAPDH (as a loading control). The anti-TGFβ antibody was the commercially available anti-TGFβ antibody (1D11) from Bio X Cell. An isotype control (bevacizumab) was used as a negative control. A GARP reference antibody ABBV-151 analog was used as a positive control. [Figure 12] This figure shows that the GA1 mutant can inhibit tumor growth in a syngeneic mouse model of MC38 (mouse colon cancer). MC38 cells were subcutaneously transplanted into C57BL / 6 mice (n = 6 mice / group). Four days after tumor inoculation, the first drug of each test was administered. Mice were treated intraperitoneally with the specified antibody twice a week for three weeks. All data points are mean ± SEM. [Figure 13] This figure shows that GA1#8 can inhibit tumor growth both alone and in combination with anti-PD1 antibodies. In a syngeneic mouse model of MC38 (mouse colon cancer), MC38 cells were subcutaneously transplanted into C57BL / 6 mice (n = 10 mice / group). Four days after tumor inoculation, the first agent of each test drug was administered. Mice were treated intraperitoneally with the specified antibody twice a week for three weeks. RMP1-14 is a commercially available anti-mouse PD1 antibody. All data points represent the mean ± SEM. [Figure 14]This figure shows that GA1#8 can inhibit tumor growth both alone and in combination with anti-PD1 antibodies. In a syngeneic mouse model of CT26 (mouse colon cancer), CT26 cells were subcutaneously transplanted into C57BL / 6 mice (n = 10 mice / group). Three days after tumor inoculation, the first agent of each test drug was administered. Mice were treated intraperitoneally with the specified antibody twice a week for three weeks. RMP1-14 is a commercially available anti-mouse PD1 antibody. All data points represent the mean ± SEM. [Figure 15] This figure shows the binding of anti-GARP / TGFβ antibodies to the human GARP / TGFβ complex (15A) and to human GARP alone (15B), as evaluated by ELISA. [Figure 16] This figure shows the whole-cell binding of anti-GARP / TGFβ antibody to Hs 578T cells (16A), human GARP-transfected CHO-S cells (16B), human platelets (16C), and human Treg cells (16D), as evaluated by flow cytometry. [Figure 17] A figure showing that anti-GARP / TGFβ antibodies inhibit the ability of thrombin-activated platelets to release TGFβ1. [Figure 18] This figure shows the ability of anti-GARP / TGFβ antibodies to reduce Treg-mediated CD3+ T cell inhibition. [Figure 19] This figure shows the ADCC effect of anti-GARP / TGFβ antibody on Hs 578T cells in the presence of PBMCs from donor 1 (19A) and donor 2 (19B). [Figure 20] This figure shows the ability of anti-GARP / TGFβ antibodies to deplete GARP+ Treg cells in PBMCs from four donors. [Figure 21] This figure shows the ability of anti-GARP / TGFβ antibodies to inhibit tumor growth in the MC38 mouse colon cancer model. Figure 21A shows tumor growth curves under specified anti-GARP / TGFβ antibody and control therapy. Figure 21B shows Treg cell populations in mouse blood in each treatment group. Figure 21C shows Treg cell populations in mouse spleens in each treatment group. [Modes for carrying out the invention]

[0030] This disclosure provides isolated monoclonal antibodies and antibody derivatives that bind specifically to the GARP / TGFβ complex with high affinity, including monospecific anti-GARP / TGFβ antibodies and multispecific antibodies that bind to the GARP / TGFβ complex and one or more other targets. In some examples, the antibodies or antibody derivatives disclosed herein include full-length antibodies that bind to the GARP / TGFβ complex. In some examples, the antibodies or antibody derivatives disclosed herein include scFv that bind to the GARP / TGFβ complex. This disclosure further provides the antibodies and antibody derivatives disclosed herein, as well as methods for preparing and using drug compositions comprising these antibodies and antibody derivatives, for example, in the treatment of diseases and disorders such as cancer. The present invention is partly based on the discovery of novel antibodies that bind to the GARP / TGFβ complex, which can target tumor cells and / or increase the immune response against tumor cells.

[0031] To clarify, and not to limit, specific embodiments of the themes of this disclosure can be categorized as follows: 1. Definition, 2. Antibodies and antibody derivatives, 3. How to use; 4. Drug preparations, and 5. Products.

[0032] 1. Definition

[0033] As used herein, the term “antibody” includes full-length antibodies and any antigen-binding fragments thereof (i.e., antibody fragments). “Antibody” may be an independent molecule or part of an antibody derivative. Exemplary antibody derivatives include, but are not limited to, multifunctional antibodies, e.g., multispecific antibodies (e.g., bispecific antibodies), antigen-recognition receptors (e.g., chimeric antigen receptors), antibody conjugates containing another protein or non-protein portion (e.g., antibody-drug conjugates or antibodies coated with polymers), and other multifunctional molecules containing antibodies.

[0034] "Full-length antibody," "complete antibody," and "whole antibody" refer to antibodies having a heavy chain that is similar in structure to that of a natural antibody or that includes an Fc region as defined herein. In some examples, a full-length antibody comprises two heavy chains and two light chains. In some examples, the variable regions of the light chains and heavy chains are responsible for antigen binding. The variable regions of the heavy chains and light chains may be referred to as "VH" and "VL," respectively. The variable regions in the two chains typically contain three highly variable loops, which are called complementarity-determining regions (CDRs) (light chain (LC)CDRs including LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC)CDRs including HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibody and antigen-binding fragment disclosed herein may be defined or identified by well-known conventions, such as those of Kabat, Chothia, MacCallum, IMGT, and AHo, described below. Three CDRs of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conserved than CDRs and form a scaffold supporting highly variable loops. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit multiple effector functions. Antibodies are classified based on the amino acid sequence of the antibody heavy chain constant region. The five main classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, each characterized by the presence of α, δ, ε, γ, and μ heavy chains. Some major antibody classes are divided into subclasses, e.g., IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain), or IgA2 (α2 heavy chain). In some examples, the full-length antibody is glycosylated. In some examples, the full-length antibody contains a glycan linked to its Fc region. In some examples, the full-length antibody contains branched-chain glycans.

[0035] As used herein, the terms “antigen-binding moiety,” “antibody fragment,” and “antibody moiety” refer to one or more fragments of an antibody that possess the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv and scFv-Fc), single-domain antibodies, VHH, VHH-Fc, nanoantibodies, domain antibodies, bivalent domain antibodies, or any other fragments or combinations thereof of an antibody that binds to an antigen. “VHH” refers to a single-domain antibody isolated from a camelid animal. In some examples, VHH contains the heavy chain variable region of a camelid heavy-chain antibody. In some examples, the size of VHH does not exceed about 25 kDa. In some examples, the size of VHH does not exceed about 20 kDa. In some embodiments, the size of VHH does not exceed approximately 15 kDa.

[0036] A "reference antibody and an antibody that cross-competes for binding" refers to an antibody that blocks the binding of the reference antibody to its antigen by 50% or more in a competitive assay, and conversely, in a competitive assay, the reference antibody blocks the binding of the antibody to its antigen by 50% or more. Exemplary competitive assays are described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).

[0037] "Fv" is the smallest antibody fragment, which contains a complete antigen recognition site and an antigen binding site. The fragment consists of a dimer of one heavy chain variable region and one light chain variable region that are tightly non-covalently bound. From the folding of these two domains, six hypervariable loops (three loops each of the heavy and light chains) are released, and the hypervariable loops provide amino acid residues for antigen binding and confer the binding specificity of the antibody to the antigen. However, even when it occurs with a lower affinity than the complete binding site, even a single variable domain (or half of the Fv containing only the three CDRs specific for the antigen) can recognize and bind the antigen.

[0038] "Single-chain Fv" (the abbreviation is also "sFv" or "scFv") is an antibody fragment containing V H and V L antibody domains linked in a single polypeptide chain. In some embodiments, the scFv polypeptide further includes a polypeptide linker between the V H and V L domains, and the polypeptide linker enables the scFv to form the desired structure for antigen binding. For an overview of scFv, see Plueckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0039] For the purposes of this specification, “receptor human framework” or “human framework” is a framework comprising the amino acid sequence of a light chain variable domain (VL) framework or heavy chain variable domain (VH) framework derived from a human immunoglobulin framework or human covalent framework. A receptor human framework “derived” from a human immunoglobulin framework or human covalent framework may include the same amino acid sequence or may include amino acid sequence variations. In some examples, the number of amino acid variations may be 10 or less, 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, or 2 or less. In some examples, the VL receptor framework and the VL human immunoglobulin framework sequence or human covalent framework sequence are identical in terms of sequence.

[0040] "Affinity" refers to the sum of the non-covalent interactions between a single binding site of a molecule (e.g., an antibody) and its binding partner (e.g., an antigen). Unless otherwise specified, as used herein, "binding affinity" refers to internal binding affinity, which reflects the 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of molecule X to partner Y may usually be expressed by the dissociation constant (KD). Affinity may be measured by common methods known in the art (including those described herein). Specific descriptions and exemplary examples for measuring binding affinity are provided below.

[0041] An antibody with "mature affinity" refers to an antibody that has one or more modifications in one or more CDRs or highly variable regions (HVRs) compared to a parent antibody without such modifications, and these modifications provide improved affinity of the antibody to the antigen.

[0042] As used herein, “GARP,” “GARP protein,” or “GARP polypeptide” means any GARP polypeptide or any fragment thereof derived from any vertebrate (mammal, e.g., primates (e.g., humans and cynomolgus monkeys)) and may optionally include at most one, two, three, four, five, six, seven, eight, nine, or ten amino acid substitutions, additions, and / or deletions. The term includes full-length GARP, unprocessed GARP, and any form of GARP produced by intracellular processing. The term further includes naturally occurring GARP variants, e.g., splice variants or allele variants. In some examples, the GARP polypeptide contains or has an amino acid sequence having at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% homology or identity with a sequence having the following NCBI reference numbers: NP_001122394.1, NP_001357116.1, NP_001357117.1, NP_001357118.1, NP_001357119.1, NP_001357120.1, or NP_005503.1 (homology as defined herein can be determined using standard software such as BLAST or FASTA). In some examples, the GARP polypeptide contains or has an amino acid sequence as the whole or a continuous portion of SEQ ID NO: 85. In some examples, the GARP protein is present in the GARP / TGFβ complex. In some examples, the GARP protein is not present in the GARP / TGFβ complex, but is, for example, an isolated GARP protein.

[0043] The term "ECD of GARP" refers to the extracellular domain of GARP. In some examples, the extracellular domain of GARP is the N-terminal extracellular domain of GARP. In some examples, the N-terminal ECD of the exemplary GARP polypeptide may include the amino acid sequence shown in SEQ ID NO: 86.

[0044] The terms "anti-GARP / TGFβ antibody" and "antibody that binds to the GARP / TGFβ complex" refer to antibodies that can bind to the GARP / TGFβ complex with sufficient affinity so that they can be used as diagnostic and / or therapeutic agents targeting the GARP / TGFβ complex. In one example, the degree of binding of the anti-GARP / TGFβ antibody to non-GARP / TGFβ proteins unrelated to the antibody is less than about 10% of the binding of the antibody to the GARP / TGFβ complex, for example, BIACORE (登録商標) It is measured by surface plasmon resonance assay. In some examples, the antibody that binds to the GARP / TGFβ complex has the following dissociation constants (KD): < approximately 1 μM, < approximately 100 nM, < approximately 10 nM, < approximately 1 nM, < approximately 0.1 nM, < approximately 0.01 nM, or < approximately 0.001 nM (e.g., 10 nM). -8 M or less, for example, 10 -8 M~10 -12 M, for example, 10 -9 M~10 -10 M) is present. In some examples, the anti-GARP / TGFβ antibody binds to GARP / TGFβ complex epitopes conserved in GARP / TGFβ complexes from different species. In some examples, the anti-GARP / TGFβ antibody binds to epitopes on GARP proteins in the ECD of the protein. In some examples, the anti-GARP / TGFβ antibody binds to GARP proteins in the GARP / TGFβ complex. In some examples, the anti-GARP / TGFβ antibody binds to GARP proteins that are not present in the GARP / TGFβ complex (e.g., isolated GARP proteins). In some examples, the anti-GARP / TGFβ antibody does not bind to GARP proteins that are not part of the GARP / TGFβ complex.

[0045] The term "chimeric" antibody refers to an antibody in which a portion of the heavy chain and / or light chain originates from a particular source or species, but the remaining portion of the heavy chain and / or light chain originates from a different source or species. In some examples, the chimeric antibodies disclosed herein include a camelid heavy chain variable region and a human Fc region.

[0046] As used herein, “CDR” or “complementarity-determining region” refers to discontinuous antigen-binding sites within the variable regions of the heavy chain and / or light chain. These specific regions are described in Kabat et al., J. Biol. Chem. 252:6609-6616 (1977), Kabat et al., US Dept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991), Chothia et al., J. Mol. Biol. 196:901-917 (1987), Al-Lazikani B. et al., J. Mol. Biol., 273: 927-948 (1997), MacCallum et al., J. Mol. Biol. 262:732-745 (1996), Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008), Lefranc MP et al., Dev. Comp. Immunol., 27: 55-77 (2003), and Honegger and Plueckthun, J. Mol. Biol., 309:657-670 (2001), where overlaps or subsets containing amino acid residues are defined for comparison purposes. However, any CDR referring to an antibody, transplanted antibody, or its variant is intended to be within the scope of the terminology defined and used herein, applying any one of these definitions. The amino acid residues covering the CDRs as defined in each of the above references are included in Table 1 below for comparison. The CDR prediction algorithms and interfaces are known in this field, including, for example, Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008), Ehrenmann F. et al., Nucleic Acids Res., 38: D301-D307 (2010), and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43: D432-D438 (2015).The contents of the referenced documents referenced in this chapter and section are incorporated into this specification by reference, used in this application, and may be included in one or more of the claims herein.

[0047] [Table 1] 1 Residue numbering follows the nomenclature of Kabat et al. (ibid.). 2 Residue numbering follows the nomenclature of Chothia et al. (ibid.). 3 Residue numbering follows the nomenclature of MacCallum et al. (ibid.). 4 Residue numbering follows the nomenclature of Lefranc et al. (ibid.). 5 Residue numbering follows the nomenclature of Honegger and Plueckthun (ibid.).

[0048] The expressions “e.g., Kabat variable domain residue numbering” or “e.g., Kabat amino acid position numbering” and their variants refer to the numbering system used for the heavy chain variable domain or light chain variable domain of the Kabat et al. antibody assembler described above. Using this numbering system, the actual linear amino acid sequence may contain fewer or different amino acids corresponding to the shortening or insertion of the variable domain FR or CDR. For example, the heavy chain variable domain may include a single amino acid insertion after H2 residue 52 (based on Kabat residue 52a) and an insertion residue after heavy chain FR residue 82 (e.g., based on Kabat residues 82a, 82b, and 82c, etc.). The Kabat numbering of residues in a given antibody can be determined by aligning the antibody sequence with homology regions in the “standard” Kabat numbering sequence.

[0049] In some examples, the amino acid residues covering the CDR of a single-domain antibody are defined based on the IMGT nomenclature of Lefranc et al. In some examples, the amino acid residues covering the CDR of a full-length antibody or scFv are defined based on the Kabat nomenclature of Kabat et al. In some examples, the residue numbering in the immunoglobulin heavy chain, e.g., the Fc region, is the EU index numbering described by Kabat et al. "Kabat EU index" refers to the residue numbering of a human IgG1 EU antibody.

[0050] "Framework" or "FR" refers to the variable domain residues other than the CDR residues as defined herein.

[0051] A "humanized" antibody refers to a chimeric antibody containing amino acid residues from a non-human CDR / HVR or from a human FR. In some examples, a humanized antibody contains all of at least one, typically two, variable domains, of which all or virtually all of the HVR / CDR corresponds to that of a non-human antibody, and all or virtually all of the FR corresponds to that of a human antibody. A humanized antibody optionally contains at least a portion of the antibody constant region derived from a human antibody. The "humanized form" of an antibody (e.g., a non-human antibody) refers to a humanized antibody.

[0052] A “human antibody” is an antibody having an amino acid sequence, the amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human, and / or prepared by any technique for the preparation of human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies may be produced using various techniques known in the art (including phage display libraries). See Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991), Marks et al., J. Mol. Biol., 222:581 (1991). The method described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985), and Boerner et al., J. Immunol., 147(1):86-95 (1991) can also be used for the preparation of human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies are modified to produce such antibodies in response to antigen attack, but they can also be prepared by administering the antigen to transgenic animals (e.g., immunized xenomices) in which the endogenous gene locus has been rendered inactive (e.g., XENOMOUSE). (商標) See U.S. Patents 6,075,181 and 6,150,584 for details on the technology. Furthermore, for example, human antibodies produced by human B-cell hybridoma technology, see Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0053] "Percent amino acid sequence identity (%)" or "homology" relating to the polypeptide identified herein and the antibody sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the polypeptide being compared, after sequence alignment (considering any conservative substitutions as part of sequence identity). For the purpose of determining the percentage amino acid sequence identity, alignment can be achieved by various methods in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine appropriate parameters to be used for measurement alignment, including any algorithm that achieves maximum alignment within the full-length range of the sequences being compared. However, for the purposes of this specification, the sequence comparison computer program MUSCLE is used to produce the value of amino acid sequence identity % (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0054] "Homologie" refers to the sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. Two comparison sequences are homologous at a given position when one position is occupied by the same base or amino acid monomer subunit; for example, when one position in each of two DNA molecules is occupied by adenine. The percentage of homology between two sequences is a function of the number of matching or homologous positions shared by the two sequences divided by the number of positions being compared, multiplied by 100. For example, if 6 out of 10 positions in two sequences match or are homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC have 50% homology. Comparisons are typically performed when the two sequences are aligned to give maximum homology.

[0055] The term "constant domain" refers to a part of an immunoglobulin molecule that has a more conserved amino acid sequence than another part of the immunoglobulin, namely the variable domain, and it contains the antigen-binding site. The constant domain is the C of the heavy chain. H 1, C H 2 and C H 3 domains (C H (collectively referred to as) and the C of the light chain L Includes the domain.

[0056] The "light chain" of any mammalian antibody (e.g., immunoglobulin) can be designated as one of two distinctly different types, depending on the amino acid sequence of its constant domain, and these are called kappa ("κ") and lambda ("λ"), respectively.

[0057] The "CH1 domain" (also known as the "C1" in the "H1" domain) typically extends from approximately 118 amino acids to approximately 215 amino acids (according to the EU numbering system).

[0058] The "hinge region" is usually defined as the region in IgG corresponding to Glu216 to Pro230 in human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). The hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form the disulfide bond between the heavy chains in the same position.

[0059] The "CH2 domain" of the human IgG Fc region (also called the "C2" domain) typically extends from approximately amino acid 231 to approximately amino acid 340. The CH2 domain is unique because it is not tightly paired with another domain. Instead, two N-linked branched carbohydrate chains are inserted between the two CH2 domains of a complete, natural IgG molecule. It is speculated that the carbohydrates may provide a domain-domain pairing substitute, contributing to the stabilization of the CH2 domain. (Burton, Molec Immunol. 22:161-206 (1985)).

[0060] The "CH3 domain" (also called the "C2" domain) includes the residues between the CH2 domain and the C-terminus of the Fc region (i.e., from approximately amino acid residue 341 to the C-terminus of the antibody sequence), and is typically located at amino acid residue 446 or 447 of IgG.

[0061] In this specification, “Fc region” or “fragment crystallizable region” is used to define the C-terminal region of an immunoglobulin heavy chain and includes both native sequence Fc regions and mutant Fc regions. While the boundaries of the Fc region of an immunoglobulin heavy chain are variable, the Fc region of a human IgG heavy chain is typically defined as extending from the amino acid residue at position Cys226 or from Pro230 to its carboxyl group terminus. For example, the C-terminal lysine of the Fc region (based on residue 447 in the EU numbering system) can be removed during antibody production or purification or by recombinant engineering of the nucleic acid encoding the antibody heavy chain. Therefore, a complete antibody composition may include antibody groups with all K447 residues removed, antibody groups with and without K447 residues, and antibody mixtures with or without K447 residues. Suitable native sequence Fc regions used in the antibodies described herein include human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0062] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. The preferred FcR is the natural human FcR. The preferred FcR is an FcR that binds to an IgG antibody (γ receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allele variants or splice forms of these receptors. The FcγRII receptor includes FcγRIIA ("activating receptor") and FcγRIIB ("inhibitory receptor"), which have similar amino acid sequences, with the main distinction being in their cytoplasmic domain. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain. (See M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997). FcR is outlined in Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991), Capel et al., Immunomethods 4: 25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126: 330-41 (1995). The term “FcR” as used herein covers other FcRs and includes FcRs identified in the future.)

[0063] As used herein, the term “epitope” refers to a specific atom or amino acid group on an antigen to which an antibody or antibody derivative binds. Two antibody or antigen-binding moieties can bind to the same epitope within an antigen if they have competitive binding to the antigen.

[0064] As used herein, the terms “specifically bind,” “specifically recognize,” and “specific to…” refer to measurable and reproducible interactions, such as the binding of a target to an antibody or antibody moiety, which determines the presence of the target in the presence of heteromolecules (including biomolecules). For example, an antibody or antibody moiety that specifically recognizes a target (which may be an epitope) is an antibody or antibody moiety that binds to the target, and its affinity, avidity, readiness, and / or duration are longer than that of binding to other targets. In some examples, the degree of binding to an antibody unrelated target is about 10% less than the degree of binding between the antibody and the target as measured, for example, by radioimmunoassay (RIA). In some examples, the dissociation constant (K) of the antibody that specifically binds to the target is D )≦ 10 -5 M, ≤ 10 -6 M, ≤ 10 -7 M, ≤ 10 -8 M, ≤ 10 -9 M, ≤ 10- 10 M, ≤ 10 -11 M, or ≤ 10 -12 M is used. In some examples, the antibody specifically binds to a protein epitope that is conserved in proteins from different species. In some examples, specific binding may include, but is not limited to, exclusive binding. The binding specificity of the antibody or antigen-binding domain may be determined experimentally by methods known in the art. Such methods include Western blotting, ELISA, RIA, ECL, IRMA, EIA, and BIACORE. TM - This includes, but is not limited to, assays and peptide scanning.

[0065] "Isolated" antibodies (or constructs) are antibodies identified, isolated, and / or recovered from components (e.g., natural or recombinant) of the production environment. In some examples, the isolated polypeptides do not associate with or substantially associate with all other components in the production environment.

[0066] The “isolated” nucleic acid molecules encoding constructs, antibodies, or their antigen-binding fragments described herein are nucleic acid molecules that are typically identified and isolated from at least one associated contaminant nucleic acid molecule in their production environment. In some examples, the isolated nucleic acids do not associate with or substantially associate with all components associated with the production environment. The forms of the polypeptides and antibodies encoding the isolated nucleic acid molecules described herein differ from those found in nature or in the background. Therefore, the isolated nucleic acid molecules differ from the nucleic acids encoding the polypeptides and antibodies described herein that are naturally present in cells. The isolated nucleic acids include the nucleic acid molecules typically found in cells, but the nucleic acid molecules are located outside the chromosome or at chromosomal locations different from their natural chromosomal locations.

[0067] When a nucleic acid has a functional relationship with another nucleic acid sequence, the nucleic acid is said to be "operably linked" or "operatively linked." For example, if the DNA of a pre-sequence or secretion leader sequence is expressed as a preprotein involved in polypeptide secretion, the DNA of the pre-sequence or secretion leader sequence is operably linked to the DNA of the polypeptide; if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer is operably linked to the sequence; or if a ribosome binding site is localized to facilitate translation, the ribosome binding site is operably linked to the coding sequence. Typically, "operably linked" means that the linked DNA sequences are contiguous, and in the case of a secretion leader sequence, contiguous and within the reading frame. However, enhancers do not necessarily have to be contiguous. Linking can be achieved by linking at a convenient restriction site. If such a site does not exist, a synthetic oligonucleotide adapter or linker is used, based on common practice.

[0068] As used herein, the term “vector” refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is ligated. The term includes vectors that form self-replicating nucleic acid structures, and vectors that are incorporated into the genome of a host cell into which they are introduced. Some vectors can guide the expression of a nucleic acid to which they are manipulably ligated. Such vectors are referred herein to as “expression vectors.”

[0069] As used herein, the terms “transfected,” “transformed,” or “transduced” refer to the process of transferring or introducing foreign nucleic acids into host cells. A “transfected,” “transformed,” or “transduced” cell is a cell that has been transfected, transformed, or transduced with foreign nucleic acids, and such cell includes primary test cells and their offspring.

[0070] The terms “host cell,” “host cell line,” and “host cell culture” are interchangeable and refer to cells into which foreign nucleic acids have been introduced, including the offspring of such cells. Host cells include “transformed organisms” and “transformed cells,” which include primary transformed cells and their derived offspring, regardless of the number of passages. The nucleic acid content of offspring may differ from that of parent cells and may contain mutations. Mutant offspring having the same function or biological activity as those screened or selected in the original transformed cells are included herein.

[0071] The terms “subject,” “individual,” and “patient” are interchangeable herein and refer to mammals, including but not limited to humans, cattle, horses, cats, dogs, rodents, or primates. In some examples, the subject is human.

[0072] The “effective dose” of a drug refers to the amount that effectively achieves the required therapeutic or prophylactic effect within the required dosage and duration. The specific dosage may vary depending on one or more of the selected drug, the subsequent administration scheme (whether or not it is combined with other compounds), the administration time, the imaging tissue, and the associated physical delivery system.

[0073] The "therapeutic effective dose" of the substance / molecule, agonist, or antagonist of this application may vary depending on factors such as disease state, age, sex, and body weight of the individual, and the ability of the substance / molecule, agonist, or antagonist to elicit a desired response in the individual. The therapeutic effective dose is further defined as the amount at which any toxic or adverse effects of the substance / molecule, agonist, or antagonist are offset by the therapeutically beneficial effects. The therapeutic effective dose can be delivered in one or more doses.

[0074] As used herein, “treatment” is a method for obtaining a beneficial or desired outcome (including clinical outcomes). For the purposes of this application, beneficial or desired clinical outcomes include, but are not limited to, reducing one or more symptoms caused by a disease, reducing the severity of the disease, stabilizing the disease (e.g., preventing or delaying disease exacerbation), preventing or delaying disease transmission (e.g., metastasis), preventing or delaying disease recurrence, slowing or mitigating the rate of disease progression, improving the disease state, providing some or all relief of the disease, reducing the dosage of one or more other drugs necessary for the treatment of the disease, delaying disease progression, increasing or improving quality of life, increasing weight gain, and / or extending survival. “Treatment” further covers reducing the pathological outcomes of cancer (e.g., tumor volume). The methods of this application consider one or more of these aspects of treatment. “Treatment” does not necessarily mean that the disease being treated is cured.

[0075] It should be understood that the embodiments of this application described herein include "consisting of embodiments" and / or "substantially consisting of embodiments."

[0076] As used herein, “about” or “approximately” means that a particular value, as determined by those skilled in the art, is within an acceptable margin of error, which is in part determined by how the value is measured or determined, i.e., limited by the measuring system. In some embodiments, “about” may mean that, according to the practice of the art, it is within three or more standard differences. In some embodiments, “about” may represent a range of at most 20% of a given value (e.g., at most 10%, at most 5%, or at most 1%). In some embodiments, particularly with respect to biological systems and methods, the term may mean that it is within an order of magnitude of a certain value, e.g., within five times or twice.

[0077] As used herein, the term “adjustment” refers to changing in a positive or negative direction. Exemplary adjustments include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0078] As used herein, the term “increase” means a change of at least about 5% in the positive direction. The change may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100%, or more.

[0079] As used herein, the term “decrease” means a change of at least about 5% in the negative direction. The change may be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0080] The term "approximately XY" used in this statement has the same meaning as "approximately X to approximately Y".

[0081] When used in this specification and the appended claims, the singular forms “a,” “or,” and “the” include multiple referents unless the context clearly indicates otherwise.

[0082] "Effector function" refers to the biological activity of an antibody by its Fc region, which varies depending on the antibody isotype. Examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC), Fc receptor binding, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, downregulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0083] An "immunoconjugate" refers to an antibody that has been conjugated to one or more heterologous molecules (including, but not limited to, cytotoxic agents).

[0084] The term "drug preparation" refers to a preparation in which the biological activity of the active ingredient contained therein is permitted, and which does not contain other ingredients that are unacceptably toxic to the subject to which the preparation is administered.

[0085] As used herein, “pharmaceutically acceptable carrier” refers to a component in a drug formulation that is non-toxic to the subject other than the active ingredient. pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0086] The term "variable region" or "variable domain" refers to a domain in the antibody heavy or light chain that is involved in the binding of the antibody to the antigen. In some examples, the variable domains of the heavy and light chains of native antibodies (VH and VL, respectively) typically have similar structures, with each domain containing four conserved framework regions (FRs) and three CDRs. (See, for example, Kindt et al. Kuby Immunology, 61st ed., WH Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, VH or VL domains can be used to isolate antibodies that bind to a specific antigen from antibodies that bind to an antigen, and complementary VL or VH domain libraries can be screened, respectively. For example, see Portolano et al., J. Immunol. 150:880-887 (1993) and Clarkson et al., Nature 352:624-628 (1991).

[0087] As used herein, the term “antigen-recognizing receptor” refers to a receptor that can activate immune-responsive cells (e.g., T cells) in response to its binding to an antigen. Non-exclusive examples of antigen-recognizing receptors include native and modified T cell receptors ("TCRs") and chimeric antigen receptors ("CARs").

[0088] As used herein, the terms “chimeric antigen receptor” or “CAR” refer to a molecule comprising an extracellular antigen-binding domain and a transmembrane domain, wherein the extracellular antigen-binding domain is fused with an intracellular signaling domain that can activate or stimulate immune-responsive cells. In some examples, the extracellular antigen-binding domain of the CAR comprises an antibody or antibody fragment, e.g., VHH or scFv. In some examples, the antibody (e.g., VHH or scFv) is fused with the transmembrane domain, which is then fused with the intracellular signaling domain. In some examples, a CAR having high binding affinity or avidity to an antigen is selected.

[0089] "Immune-responsive cells" refer to cells that function in the immune response, or their ancestors or descendants.

[0090] 2. Antibodies and antibody derivatives

[0091] This disclosure provides antibodies and antibody derivatives. In some examples, this disclosure is based in part on the discovery of monoclonal antibodies that bind to the GARP / TGFβ complex, which can be used in antitumor therapy, where these antibodies induce beneficial antitumor effects on tumor cells by selectively targeting tumor cells and / or inhibiting the GARP / TGFβ complex-mediated signaling pathway. In some examples, the antibodies disclosed herein are antagonist antibodies that inhibit the function of the GARP / TGFβ complex. In some examples, anti-GARP / TGFβ antibodies inhibit the interaction between GARP and one or more TGFβ molecules. In some examples, anti-GARP / TGFβ antibodies block the signaling pathway related to the GARP / TGFβ complex. In some examples, anti-GARP / TGFβ antibodies block the release of mature TGFβ from the GARP / TGFβ complex. In some examples, anti-GARP / TGFβ antibodies inhibit the TGFβ signaling pathway in tumor cells. In some examples, anti-GARP / TGFβ antibodies inhibit the TGFβ signaling pathway in immune cells, e.g., Treg cells. In some examples, anti-GARP / TGFβ antibodies reduce the immunosuppressive effect of Treg cells. In some examples, anti-GARP / TGFβ antibodies increase the secretion of antitumor cytokines in immune cells (e.g., effector T cells). In some examples, anti-GARP / TGFβ antibodies show a superior ability to increase the secretion of antitumor cytokines in immune cells (e.g., effector T cells) compared to reference antibodies (e.g., ABBV-151 analogs). In some examples, anti-GARP / TGFβ antibodies exhibit antitumor effects in subjects. In some examples, anti-GARP / TGFβ antibodies show superior antitumor effects compared to reference antibodies (e.g., ABBV-151 analogs or DS-1005a analogs). ABBV-151, also known as LHG10.6, is a clinically-stage anti-GARP / TGFβ therapeutic antibody, and its sequence is publicly disclosed in US 2016 / 0251438.DS-1005a, also known as H151D-H1L1, is a clinical-stage anti-GARP / TGFβ IgG1 antibody, and its sequence is disclosed in US 2018 / 0258184.

[0092] In some examples, the antibodies disclosed herein may be monoclonal antibodies (including chimeric antibodies, humanized antibodies, or human antibodies), or may contain such antibodies. In some examples, the antibodies disclosed herein include humanized antibodies. In some examples, the antibodies include human receptor frameworks, such as human immunoglobulin frameworks or human covalent frameworks. In some examples, the antibodies disclosed herein include human antibodies.

[0093] In some embodiments, the antibodies of the Disclosure may be antibody fragments, e.g., Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragments. In some embodiments, the antibody may be a full-length antibody, e.g., a complete IgG4 antibody, or another antibody type or isotype as defined herein. In some embodiments, the antibodies or antibody derivatives of the Disclosure may incorporate any of the features described in this application (e.g., Sections 2.1-2.12 described in detail herein) individually or in combination.

[0094] The antibodies and antibody derivatives of this disclosure can be used, for example, in the diagnosis or treatment of neoplasms or cancer. In some examples, tumors and cancers whose proliferation can be inhibited by using the antibodies of this disclosure typically include tumors and cancers that respond to immunotherapy. In some examples, tumors and cancers include breast cancer (e.g., mammary gland cell carcinoma), ovarian cancer (e.g., ovarian cell carcinoma), and renal cell carcinoma (RCC). Other cancers that can be treated by the methods of this disclosure include melanoma (e.g., metastatic melanoma), prostate cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, brain tumors, chronic or acute leukemia (including acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, and chronic lymphocytic leukemia), lymphoma (e.g., Hodgkin lymphoma or non-Hodgkin lymphoma, lymphocytic lymphoma, primary CNS lymphoma, and T-cell lymphoma), nasopharyngeal carcinoma, head or cervical cancer, skin cancer, or intraocular melanoma, uterine cancer, rectal cancer, anal cancer, gastric tumors, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, external vaginal cancer, esophageal cancer, small intestine cancer, endocrine cancer, thyroid cancer, parathyroid cancer, and breast cancer. Examples include cancers induced by the environment, including asbestos-induced cancers (e.g., mesothelioma), soft tissue sarcomas, urethral cancers, penile cancers, pediatric solid tumors, bladder cancers, kidney cancers or ureteral cancers, breast cancers, pelvic cancers, central nervous system (CNS) neoplasms, tumor angiogenesis, spinal tumors, brainstem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermal carcinoma, squamous cell carcinoma, asbestos-induced cancers (e.g., mesothelioma), and combinations of the above cancers.

[0095] 2.1.1 Exemplary anti-GARP / TGFβ antibodies

[0096] This disclosure provides isolated antibodies that bind to the GARP / TGFβ complex. In some examples, the anti-GARP / TGFβ antibodies of this disclosure bind to the ECD of GARP. In some examples, the anti-GARP / TGFβ antibodies include an N-terminal ECD that binds to GARP in the amino acid sequence shown in SEQ ID NO: 86. In some examples, the anti-GARP / TGFβ antibodies bind to the GARP protein in the GARP / TGFβ complex. In some examples, the anti-GARP / TGFβ antibodies bind to a GARP protein that is not present in the GARP / TGFβ complex (e.g., isolated GARP protein). In some examples, the anti-GARP / TGFβ antibodies do not bind to a GARP protein that is not present in the GARP / TGFβ complex. In some examples, the anti-GARP / TGFβ antibodies bind to the same epitope as the anti-GARP / TGFβ antibodies described herein (e.g., clone GA1, clone hGA17, or their variants, e.g., GA1#7, GA1#8, or GA1#9). In some examples, the anti-GARP / TGFβ antibody binds to the human GARP / TGFβ complex. In some examples, the anti-GARP / TGFβ antibody binds to the cynomolgus monkey GARP / TGFβ complex. In some examples, the anti-GARP / TGFβ antibody binds to the mouse GARP / TGFβ complex. In some examples, the anti-GARP / TGFβ antibody binds to the human GARP / TGFβ complex, the cynomolgus monkey GARP / TGFβ complex, and the mouse GARP / TGFβ complex.

[0097] In some examples, the anti-GARP / TGFβ antibodies disclosed herein may act as antagonists based on the GARP / TGFβ signaling pathway. In some examples, the anti-GARP / TGFβ antibodies can block or reduce the interaction between GARP and one or more TGFβ molecules (e.g., TGFβ1, TGFβ2, or TGFβ3). In some examples, the anti-GARP / TGFβ antibodies can reduce the interaction between GARP and TGFβ molecules by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, or about 99.9%. In some examples, the anti-GARP / TGFβ antibodies block the function of the GARP / TGFβ complex. In some examples, the anti-GARP / TGFβ antibodies block the release of mature TGFβ from the GARP / TGFβ complex.

[0098] In some examples, anti-GARP / TGFβ antibodies inhibit the TGFβ signaling pathway in target cells, for example, by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 99%, or about 99.9%. In some examples, the target cells are tumor cells. In some examples, the target cells are immune cells, for example, Treg cells. In some examples, anti-GARP / TGFβ antibodies reduce the immunosuppressive effect of Treg cells. In some examples, anti-GARP / TGFβ antibodies increase the secretion of antitumor cytokines in immune cells (e.g., effector T cells). In some examples, anti-GARP / TGFβ antibodies show a superior ability to increase the secretion of antitumor cytokines in immune cells (e.g., effector T cells) compared to a reference antibody (e.g., ABBV-151 analog).

[0099] In several examples, treatment with anti-GARP / TGFβ antibodies exhibits antitumor effects in subjects, thereby reducing tumor growth and / or extending subject survival. In several examples, anti-GARP / TGFβ antibodies enhance the immune response and / or antitumor activity of immune cells (e.g., effector T cells and / or NK cells). In several examples, anti-GARP / TGFβ antibodies exhibit superior antitumor effects compared to reference anti-GARP / TGFβ antibodies (e.g., ABBV-151 analog or DS-1055a analog).

[0100] In some examples, the antibody was approximately 1 x 10⁶ -7 The antibody binds to the GARP / TGFβ complex with a KD of M or less. In some examples, the antibody was approximately 1 x 10⁻¹⁶ -8 The antibody binds to the GARP / TGFβ complex with a KD of M or less. In some examples, the antibody was approximately 5 x 10⁻⁶ -9 The antibody binds to the GARP / TGFβ complex with a KD of M or less. In some examples, the antibody was approximately 1 x 10⁻¹⁶ -9 The antibody binds to the GARP / TGFβ complex with a KD of M or less. In some examples, the antibody was approximately 1 x 10⁻¹⁶ -10 The antibody binds to the GARP / TGFβ complex with a KD of M or less. In some examples, the antibody was approximately 1 x 10⁻¹⁶ -12 From M to approximately 1x10 -7 The antibody binds to the GARP / TGFβ complex at KD of M. In some examples, the antibody was approximately 1 x 10⁶ -11 From M to approximately 1x10 -8 The antibody binds to the GARP / TGFβ complex at KD of M. In some examples, the antibody was approximately 1 x 10⁶ -10 From M to approximately 1x10 -8 The antibody binds to the GARP / TGFβ complex at KD of M. In some examples, the antibody was approximately 1 x 10⁶ -10 M to approximately 5x10 -8 The antibody binds to the GARP / TGFβ complex at KD of M. In some examples, the antibody was approximately 5 x 10 -10 From M to approximately 1x10 -9The antibody binds to the GARP / TGFβ complex at KD of M. In some examples, the antibody was approximately 1 x 10⁶ -9 M to approximately 5x10 -8 The antibody binds to the GARP / TGFβ complex at KD of M. In some examples, the antibody was approximately 1 x 10⁶ -10 M to approximately 5x10 -9 M binds to the GARP / TGFβ complex via KD.

[0101] In some examples, the anti-GARP / TGFβ antibody comprises a) heavy chain variable region including (1) heavy chain variable region CDR-H1 containing one amino acid sequence from SEQ ID NO: 1, 11, 21, 31, 41, 51, 61 and 105, or a variant of said amino acid sequence containing at least approximately three amino acid substitutions; (2) heavy chain variable region CDR-H2 containing one amino acid sequence from SEQ ID NO: 2, 12, 22, 32, 42, 52, 62 and 106, or a variant of said amino acid sequence containing at least three amino acid substitutions; and (3) heavy chain variable region CDR-H3 containing one amino acid sequence from SEQ ID NO: 3, 13, 23, 33, 43, 53, 63 and 107, or a variant of said amino acid sequence containing at least three amino acid substitutions; and b) (1) SEQ ID NO: The light chain variable region comprises: (2) a light chain variable region CDR-L1 containing one amino acid sequence among 4, 14, 24, 34, 44, 54, 64, and 108, or a variant of said amino acid sequence containing at least three amino acid substitutions; (3) a light chain variable region CDR-L3 containing one amino acid sequence among SEQ ID NO: 5, 15, 25, 35, 45, 55, 65, and 109, or a variant of said amino acid sequence containing at least three amino acid substitutions; and (4) a light chain variable region CDR-L3 containing one amino acid sequence among SEQ ID NO: 6, 16, 26, 36, 46, 56, 66, and 110, or a variant of said amino acid sequence containing at least three amino acid substitutions.

[0102] In some examples, the anti-GARP / TGFβ antibody cross-competed with the reference anti-GARP / TGFβ antibody, and the reference anti-GARP / TGFβ antibody included a) heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 6, and b) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 11, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 12, and (3) c) A heavy chain variable domain (VH) sequence including CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 14, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 15, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 16, c) A heavy chain variable domain (VH) sequence including CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 21, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 22, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 23, and (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 24, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 25, and (3) SEQ ID NO: CDR-L3 containing the amino acid sequence shown in 26 and a light chain variable domain (VL) sequence, d) (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 31, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 32, (3) SEQ ID NO:e) A heavy chain variable domain (VH) sequence including CDR-H3 containing the amino acid sequence shown in 33, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 34, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 35, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 36, e) A heavy chain variable domain (VH) sequence including CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 41, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 42, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 43, and (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 44, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 45, and (3) SEQ ID NO: f) A light chain variable domain (VL) sequence including CDR-L3 containing the amino acid sequence shown in 46, (1) a heavy chain variable domain (VH) sequence including CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 51, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 52, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 53, and (1) a light chain variable domain (VL) sequence including CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 54, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 55, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 56, g) a light chain variable domain (VL) sequence including CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 61, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 62, and (3) SEQ ID NO: A heavy chain variable domain (VH) sequence including CDR-H3 containing the amino acid sequence shown in 63, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 64, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 65, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 66, or h)The heavy chain variable domain (VH) sequence includes (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 105, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 106, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 107, and the light chain variable domain (VL) sequence includes (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 108, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 109, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 110.

[0103] In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain, and the light chain variable region comprises the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain, where the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain included in the reference heavy chain variable region, and the reference heavy chain variable region has a SEQ ID NO: The amino acid sequence is selected from the group consisting of 7, 17, 27, 37, 47, 57, 67, 85, 89, 93, 97, 101, and 111, and the CDR-L1 domain, CDR-L2 domain, and CDR-L3 domain each include the CDR-L1 domain, CDR-L2 domain, and CDR-L3 domain contained in the reference light chain variable region, and the reference light chain variable region contains an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 83, 84, 86, 90, 94, 98, 102, and 112.

[0104] In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain and a CDR-H3 domain, and the light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain and a CDR-L3 domain, where the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain included in the reference heavy chain variable region, the reference heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 7, and the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain included in the reference light chain variable region, the reference light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 8.

[0105] In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain and a CDR-H3 domain, and the light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain and a CDR-L3 domain, where the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain included in the reference heavy chain variable region, the reference heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 17, and the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain included in the reference light chain variable region, the reference light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 18.

[0106] In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR-H1 domain, a CDR-H2 domain and a CDR-H3 domain, and the light chain variable region comprises a CDR-L1 domain, a CDR-L2 domain and a CDR-L3 domain, where the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L2 domain and CDR-L3 domain each comprise the reference light chain variable region, and the reference light chain variable region comprises the amino acid sequence each comprised of the SEQ ID NO: 28.

[0107] In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR-H1 domain, a CDR-H2 domain and a CDR-H3 domain, and the light chain variable region comprises a CDR-L1 domain, a CDR-L2 domain and a CDR-L3 domain, where the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L2 domain and CDR-L3 domain each comprise the reference light chain variable region, and the reference light chain variable region comprises the amino acid sequence each comprised of the SEQ ID NO: 38.

[0108] In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain and a CDR-H3 domain, and the light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain and a CDR-L3 domain, where the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L2 domain and CDR-L3 domain each comprise the reference light chain variable region, and the reference light chain variable region comprises the amino acid sequence each comprised of a SEQ ID NO: 83.

[0109] In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR-H1 domain, a CDR-H2 domain and a CDR-H3 domain, and the light chain variable region comprises a CDR-L1 domain, a CDR-L2 domain and a CDR-L3 domain, where the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L2 domain and CDR-L3 domain each comprise the reference light chain variable region, and the reference light chain variable region comprises the amino acid sequence each comprised of the SEQ ID NO: 48.

[0110] In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain and a CDR-H3 domain, and the light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain and a CDR-L3 domain, where the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L2 domain and CDR-L3 domain each comprise the reference light chain variable region, and the reference light chain variable region comprises the amino acid sequence each comprised of a SEQ ID NO: 84.

[0111] In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain and a CDR-H3 domain, and the light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain and a CDR-L3 domain, where the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain included in the reference heavy chain variable region, the reference heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 57, and the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain included in the reference light chain variable region, the reference light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 58.

[0112] In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR-H1 domain, a CDR-H2 domain and a CDR-H3 domain, and the light chain variable region comprises a CDR-L1 domain, a CDR-L2 domain and a CDR-L3 domain, where the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L2 domain and CDR-L3 domain each comprise the reference light chain variable region, and the reference light chain variable region comprises the amino acid sequence each comprised of the SEQ ID NO: 68.

[0113] In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises a CDR-H1 domain, a CDR-H2 domain and a CDR-H3 domain, and the light chain variable region comprises a CDR-L1 domain, a CDR-L2 domain and a CDR-L3 domain, where the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-H1 domain, CDR-H2 domain and CDR-H3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L1 domain, CDR-L2 domain and CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L3 domain each comprise the CDR-L2 domain and CDR-L3 domain each comprise the reference light chain variable region, and the reference light chain variable region comprises the amino acid sequence each comprised of the SEQ ID NO: 112.

[0114] In some examples, the anti-GARP / TGFβ antibody includes a heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 6. In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 11, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 12, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 14, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 15, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 16. In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 21, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 22, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 24, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 25, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 26.In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 31, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 32, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 33, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 34, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 35, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 36. In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 41, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 42, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 43, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 44, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 45, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 46. In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 51, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 52, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 53, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 54, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 55, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 56.In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 61, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 62, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 64, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 65, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 66. In some examples, the anti-GARP / TGFβ antibody includes a heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 105, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 106, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 107, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 108, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 109, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 110.

[0115] In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising an amino acid sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 85, 89, 93, 97, 101, and 111, and the light chain variable region comprising SEQ ID NO: It includes an amino acid sequence having at least approximately 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with an amino acid sequence selected from the group consisting of 8, 18, 28, 38, 48, 58, 68, 83, 84, 86, 90, 94, 98, 102, and 112. In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 85, 89, 93, 97, 101, and 111, and the light chain variable region comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 83, 84, 86, 90, 94, 98, 102, and 112.

[0116] In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8. In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18. In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28. In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 37, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 38. In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 37, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 83. In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 47, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 48. In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 47, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 84. In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 57, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 58.In some examples, the anti-GARP / TGFβ antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 67, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 68. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 85, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 86. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 89, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 90. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 93, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 94. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 97, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 98. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 101, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 102. In some examples, the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 111, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO: 112.

[0117] In some examples, any amino acid sequence included in the heavy chain variable region may contain at most about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions. In some examples, the amino acid substitutions are conservative substitutions.

[0118] In some examples, the antibody comprises a human framework. In some examples, the antibody is a human antibody. In some examples, the antibody isolates a human-derived phage display library.

[0119] In some examples, the anti-GARP / TGFβ antibody does not contain an Fc region. In some examples, the anti-GARP / TGFβ antibody further contains an Fc region. In some examples, the Fc region contains a human Fc region. In some examples, the Fc region contains an Fc region selected from the group consisting of IgG, IgA, IgD, IgE, and IgM Fc regions. In some examples, the Fc region contains an Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions. In some examples, the Fc region contains an IgG1 Fc region. In some examples, the IgG1 Fc region contains one or more mutations that modify antibody-dependent cell-mediated cytotoxicity (ADCC). In some examples, the IgG1 Fc region contains one or more mutations that reduce antibody-dependent cell-mediated cytotoxicity (ADCC). In some examples, the IgG1 Fc region contains one or more mutations that enhance antibody-dependent cell-mediated cytotoxicity (ADCC). In some examples, the Fc region includes the IgG4 Fc region. In some examples, the IgG4 Fc region includes the S228P mutation. In some examples, the Fc region includes the C-terminal lysine. In some examples, the Fc region includes the deletion of the C-terminal lysine.

[0120] In some examples, the anti-GARP / TGFβ antibody contains a heavy chain and a light chain (GA1#7K) containing the amino acid sequences shown in SEQ ID NO: 71 and 72, respectively. In some examples, the anti-GARP / TGFβ antibody contains a heavy chain and a light chain (GA1#7K (LC_FS / IT)) containing the amino acid sequences shown in SEQ ID NO: 73 and 74, respectively. In some examples, the anti-GARP / TGFβ antibody contains a heavy chain and a light chain (GA1#7 (LC_FS / IT)) containing the amino acid sequences shown in SEQ ID NO: 75 and 76, respectively. In some examples, the anti-GARP / TGFβ antibody contains a heavy chain and a light chain (GA1#8K) containing the amino acid sequences shown in SEQ ID NO: 77 and 78, respectively. In some examples, the anti-GARP / TGFβ antibody contains a heavy chain and a light chain (GA1#8K (LC_FS / IT)) containing the amino acid sequences shown in SEQ ID NO: 79 and 80, respectively. In some examples, the anti-GARP / TGFβ antibody contains a heavy chain and a light chain (GA1#8 (LC_FS / IT)) containing the amino acid sequences shown in SEQ ID NO: 81 and 82, respectively. In some examples, the anti-GARP / TGFβ antibody contains a heavy chain and a light chain (GA1#8_14) containing the amino acid sequences shown in SEQ ID NO: 87 and 88, respectively. In some examples, the anti-GARP / TGFβ antibody contains a heavy chain and a light chain (GA1#8_17) containing the amino acid sequences shown in SEQ ID NO: 91 and 92, respectively. In some examples, the anti-GARP / TGFβ antibody contains a heavy chain and a light chain (GA1#8_18) containing the amino acid sequences shown in SEQ ID NO: 95 and 96, respectively. In some examples, the anti-GARP / TGFβ antibody contains a heavy chain and a light chain (GA1#8_20) containing the amino acid sequences shown in SEQ ID NO: 99 and 100, respectively. In some examples, the anti-GARP / TGFβ antibody contains a heavy chain and a light chain (GA1#8_21) containing the amino acid sequences shown in SEQ ID NO: 103 and 104, respectively.In some examples, the anti-GARP / TGFβ antibody contains a heavy chain and a light chain (hGA17) having the amino acid sequences shown in SEQ ID NO: 113 and 114, respectively.

[0121] In some examples, the anti-GARP / TGFβ antibody comprises full-length immunoglobulin, single-stranded Fv(scFv) fragment, Fab fragment, Fab' fragment, F(ab')2, Fv fragment, disulfide-bonded stable Fv fragment (dsFv), (dsFv)2, VHH, Fv-Fc fusion, scFv-Fc fusion, VHH-Fv fusion, diabody, triabody, tetrabody, or any combination thereof.

[0122] In some examples, the antibody is contained in a larger molecule as an antibody derivative. In some examples, the antibody derivative is a multispecific antibody, for example, a bispecific antibody, where the multispecific antibody includes a second antibody moiety that specifically binds to a second antigen. In some examples, the second antigen is a tumor-associated antigen. In some examples, tumor-associated antigens include Her-2, B7H3, EGFR, PD-L1, MSLN, c-Met, B-cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD47, CD49f, CD56, CD74, CD123, CD133, CD138, CD276 (B7H3), and epithelium. Glycoprotein (EGP2), trophoblast cell surface antigen 2 (TROP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine kinase erb-B2, 3, 4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human telomerase reverse transcriptase (hTERT), kinase insertion domain receptor (KDR), Lewis The antigen is selected from the group consisting of A (CA 1.9.9), Lewis Y (LeY), phosphatidylinositol proteoglycan-3 (GPC3), L1 cell adhesion molecule (L1CAM), mucin 16 (Muc-16), mucin 1 (Muc-1), NG2D ligand, tumor embryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), claudin 18.2 (CLDN18.2), vascular endothelial growth factor R2 (VEGF-R2), nephroblastoma protein (WT-1), type 1 tyrosine kinase transmembrane receptor (ROR1), PVR, PVRL2, and any combination thereof. In some examples, the second antigen is an immune checkpoint modulator. In some examples, the immune checkpoint modulator is selected from the group consisting of TIGIT, PD1, CTLA4, LAG-3, 2B4, BTLA, and any combination thereof.In some examples, binding of an antibody derivative or multispecific antibody to a second antigen inhibits an immune checkpoint modulator. In some examples, the second antigen is an immunocostimulatory molecule or a subunit of the T cell receptor / CD3 complex. In some examples, the immunocostimulatory molecule is selected from the group consisting of CD28, ICOS, CD27, 4-1BB, OX40, and CD40 and any combination thereof. In some examples, binding of an antibody derivative or multispecific antibody to a second antigen activates the immunocostimulatory molecule. In some examples, the subunit of the T cell receptor / CD3 complex is selected from the group consisting of CD3γ, CD3δ, CD3ε and any combination thereof. In some examples, binding of an antibody derivative or multispecific antibody to a second antigen activates the T cell receptor / CD3 complex.

[0123] In some examples, the anti-GARP / TGFβ antibody is partially linked to a second antigen via a linker. In some examples, the linker is a peptide linker. In some examples, the peptide linker contains about 4 to about 30 amino acids. In some examples, the peptide linker contains about 4 to about 15 amino acids. In some examples, the peptide linker contains an amino acid sequence selected from the group consisting of SEQ ID NO: 117-145.

[0124] In some examples, the anti-GARP / TGFβ antibody is conjugated with a therapeutic agent or label. In some examples, the label is selected from the group consisting of radioisotopes, fluorescent dyes, and enzymes. In some examples, the therapeutic agent is a cytotoxin or a radioisotope.

[0125] 2.2 Antibody affinity

[0126] In some examples, the antibodies or antibody derivatives disclosed herein have high binding affinity to their target antigens. In some examples, the antibodies or antibody derivatives have approximately 1 x 10⁻¹⁶ binding affinity. -7The antibody or antibody derivative binds to the target with a KD of M or less. In some examples, the antibody or antibody derivative is approximately 1 x 10⁻⁶ -8 The antibody or antibody derivative binds to the target with a KD of M or less. In some examples, the antibody or antibody derivative is approximately 5 x 10 -9 The antibody or antibody derivative binds to the target with a KD of M or less. In some examples, the antibody or antibody derivative is approximately 1 x 10⁻⁶ -9 The antibody or antibody derivative binds to the target with a KD of M or less. In some examples, the antibody or antibody derivative is approximately 1 x 10⁻⁶ -10 Binding to the target with a KD of M or less.

[0127] In some examples, the antibody or antibody derivative is approximately 1 x 10⁻⁶ -12 From M to approximately 1x10 -7 The antibody or antibody derivative binds to the target at M KD. In some examples, the antibody or antibody derivative is approximately 1 x 10 -11 From M to approximately 1x10 -7 The antibody or antibody derivative binds to the target at M KD. In some examples, the antibody or antibody derivative is approximately 1 x 10 -10 M to approximately 5x10 -8 The antibody or antibody derivative binds to the target at M KD. In some examples, the antibody or antibody derivative is approximately 1 x 10 -11 From M to approximately 1x10 -9 The antibody or antibody derivative binds to the target at M KD. In some examples, the antibody or antibody derivative is approximately 2 x 10 -10 M to approximately 5x10 -9 The antibody or antibody derivative binds to the target at M KD. In some examples, the antibody or antibody derivative is approximately 1 x 10 -9 M to approximately 5x10 -8 The antibody or antibody derivative binds to the target at M KD. In some examples, the antibody or antibody derivative is approximately 1 x 10 -10 From M to approximately 1x10 -9 M binds to the target via KD.

[0128] The KD of an antibody or antibody derivative may be determined by methods known in the art. Such methods include, but are not limited to, Western blotting, ELISA-, RIA-, ECL-, IRMA-, EIA-, Octet-BIACORE®-test, and peptide scanning.

[0129] In some examples, KD may be measured using a BIACORE® surface plasmon resonance assay. For example, the measurement may be performed using a BIACORE®-2000 or BIACORE® 3000 (Biacore, Piscataway, New Jersey (NJ)) at 25°C with approximately 10 response units (RUs) on an immobilized antigen CMS chip. In some examples, according to the supplier's manual, the carboxymethylated dextran sinter sensor chip (CMS, Biacore) is activated with N-ethyl-N′-(3-dimethylaminopropyl)-carbodimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The antigen is diluted to 5 μg / ml (approximately 0.2 μM) with 10 mM sodium acetate at pH 4.8 and injected at a flow rate of 5 μl / min to realize approximately 10 response units (RUs) of the conjugate protein. After injecting the antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetic measurements, Fab's twofold serial dilutions (0.78 nM to 500 nM) in PBS containing 0.05% polysorbate 20 (TWEEN-20™) surfactant (PBST) are injected at a flow rate of approximately 25 μl / min at 25°C. The association rate (k on ) and dissociation rate (k offThe aggregation rate (KD) is calculated by simultaneously fitting the aggregation and dissociation sensorgrams using a simple one-to-one Langmuir coupling model (BIACORE® evaluation software version 3.2). The equilibrium dissociation constant (KD) may be calculated as the ratio koff / kon. See, for example, Chen et al., J. Mol. Biol. 293:865-881 (1999). If the aggregation rate (on-rate) measured by surface plasmon resonance assay exceeds 10⁶ Ml s⁻¹, the aggregation rate may be determined by fluorescence quenching technique, which is used to increase the antigen concentration (e.g., a spectrometer, e.g., a pass-cut configuration spectrophotometer (Aviv Instruments)) or an 8000 series SLM-AMINCO with a stirred absorption pool. (商標) The presence of a spectrophotometer (ThermoSpectronic) is used to measure the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of a 20 nM anti-antigen antibody (Fab format) in PBS (pH 7.2) at 25°C.

[0130] 2.3 Antibody fragments

[0131] In some embodiments, the antibodies of this disclosure include antigen-binding fragments or antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab')2, VHH, Fv and scFv fragments, and other fragments described herein. For an overview of some antibody fragments, see Hudson et al. Nat. Med. 9: 129-134 (2003). For an overview of scFv fragments, see, for example, Pluckthtin, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994), and also see WO 93 / 16185 and U.S. Patents 5,571,894 and 5,587,458. For studies on the Fab and F(ab)2 fragments containing rescue receptor-binding epitope residues and exhibiting increased in vivo half-lives, please refer to U.S. Patent No. 5,869,046.

[0132] In some embodiments, the antibodies of this disclosure may be diabodies. A diabody is an antibody fragment having two antigen-binding sites, which may be bivalent or bispecific. See, for example, EP 404,097;WO 1993 / 01 161; Hudson et al., Nat. Med. 9:129-134 (2003), and Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993). Triabodies and tetrabodies are further described in Hudson et al., Nat. Med. 9: 129-134 (2003).

[0133] In some embodiments, the antibodies of this disclosure may include single-domain antibodies. A single-domain antibody is an antibody fragment containing all or part of the heavy-chain variable domains or all or part of the light-chain variable domains of an antibody. In some embodiments, the single-domain antibody is a human single-domain antibody (see, for example, U.S. Patent No. 6,248,516 Bl, Domantis, Inc., Waltham, MA). In some embodiments, the single-domain antibody is a camelid single-domain antibody. In some embodiments, the single-domain antibody is a VHH. In some embodiments, the single-domain antibody is a chimeric antibody. In some embodiments, the single-domain antibody is a humanized antibody.

[0134] Antibody fragments may be prepared by a number of techniques, including, but not limited to, complete proteolytic digestion of antibodies and the production of recombinant host cells (e.g., Escherichia coli or phages), as described herein.

[0135] 2.4 Chimeric antibodies and humanized antibodies

[0136] In some embodiments, the antibodies of this disclosure are chimeric antibodies. Some chimeric antibodies are described, for example, in U.S. Patent No. 4,816,567 and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984). In some embodiments, the chimeric antibody comprises a non-human variable region (e.g., a variable region derived from mouse) and a human constant region. In some embodiments, the chimeric antibody is a “class-converted” antibody in which a class or subclass is modified from a class or subclass of the parent antibody. The chimeric antibody comprises its antigen-binding fragment.

[0137] In some embodiments, the antibodies of this disclosure may be humanized antibodies. Typically, non-human antibodies are humanized to reduce their immunogenicity to humans while retaining the specificity and affinity of the parent non-human antibody. Typically, humanized antibodies include one or more variable domains, where HVR, e.g., CDR, (or a portion thereof) is derived from a non-human antibody, and one or more frameworks (FRs) (or any portion thereof) are derived from a human antibody sequence. The humanized antibody may optionally further include at least a portion of the human constant region. In some embodiments, some FR residues in the humanized antibody are replaced with corresponding residues from a non-human antibody (e.g., an antibody derived from HVR residues) to restore or improve, for example, antibody specificity or affinity.

[0138] Humanized antibodies and their preparation methods are described, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further, for example, in Riechmann et al., Nature 332:323-329 (1988), Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989), U.S. Patent Nos. 5,821,337, 7,527,791, 6,982,321 and 7,087,409, Kashmiri et al., Methods 36:25-34 (2005) (SDR (a-CDR) transplantation is described), and Padlan, Mol. Immunol. 28:489-498. This is described in (1991) (where "resurfacing" is described), Dall'Acqua et al., Methods 36:43-60 (2005) (where "FR shuffling" is described), and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (where the "guide selection" method for FR shuffling is described).

[0139] Human framework regions that can be used for humanization include framework regions selected using a "best-fit" method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)), framework regions derived from shared sequences of human antibodies of specific subgroups of light chain or heavy chain variable regions (see, e.g., Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992), and Presta et al. J. Immunol., 151:2623 (1993)), mature (somatically mutant) human framework regions or human germline framework regions (see, e.g., Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)), and framework regions derived from screening of FR libraries (see, e.g., Baca et al., J. Biol. Chem. 272:10678-10684). This includes, but is not limited to, (see (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0140] 2.5 Human Antibodies

[0141] In some embodiments, the antibodies of this disclosure may be human antibodies (e.g., human domain antibodies or human DAbs). Human antibodies may be produced using different techniques known in the art. Human antibodies are generally described in van Dijk and van de Winkel, Curr. Opin. Pharmacol. 5: 368-74 (2001), Lonberg, Curr. Opin. Immunol. 20:450-459 (2008), and Chen, Mol. Immunol. 47(4):912-21 (2010). Transgenic mice or rats capable of producing fully human single-domain antibodies (or DAbs) are known in the art. For example, see US 20090307787 A1, US Patent No. 8,754,287, US 20150289489 A1, US 20100122358 A1, and WO 2004049794.

[0142] Human antibodies (e.g., human DAbs) can be prepared by administering immunogens to transgenic animals modified to produce fully human antibodies or fully antibodies with human variable regions in response to antigen attack. Such animals typically contain all or some human immunoglobulin loci, which either replace endogenous immunoglobulin loci, are extrachromosomal, or are randomly incorporated into the animal's chromosomes. In such transgenic mice, endogenous immunoglobulin loci are usually inactivated. For an overview of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Furthermore, see, for example, XENOMOUSE. TM U.S. Patents 6,075,181 and 6,150,584, and HuMab describe the technology. (登録商標) The technology is described by the U.S., 5,770,429, KM MOUSE. (登録商標) U.S. Patent No. 7,041,870 and VelociMouse describe the technology. (登録商標)See U.S. Patent No. 2007 / 0061900 for a description of the technology. The human variable region from a complete antibody produced by such an animal may be further modified (for example, by binding to a different human constant region).

[0143] Human antibodies (e.g., human DAbs) may be prepared by hybridoma-based methods. Human myeloma and mouse human xenomyeloma cell lines for producing human monoclonal antibodies have been described (e.g., Kozbor J. Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987), and Boerner et al., J. Immunol., 147: 86 (1991)). Human antibodies produced by human B-cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Other methods include those described in, for example, U.S. Patent No. 7,189,826 (which describes the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (which describes human-human hybridomas). Human hybridoma technology (trioma technology) is also described in Vollmers and Brandlein, Histology and Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein, Methods and Findings in Experimental and Clinical Pharmacology, 27(3):185-91 (2005).

[0144] Human antibodies (e.g., human DAbs) may be produced by isolating a variable domain sequence of Fv clones selected from a human phage display library. Such variable domain sequences may then be conjugated to the required human constant domain. A description of the technique for selecting human antibodies from an antibody library is provided below.

[0145] 2.6 Antibodies derived from libraries

[0146] The antibodies of this disclosure can be isolated by screening a combination library for antibodies having the desired activity or multiple activities. For example, several methods are known in the art for producing a phage display library and screening such a library for antibodies having the required binding properties. Such methods are described, for example, in Hoogenboom et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and also in, for example, McCafferty et al., Nature 348:552-554, Clackson et al., Nature 352: 624-628 (1991), Marks et al., J. Mol. Biol. 222: 581-597 (1992), Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003), Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al., J. Mol. Further details are provided in Biol. 340(5): 1073-1093 (2004), Fellouse, Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004), and Lee et al., J. Immunol. Methods 284(1-2): 119-132 (2004). Methods for constructing single-domain antibody libraries are described; see, for example, U.S. Patent No. 7,371,849.

[0147] In some phage display methods, polymerase chain reaction (PCR) is used to display V H and V LEach gene library can be cloned, randomly recombined in the phage library, and antigen-binding phages can be screened according to the description in Winter et al., Ann. Rev. Immunol., 12: 433-455 (1994). The phages typically display antibody fragments as scFv or Fab fragments. Libraries from immunogens can provide high-affinity antibodies against immunogens without constructing hybridomas. Alternatively, as described in Griffiths et al., EMBO J, 12: 725-734 (1993), natural libraries (e.g., those obtained from humans) can be cloned without any immunization to provide a single antibody source against a wide range of non-self and self-antigens. Finally, as described in Hoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992), a natural library can also be synthesized by cloning an unreconstituted V gene fragment from stem cells, encoding a highly variable CDR3 region using PCR primers containing random sequences, and completing the reconstitution in vitro. Publications describing human antibody phage libraries include U.S. Patent No. 5,750,373, and U.S. Patent Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936 and 2009 / 0002360.

[0148] In this specification, antibodies or antibody fragments isolated from a human antibody library are considered to be human antibodies or human antibody fragments.

[0149] 2.7 Antibody variants

[0150] This disclosure further provides amino acid sequence variants of the disclosed antibody. For example, improvements to the binding affinity and / or other biological properties of the antibody may be required. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications to the nucleotide sequence encoding the antibody or by performing peptide synthesis. Such modifications include, but are not limited to, deletions and / or insertions and / or substitutions of residues in the amino acid sequence of the antibody. Any combination of deletions, insertions and substitutions can be used to obtain a final construct, provided that the final (i.e., modified) antibody has the desired properties (e.g., antigen binding).

[0151] 2.7.1 Substitution, insertion, and deletion variants

[0152] In several examples, antibody variants having one or more amino acid substitutions are provided. Target sites for substitutional mutagenesis include HVR (or CDR) and FR. Conservative substitutions are shown under the theme "Preferred Substitutions" in Table 2. More substantial variations are provided under the theme "Exemplary Substitutions" in Table 2 and are further described below with reference to amino acid side chain classes. Amino acid substitutions are introduced into target antibodies, and the products can be screened for desired activity (e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC).

[0153] [Table 2]

[0154] Amino acids can be grouped as follows according to their commonly observed chain properties: (1) Hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) Basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; and (6) Aromatic: Trp, Tyr, Phe. In some examples, non-conservative substitutions require replacing one member of one of these classes with another.

[0155] In some examples, one type of substitution mutant involves the substitution of one or more highly variable region residues of the parent antibody (e.g., a humanized or human antibody). Typically, the resulting mutants are selected for further study and modified (e.g., improved) with several biological properties (e.g., increased affinity, decreased immunogenicity) and / or substantially retained with several biological properties of the parent antibody. Exemplary substitution mutants are affinity-mature antibodies that are easy to produce and utilize affinity-mature techniques (e.g., those disclosed herein), such as phage display. In short, mutations are made in one or more HVR (or CDR) residues, and the mutant antibody is displayed on a phage and screened for specific biological activity (e.g., binding affinity).

[0156] In HVR (or CDR), modifications (e.g., substitutions) can be made to improve, for example, antibody affinity. Such modifications are performed in HVR (or CDR) "hot points," i.e., residues encoded by codons that frequently mutate during somatic cell maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)) and / or SDR (a-CDR), and the binding affinity of the resulting mutant VH or VL can be tested. Affinity maturation by construction and reselection from secondary libraries is described, for example, in Hoogenboom et al. Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)). In some examples of affinity maturation, diversity is introduced into the variable genes selected for maturation by one of several methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-specific mutagenesis). Then, a secondary library is produced. The library is then screened to identify any antibody variant with the desired affinity. Another method for introducing diversity relates to an HVR (or CDR) orientation method, where several HVR (or CDR) residues (e.g., 4-6 residues at a time) are randomized. For example, alanine scanning mutagenesis or modeling can be used to specifically identify HVR (or CDR) residues involved in antigen binding. In particular, CDR-H3 and CDR-L3 are always targeted.

[0157] In some examples, substitutions, insertions, or deletions may occur within one or more HVRs (or CDRs), provided that such modifications do not substantially reduce the antibody's ability to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions as defined herein) may be made within an HVR (or CDR) that do not substantially reduce binding affinity. Such modifications may be located at or outside the HVR (or CDR) "hot point". In some examples of the variant VHH sequences provided above, each HVR (or CDR) is either unmodified or contains one, two, or three or fewer amino acid substitutions.

[0158] As described by Cunningham and Wells (1989) Science, 244:1081-1085, a useful method for identifying antibody residues or regions that can be subjected to targeted mutagenesis is called "alanine scanning mutagenesis." In this method, a target residue or group of residues (e.g., charged residues, e.g., Arg, Asp, His, Lys, and Glu) is identified, and substitution with a neutral or negatively charged amino acid (e.g., alanine or polyalanine) is performed to determine whether the antibody-antigen interaction is affected. Functional sensitivity to the initial substitution can be demonstrated by introducing another substitution at the amino acid position. Alternatively or additionally, the crystal structure of the antigen-antibody complex is used to identify the contact sites between the antibody and antigen. Such contact residues and adjacent residues may be targeted or excluded as substitution candidates. Mutants may be screened to determine whether they contain desired attributes.

[0159] Amino acid sequence insertions include fusions of amino and / or carboxyl terminals within polypeptides ranging in length from one residue to one hundred or more residues, and intrasequence insertions of single or multiple amino acid residues. An example of terminal insertion is an antibody with an N-terminal methionyl residue. Other insertion variants of antibody molecules include fusions of the N-terminus or C-terminus of an antibody with an enzyme (e.g., ADEPT) or polypeptide that increases the serum half-life of the antibody.

[0160] 2.7.2 Glycosylated Mutants

[0161] In some examples, the antibody is modified to increase or decrease the degree of glycosylation of the construct. Addition or deletion of glycosylation sites to the antibody can be easily achieved by modifying the amino acid sequence to produce or remove one or more glycosylation sites.

[0162] If an antibody contains an Fc region (e.g., scFv-Fc), the carbohydrate to which it is linked is modifiable. Natural antibodies produced by mammalian cells typically contain branched bicontact angle oligosaccharides, which are usually linked to the Fc region C by N-bonding. H It is linked to Asn297 in two domains. See, for example, Wright et al. TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose and sialic acid, and fucose attached to GlcNAc in the "stem" of the bi-contact angle oligosaccharide structure. In some examples, modification of the oligosaccharide in the antibody can produce antibody variants with several improved properties.

[0163] In some examples, the antibody has a carbohydrate structure, and the carbohydrate structure lacks fucose attached (directly or indirectly) to the Fc region. For example, the fucose content in such an antibody may be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the Asn297 glycan relative to the total of all sugar structures (e.g., complexed, hybrid, and high-mannose structures) bound to Asn297 as measured by MALDI-TOF mass spectrometry, as described, for example, in WO 2008 / 077546. Asn297 refers to the aspartic acid residue (EU number of the Fc region residue) located around position 297 in the Fc region; however, due to minor sequence changes in the antibody, Asn297 may also be located approximately ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. Such fucosylated mutants may possess improved ADCC function. See, for example, U.S. Patent No. 2003 / 0157108 (Presta, L.) and U.S. Patent No. 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include US 2003 / 0157108, WO 2000 / 61739, WO 2001 / 29246, US 2003 / 0115614, US 2002 / 0164328, US 2004 / 0093621, US 2004 / 0132140, US 2004 / 0110704, US 2004 / 0110282, US 2004 / 0109865, WO 2003 / 085119, WO 2003 / 084570, WO 2005 / 035586, WO 2005 / 035778, WO Examples include 2005 / 053742, WO 2002 / 031140, Okazaki et al. J. Mol. Biol. 336:1239-1249 (2004), and Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986), US 2003 / 0157108 A1, Presta, L, and WO 2004 / 056312 A1, Adams et al.). Examples include the α-1,6-fucosyltransferase gene FUT8, and knockout cell lines (see, for example, Yamane-Ohnuki et al., Biotech. Bioeng. 87: 614 (2004), Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006), and WO 2003 / 085107).

[0164] In some examples, the antibody has a bisecting oligosaccharide, for example, a bicontact angle oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibody variants may have reduced fucosylation and / or improved ADCC function. Examples of such antibody variants are described, for example, in WO 2003 / 011878 (Jean-Mairet et al.), U.S. No. 6,602,684 (Umana et al.), and U.S. 2005 / 0123546 (Umana et al.). Further antibody variants are provided having at least one galactose residue linked to the Fc region in the oligosaccharide. Such antibody variants may have improved CDC function. Such antibody variants are described, for example, in WO 1997 / 30087 (Patel et al.), WO 1998 / 58964 (Raju, S.), and WO 1999 / 22764 (Raju, S.).

[0165] 2.7.3 Fc region variants

[0166] In some embodiments, the Fc region of the antibody or antibody derivative of the present disclosure may include a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region), which includes amino acid modifications (e.g., substitutions) at one or more amino acid positions. In some embodiments, Fc region variants can be produced by introducing one or more amino acid modifications into the Fc region of the antibody moiety (e.g., scFv-Fc or VHH-Fc).

[0167] In some examples, the Fc region possesses several (but not all) effector functions, and these functions make the region a desirable candidate for application, where the in vivo antibody half-life is important, but some effector functions (e.g., complement and ADCC) are unnecessary or harmful. Decreased / depleted CDC and / or ADCC activity can be confirmed by performing in vitro and / or in vivo cytotoxicity assays. For example, by performing an Fc receptor (FcR) binding assay, it can be confirmed that an antibody lacks FcγR binding ability (and therefore may lack ADCC activity) but retains FcRn binding ability. Primary NK cells for mediating ADCC express only FcγRIII, while mononuclear cells express FcγRI, FcγRII, and FcγRIII. FcR expression in hematopoietic cells is summarized in Table 2 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays for evaluating the ADCC activity of target molecules are described in U.S. Patent No. 5,500,362 (see, for example, Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) and Hellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985), and No. 5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361 (1987)). Alternatively, non-radioactive assay methods may be used (e.g., ACTI for flow cytometry). (商標) Non-radioactive cytotoxicity assay (CellTechnology, Inc., Mountain View, California), and CytoTox 96 (登録商標)Non-radioactive cytotoxicity assays (see Promega, Madison, Wisconsin). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, the ADCC activity of the target molecule may be evaluated in vivo, for example, in an animal model, e.g., as disclosed in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). By performing a C1q binding assay, it can be confirmed that the antibody cannot bind to C1q and therefore lacks CDC activity. See, for example, the binding of C1q and C3c to ELISA in WO 2006 / 029879 and WO 2005 / 100402. To evaluate complement activation, a CDC assay may be performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996), Cragg, MS et al., Blood 101:1045-1052 (2003), and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life assays may also be performed using methods known in this field (see, for example, Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0168] Antibodies with reduced effector function include antibodies (U.S. Patent No. 6,737,056) having one or more substitutions of Fc region residues 238, 265, 269, 270, 297, 327, and 329. Such Fc mutants include Fc mutants having two or more substitutions of amino acid positions 265, 269, 270, 297, and 327, and include the so-called "DANA" Fc mutant (U.S. Patent No. 7,332,581) in which residues 265 and 297 are substituted with alanine.

[0169] Several antibody variants exhibiting improved or decreased binding affinity to FcR have been described. (See, for example, U.S. Patent No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

[0170] In some examples, the Fc region contains one or more mutations based on the EU number of the residue. In some examples, the Fc region is the IgG1 Fc region. In some examples, the IgG1 Fc region contains the L234A mutation and / or the L235A mutation. In some examples, the Fc region is the IgG2 or IgG4 Fc region. In some examples, the Fc region is the IgG4 Fc region containing the F234A and / or L235A mutation.

[0171] In some examples, the Fc region is the IgG1 Fc region. In some examples, the IgG1 Fc region contains one or more mutations that modify antibody-dependent cell-mediated cytotoxicity (ADCC). In some examples, the IgG1 Fc region contains one or more mutations that reduce antibody-dependent cell-mediated cytotoxicity (ADCC). In some examples, the IgG1 Fc region contains one or more mutations that enhance antibody-dependent cell-mediated cytotoxicity (ADCC). In some examples, the IgG1 Fc region contains the L235V, F243L, R292P, Y300L, and P396L mutations. In some examples, the IgG1 Fc region contains the S239D, A330L, and I332E mutations. In some examples, the IgG1 Fc region contains the L235V, F243L, R292P, and Y300L mutations. In some embodiments, the IgG1 Fc region includes substitution at positions 298, 333 and / or 334 of the Fc region.

[0172] In some examples, the Fc region includes the IgG4 Fc region. In some examples, the IgG4 Fc region includes the S228P mutation.

[0173] In some examples, the Fc region includes C-terminal lysine. In some examples, the Fc region includes a deletion of C-terminal lysine.

[0174] In some examples, modifications within the Fc region have resulted in alterations (i.e., increases or decreases) to C1q binding and / or complement-dependent cytotoxicity (CDC), as described, for example, in U.S. Patent No. 6,194,551, WO 99 / 51642 and Idusogie et al. J. Immunol. 164: 4178-4184 (2000).

[0175] In some examples, antibody variants (e.g., scFv-Fc or VHH-Fc) include a variant Fc region containing one or more amino acid substitutions that alter the half-life and / or the binding to the neonatal Fc receptor (FcRn). Antibodies with an extended half-life and improved binding to the neonatal Fc receptor (FcRn) are responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), as described in US 2005 / 0014934 A1 (Hinton et al.). These antibodies include an Fc region with one or more substitutions, where these substitutions alter the binding between the Fc region and FcRn. Such Fc variants include those variants (U.S. Patent No. 7,371,826) that have substitutions in one or more Fc region residues (e.g., substitution of Fc region residue 434).

[0176] Furthermore, see Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO94 / 29351, which relates to other examples of Fc region variants.

[0177] 2.7.4 Cysteine-engineered antibody variants

[0178] In some examples, it may be necessary to produce a cysteine-engineered antibody moiety, e.g., "thioMAb," in which one or more residues of the antibody are substituted with cysteine ​​residues. In some examples, the substituted residues are located in an accessible site of the antibody. By substituting these residues with cysteine ​​residues, the reactive thiol group is located in an accessible site of the antibody and may be used to conjugate the antibody with other moieties, e.g., a drug moiety or a linker-drug moiety, to produce an immunoconjugate, as further described herein. In some examples, any one or more residues of A118 (EU number) of the heavy chain and S400 (EU number) of the heavy chain Fc region may be substituted with cysteine. The cysteine-engineered antibody moiety may be produced, for example, as described in U.S. Patent No. 7,521,541.

[0179] 2.8 Antibody derivatives

[0180] In some examples, the antibodies described herein may be further modified into antibody derivatives containing other protein or non-protein moieties known and readily available in the art. Non-protein moieties suitable for antibody derivatization 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, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymer, polyamino acids (homopolymer or random copolymer) with dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in preparation due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers linked to the antibody may vary, and if there are more than one type of polymer linked, they may be the same or different molecules. Generally, the number and / or type of polymers used in derivatization may be determined based on the following considerations, including, but not limited to, the specific properties or functions of the antibody to be improved, and whether or not the antibody derivative will be used for the determined diagnostic conditions.

[0181] In some embodiments, the antibody may be further modified into an antibody derivative comprising one or more bioactive proteins, polypeptides, or fragments thereof. As used interchangeably herein, “biologically active” or “having biological activity” refers to the biological activity demonstrated in vivo to perform a particular function. For example, it may mean binding to a particular biomolecule (e.g., protein, DNA, etc.) and promoting or inhibiting the activity of such biomolecule. In some embodiments, the bioactive proteins or fragments thereof include proteins and polypeptides administered to a patient as active drug substances, proteins and polypeptides used for the prevention or treatment of a disease or condition and for diagnosis (e.g., enzymes used in diagnostic tests or in vitro assays), and proteins and polypeptides administered to a patient to prevent a disease (e.g., vaccines).

[0182] 2.9 Production Method

[0183] The antibodies and antibody derivatives disclosed herein can be produced using any available or known techniques in the art. For example, antibodies and antibody derivatives can be produced using, for example, the recombinant methods and compositions described in U.S. Patent No. 4,816,567. Detailed procedures for producing antibodies and antibody derivatives are described in detail in the following examples.

[0184] The subject of this disclosure further provides isolated nucleic acids encoding antibodies or antibody derivatives disclosed herein. For example, isolated nucleic acids may encode amino acid sequences including the VL of an antibody and / or amino acid sequences including the VH of an antibody, such as the light chain and / or heavy chain of an antibody.

[0185] In some embodiments, the nucleic acid may be present in one or more vectors (e.g., expression vectors). As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it can be ligated. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop to which another DNA segment can be ligated. Another type of vector is a viral vector, to which another DNA segment can be ligated into a viral genome. Some vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors with bacterial replication origins and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors), after being introduced into a host cell, are integrated into the host cell's genome and thereby replicate with the host genome. Also, some vector expression vectors can guide the expression of genes to which they are manipulably ligated. Generally, expression vectors used in recombinant DNA technology are always in plasmid (vector) form. However, the topics disclosed are intended to include other expression vectors with equivalent function, such as viral vectors (e.g., replication-deficient retroviruses, adenoviruses, and adeno-associated viruses).

[0186] Different portions of the antibodies or antibody derivatives disclosed herein can be constructed in a single polycistronic expression cassette, multiple expression cassettes of a single vector, or multiple vectors. Examples of elements that produce polycistronic expression cassettes include, but are not limited to, various viral and nonviral internal ribosome entry sites (IRES, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-κB IRES, RUNX1 IRES, p53 IRES, hepatitis A IRES, hepatitis C IRES, pestivirus IRES, foot-and-mouth disease virus IRES, picornanilus IRES, poliovirus IRES, and encephalomyocarditis virus IRES), and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). A combination of a retroviral vector and a suitable packaging thread is also preferred, where the capsid protein has the function of infecting human cells. Cell lines that produce various amphiphilic viruses are known, including, but are not limited to, PA12 (Miller, et al. (1985) Mol. Cell. Biol. 5:431-437), PA317 (Miller, et al. (1986) Mol. Cell. Biol. 6:2895-2902), and CRIP (Danos, et al. (1988) Proc. Natl. Acad. Sci. USA 85:6460-6464). Non-amphiphilic particles are also preferred, for example, VSVG, RD114, or GALV envelopes and any other known pseudotyped particles in the art.

[0187] In some embodiments, one or more vectors comprising nucleic acids encoding the antibodies or antibody derivatives of the Disclosure may be introduced into host cells. In some embodiments, nucleic acids may be introduced into cells by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a viral or phage vector comprising a nucleic acid sequence, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, and the like. In some embodiments, the host cells may include, for example, host cells transformed with the following vectors, which comprise a single-domain antibody and / or a nucleic acid encoding an amino acid sequence comprising the VH of the single-domain antibody. In some embodiments, the host cells may include, for example, host cells transformed with a vector comprising (1) a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and an amino acid sequence comprising the VH of the antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of the antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of the antibody. In some embodiments, the host cells are eukaryotic cells, such as Chinese hamster ovary (CHO) cells or lymphocytes (e.g., YO, NSO, Sp20 cells).

[0188] In some examples, the method for preparing an antibody or antibody derivative disclosed herein may include culturing host cells into which the nucleic acid encoding the antibody or antibody derivative has been introduced under conditions suitable for antibody or antibody derivative expression, and optionally recovering the antibody or antibody derivative from the host cells and / or host cell culture medium. In some examples, the antibody or antibody derivative is recovered from the host cells by chromatographic technique.

[0189] To recombinantly produce the antibodies or antibody derivatives of this disclosure, for example, the nucleic acids encoding the antibodies or antibody derivatives described above can be isolated, inserted into one or more vectors, and then cloned and / or expressed in host cells. These nucleic acids can be easily isolated and sequenced using conventional procedures (for example, by using oligonucleotide probes that can specifically bind to the genes encoding the heavy and light chains of the antibody or antibody derivative). Suitable host cells for cloning or expressing antibody-encoding vectors include prokaryotic or eukaryotic cells as described herein. For example, antibodies or antibody derivatives may be produced in bacteria, particularly when fucosylation and Fc effector function are not required. For expression of antibody fragments and polypeptides in bacteria, see, for example, U.S. Patents 5,648,237, 5,789,199 and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254 for further reference on antibody fragment expression in Escherichia coli.) After expression, the antibody or antibody derivative can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0190] In addition to prokaryotes, eukaryotic microorganisms (e.g., filamentous fungi or yeasts) are also suitable cloning or expression hosts for antibody-encoding vectors, and by including fungal and yeast strains in which the glycosylation pathway has been "humanized," they produce antibodies or antibody derivatives with a partially or completely human glycosylation pattern. See Gemgross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:21 0-215 (2006). Suitable host cells for expressing glycosylated antibodies may be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Many baculovirus strains have been identified, and these can be used in combination with insect cells, particularly for transfection of fall armyworm (Spodoptera frugiperda) cells. In some examples, plant cell cultures may be used as host cells. For example, U.S. Patent Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978 and 6,417,429 (PLANTIBODIES for producing antibodies in transgenic plants) (商標) Please refer to the technical description.

[0191] In some embodiments, vertebrate cells may be used as host cells. For example, mammalian cell lines suitable for suspension growth may be useful, but are not limited to them. Non-limiting examples of useful mammalian host cell lines include the SY40(COS-7) transformed monkey kidney CV1 cell line, human fetal kidney cell line (293 or 293 cells, e.g., described in Graham et al., J Gen Viral. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells, e.g., described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV 1), African green monkey kidney cells (VERO-76), human cervical cancer cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep 02), mouse mammary tumor cells (MMT 060562), and TRI cells (e.g., Mather et al., These include MRC 5 cells and FS4 cells (described in Annals NY Acad. Sci. 383:44-68 (1982)). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, which include DHFK CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:42 I6 (1980)) and myeloma cell lines (e.g., YO, NSO, and Sp2 / 0). For an overview of several mammalian host cell lines suitable for antibody or antibody derivative production, see, for example, Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKCLo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0192] In some examples, techniques for preparing bispecific and / or multispecific antibodies include, but are not limited to, the recombinant expression of two immunoglobulin heavy-chain-light-chain pairs having the same specificity, with one or both of the heavy or light chains being fused to an antigen-binding moiety having different specificity (e.g., VHH or scFv), the recombinant co-expression of two immunoglobulin heavy-chain-light-chain pairs having different specificity (see Milstei n and Cuello, Nature 305: 537 (1983)), PCT Patent Application No. WO 93 / 08829 and Traunecker et al., EMBO J 10: 3655 (1991)), and “knob-hole structure” engineering (see, for example, U.S. Patent No. 5,731,168). Bispecific antibodies can be produced by engineering the electrostatic steering effect to prepare antibody Fc-heterodimer molecules (WO 2009 / 089004 A1), crosslinking two or more antibodies or fragments (see, e.g., U.S. Patent No. 4,676,980 and Brennan et al., Science, 229: 81 (1985)), producing bispecific antibodies using a leucine zipper (see, e.g., Kostelny et al., J Immunol., 148(5): 1547-1553 (1992)), preparing bispecific antibody fragments using "diabody" technology (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), or using single-stranded Fv(sFv) dimers (see, e.g., Gruber et al., J. Immunol. See 152:5368 (1994), and may also be prepared by using a trispecific antibody, for example, as described in Tutt et al. J Immunol. 147: 60 (1991).

[0193] The bispecific and multispecific molecules of this disclosure may be prepared by chemical techniques (see, for example, Kranz (1981) Proc. Natl. Acad. Sci. USA 78:5807), "polydoma" techniques (see, for example, U.S. Patent No. 4,474,893), or recombinant DNA techniques. Furthermore, the bispecific and multispecific molecules of the topic now disclosed may be prepared by conjugating constitutive binding specificities, e.g., a first epitope and a second epitope binding specificity, by methods known in the art and those described herein. For example, each binding specificity of the bispecific and multispecific molecules may be prepared together by recombinant fusion protein techniques, or separately and then conjugated to each other. When the binding specificity is a protein or peptide, covalent bonding may be performed using various coupling or crosslinking agents. Non-restrictive examples of crosslinking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) (see, for example, Karpovsky (1984) J. Exp. Med. 160:1686 and Liu (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus (Behring Ins. Mitt. (1985) No. 78, 1 18-132, Brennan (1985) Science 229:81-83), Glennie (1987) J Immunol. 139: 2367-2375). When the binding specificity is an antibody (e.g., two humanized antibodies), they may be conjugated by mercapto linkage of the C-terminal hinge regions of the two heavy chains. In some examples, prior to conjugation, the hinge region may be modified to contain an odd number (e.g., one) mercapto residues.

[0194] In some embodiments, the two binding specificities of a bispecific antibody may be encoded in the same vector and expressed and assembled in the same host cell. The method is particularly useful when the bispecific and multispecific molecules are MAb x Mab, MAb x Fab, Fab x F(ab')2, or ligand x Fab fusion proteins. In some embodiments, the bispecific antibody of the Disclosure may be a single-chain molecule, e.g., a single-chain bispecific antibody, a single-chain bispecific molecule comprising one single-chain antibody and a binding determination cluster, or a single-chain bispecific molecule comprising two binding determination clusters. The bispecific and multispecific molecules may be single-chain molecules, or may comprise at least two single-chain molecules. Methods for preparing bispecific and multispecific molecules are described, for example, in U.S. Patents 5,260,203, 5,455,030, 4,881,175, 5,132,405, 5,091,513, 5,476,786, 5,013,653, 5,258,498, and 5,482,858. This specification further includes engineered antibodies having three or more functional antigen-binding sites (e.g., epitope-binding sites), and includes "octopus antibodies" (see, for example, US 2006 / 0025576 A1).

[0195] In some examples, animal systems can be used to produce the antibodies or antibody derivatives of the present disclosure. The animal system used to prepare hybridomas is a mouse system.

[0196] The production of hybridomas in mice is a very well-established procedure. Immunotherapy schemes and techniques for isolating immunized splenocytes for fusion are known in this field. Fusion partners (e.g., mouse myeloma cells) and fusion procedures are also known (see, e.g., Harlow and Lane (1988), Antibodies, A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York).

[0197] 2.10 Assay

[0198] The antibodies and antibody derivatives disclosed herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by known assays in the art and by several assays provided herein.

[0199] In some embodiments, the antigen-binding activity of the antibodies or antibody derivatives of this disclosure can be tested by known methods (e.g., enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or Western blot assay). Each of these assays typically detects the presence of a protein-antibody complex of particular interest using a labeled reagent (e.g., an antibody) that is specific to the complex of interest. For example, an antibody or antibody derivative can be detected by an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the antibody or antibody derivative. Alternatively, an antibody or antibody derivative may be detected by any one of several other immunoassays. For example, the antibody or antibody derivative may be radiolabeled and used in a radioimmunoassay (RIA) (see, for example, Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, which is incorporated herein by reference). For example, radioactive isotopes can be detected using a Geiger counter, a scintillation counter, or by means such as autoradiography.

[0200] In some examples, a competitive assay may be used to identify antibodies or antibody derivatives that compete with the antibodies disclosed herein for binding to the GARP / TGFβ complex. In some examples, such competitive antibodies bind to the same epitopes (e.g., linear or structural epitopes) that bind to the antibodies disclosed herein. Detailed exemplary methods for positioning antibody-binding epitopes are provided in Morris (1996) "Epitope Mapping Protocols," Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ).

[0201] In a non-limiting example of a competitive assay, an immobilized GARP / TGFβ complex can be incubated in a solution containing a first labeled antibody or antibody derivative that binds to the GARP / TGFβ complex and a second unlabeled antibody to test the ability of the second unlabeled antibody to compete with the first antibody for binding to the GARP / TGFβ complex. The second antibody may be present in the hybridoma supernatant. As a control, the immobilized GARP / TGFβ complex is incubated in a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow binding of the first antibody to the GARP / TGFβ complex, excess unbound antibody is removed and the amount of labeling associated with the immobilized GARP / TGFβ complex is measured. If the amount of labeling associated with the immobilized GARP / TGFβ complex in the test sample is significantly reduced compared to the control sample, it indicates that the second antibody is competing with the first antibody for binding to the GARP / TGFβ complex. See Harlow and Lane (1988) Antibodies: A Laboratory Manual ch.14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0202] This disclosure provides assays for identifying biologically active anti-GARP / TGFβ antibodies or antibody derivatives thereof. The biological activity may include, for example, immune cells or immune activation reporter genes (e.g., NFAT reporter genes or NF-κB reporter genes). Further disclosures provide antibodies having such biological activity in vivo and / or in vivo.

[0203] 2.11 Immune Conjugates

[0204] The Theme of this Disclosure further provides an immunoconjugate comprising an antibody or antibody derivative disclosed herein that is conjugated to one or more detection probes and / or cytotoxic agents (e.g., chemotherapeutic agents or drugs, growth inhibitors, toxins (e.g., protein toxins, enzyme-active toxins of bacterial, fungal, plant or animal origin, or fragments thereof)), or radioisotopes. For example, the antibody or antigen-binding portion of the Theme of the Disclosure may be functionally linked to one or more other binding molecules (e.g., another antibody, antibody fragment, peptide or binding mimetic) (e.g., by chemical coupling, gene fusion, non-covalent association or other means).

[0205] In some embodiments, the immunoconjugate is an antibody-drug conjugate (ADC), where the antibody is conjugated to one or more drugs, and maytansinoids (U.S. Patents No. 5,208,020, 5,416,064 and European Patent No. EP 0 425). See Patent No. 235), auristatin, e.g., monomethyl auristatin drug parts DE and DF (MMAE and MMAF) (see U.S. Patents 5,635,483, 5,780,588, and 7,498,298), dolastatin, calicheamicin or its derivatives (see U.S. Patents 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710, 5,773,001, and 5,877,296, Hinman et al., Cancer Res. 53:3336-3342 (1993), and Lode et al., Cancer Res. 58:2925-2928) (See 1998), anthracyclines such as donomycin or doxorubicin (Kratz et al., Current Med Chem. 13:477-523 (2006), Jeffrey et al., Bioorganic & Med. Chem. Letters 16:358-362 (2006), Torgov et al., Bioconj. Chem. 16:717-721 (2005), Nagy et al., Proc. Natl. Acad. Sci. USA 97:829-834 (2000), Dubowchik et al., Bioorg. & Med. Chem. Letters 12:1529-1532 (2002), King et al., J Med. Chem. 45:4336-4343 See (2002) and U.S. Patent No. 6,630,579), including, but not limited to, methotrexate, vindesine, taxanes such as docetaxel, paclitaxel, lalotaxel, tesetaxel and ortaxel, trichothecenes, and CC1065.

[0206] In some embodiments, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, and the enzymatically active toxin or fragment thereof comprises, but is not limited to, diphtheria A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain (derived from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, abrin, dianthin, Phytolaca americana (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor, jatrophin, curcin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and trichothecenes.

[0207] In some embodiments, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioactive conjugate. A plurality of radioisotopes may be used in the production of the radioactive conjugate. Non-limiting examples include At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 and radioisotopes of Lu. When the radioactive conjugate is used for detection, it may comprise a radioactive atom used in scintillation studies, such as tc99m or 1123, or a spin label used in nuclear magnetic resonance (NMR) imaging (also referred to as magnetic resonance imaging, MRI), such as iodine 123, iodine 131, indium 11, fluorine 19, carbon 13, nitrogen 15, oxygen 17, gadolinium, manganese, or iron.

[0208] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents (e.g., N-succinimidyl-3-(2-pyridine dimercapto)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminosulfan (IT), bifunctional derivatives of imide esters (e.g., dimethyladipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), disazo compounds (e.g., bis(p-azidobenzoyl)hexanediamine), binitrogen derivatives (e.g., bis-(p-diazobenzoyl)-ethylenediamine), diisocyanates (e.g., tolylene 2,6-diisocyanate), and bisactive fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene)). For example, lysine immunotoxins can be prepared as described in Vitetta et al., Science 238: 1098 (1987). Carbon-4-labeled l-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radioactive nucleotides and antibodies. See WO 94 / 11026. The linker may be a “cleavable linker” that facilitates the release of cytotoxic drugs in cells. For example, acid-unstable linkers, peptidase-sensitive linkers, photo-unstable linkers, dimethyl linkers, or disulfide-containing linkers can be used (Chari et al., Cancer Res. 52:127-1 31 (1992), U.S. Patent No. 5,208,020).

[0209] The immunoconjugates or ADCs described herein clearly cover, but are not limited to, such conjugates prepared using crosslinking agents, and include, but are not limited to, commercially available BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB, SMCC, SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB, sulfo-SMCC, sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) (e.g., from Pierce Biotechnology, Inc., Rockford, IL., USA).

[0210] 2.12 Antigen Recognition Receptors

[0211] The theme of this publication further provides antigen-recognizing receptors, including antibodies or antibody fragments disclosed herein. Antigen-recognizing receptors are receptors that are activated in response to their binding to an antigen and can stimulate or inhibit immune-responsive cells (e.g., T cells). Non-limiting examples of antigen-recognizing receptors include native and recombinant T cell receptors (TCRs), chimeric costimulatory receptors (CCRs), chimeric antigen receptors (CARs), and inhibitory CARs (iCARs). The design and use of antigen-recognition receptors are well-known in this field and are described in the literature, for example, the international publications WO 2018 / 027155, WO 2019 / 099483, WO 2019 / 157454, WO 2019 / 133969, WO 2019 / 099993, WO 2015 / 142314, WO 2018 / 027197, and WO 2014055668.

[0212] In some embodiments, the theme of this disclosure provides chimeric antigen receptors (CARs) comprising antibodies or antibody fragments disclosed herein. CARs are engineered receptors that can be transplanted or conferred with specificity of interest to immune effector cells. In some embodiments, CARs can be used to transplant the specificity of monoclonal antibodies into T cells, with a vector facilitating the transfer of their coding sequence. In some embodiments, the CARs are “first-generation” CARs, which typically consist of an extracellular antigen-binding domain (e.g., scFv or VHH) fused to a transmembrane domain, the transmembrane domain fused to a cytoplasmic / intracellular signaling domain. “First-generation” CARs provide de novo antigen recognition regardless of HLA-mediated antigen presentation and can induce activation of immune-responsive cells (e.g., CD4+ and CD8+ T cells) by their CD3z chain signaling domain in a single fusion molecule. In some embodiments, the CAR is a “second-generation” CAR, which further includes an intracellular signaling domain from various co-stimulatory molecules (e.g., CD28, 4-1BB, ICOS, 0X40, CD27, CD40 / My88, and NKGD2) to the cytoplasmic tail region of the CAR to provide additional signaling to immune-responsive cells, thereby including those that provide both co-stimulation (e.g., CD28 or 4-1BB) and activation (CD3z). In some embodiments, the CAR is a “third-generation” CAR, which includes multiple co-stimulatory domains (e.g., CD28 and 4-1BB) and activation (CD3z). In some embodiments, the CAR is a second-generation CAR. In some embodiments, the CAR includes an extracellular antigen-binding domain that binds to an antigen, a transmembrane domain, and an intracellular signaling domain, where the intracellular signaling domain includes a co-stimulatory signaling domain. In some embodiments, the CAR further includes a hinge / spacing region between the extracellular antigen-binding domain and the transmembrane domain. In some examples, the extracellular antigen-binding domain comprises an antibody or antibody fragment disclosed herein. In some examples, the antibody or antibody fragment comprises VHH, Fab, or scFv.

[0213] In some embodiments, the theme of this disclosure provides recombinant TCRs comprising antibodies or antibody fragments disclosed herein. Native TCRs are protein complexes comprising a heterodimer protein linked by disulfide bonds, the heterodimer protein consisting of two variable chains expressed as part of a complex with a CD3 chain molecule. Native TCRs reside on the T cell surface and play a role in recognizing antigens as peptides that bind to major histocompatibility complex (MHC) molecules. In some embodiments, native TCRs comprise α and β chains (encoded by TRA and TRB genes, respectively). In some embodiments, TCRs comprise γ and δ chains (encoded by TRG and TRD genes, respectively). The α, β, γ, and δ chains each comprise two extracellular domains: a variable (V) region and a constant (C) region. The constant region is close to the cell membrane, followed by a transmembrane region and a short cytoplasmic tail region. The variable region binds to the peptide / MHC complex. Each variable region comprises three complementarity-determining regions (CDRs). In some examples, the TCR includes receptor complexes with CD3δ, CD3γ, CD3ε, and CD3ζ. When the TCR complex binds to its antigen and MHC (peptide / MHC), the T cells expressing the TCR complex are activated.

[0214] In some examples, the recombinant TCR is a non-natural TCR. In some examples, the recombinant TCR comprises a recombinant α chain and / or a recombinant b chain, where part or all of the variable region of the recombinant α chain and / or recombinant b chain is replaced with an antibody or antibody fragment disclosed herein. In some examples, the antibody or antibody fragment comprises VHH, VH, VL, or scFv. In some examples, the antibody or antibody fragment comprises VHH. In some examples, the recombinant TCR binds to an antigen of interest in an MHC / HLA-independent manner. In some non-limiting examples, antigen binding can activate immune-responsive cells containing the recombinant TCR.

[0215] The subject of this disclosure provides immune-responsive cells, which (a) include antigen-recognizing receptors (e.g., CARs or TCRs) disclosed herein. In some embodiments, antigen-recognizing receptors can activate immune-responsive cells. The immune-responsive cells of the subject of this disclosure may also be lymphoid cells. Lymphoid cells, including B, T, and natural killer (NK) cells, provide functions such as antibody production, regulation of the cellular immune system, detection of exogenous reagents in the blood, and detection of exogenous host cells. Non-limiting examples of lymphoid immune-responsive cells include T cells, natural killer (NK) cells, embryonic stem cells, and pluripotent stem cells (e.g., from which lymphocytes can differentiate). T cells may be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells are involved in the adaptive immune system. The T cells of the subject of this disclosure may be any type of T cell, including auxiliary T cells, cytotoxic T cells, memory T cells (central memory T cells, stem-cell-like memory T cells / stem-like memory T cells), and two types of effect memory T cells, e.g., TEM cells and TEMRA cells, regulatory T cells (also called inhibitory T cells), natural killer T cells, mucosa-associated invariant T cells, and gd T cells. Cytotoxic T cells (CTLs or killer T cells) are a subset of T lymphocytes that can induce the death of infected somatic or tumor cells. The patient's own T cells can be genetically modified to target specific antigens by introducing antigen-recognizing receptors (e.g., CARs or TCRs). In some embodiments, the immune-responsive cells are T cells. The T cells may be CD4+ T cells or CD8+ T cells. In some embodiments, the T cells are CD4+ T cells. In some embodiments, the T cells are CD8+ T cells. Natural killer (NK) cells may be lymphocytes that function as part of cell-mediated immunity during innate immune responses. NK cells do not need to be activated beforehand to exert cytotoxic effects against target cells.The types of human lymphocytes that are the subject of this disclosure include, but are not limited to, peripheral donor lymphocytes, for example, Sadelain, M., et al. 2003 Nat Rev Cancer 3:35-45 (peripheral donor lymphocytes genetically modified to express a CAR are disclosed), Morgan, R.A., et al. 2006 Science 314: 126-129 (peripheral donor lymphocytes genetically modified to express a full-length tumor antigen recognition T cell receptor complex containing an a and b heterodimer are disclosed), Panelli, M.C., et al. 2000 J Immunol 164:495-504, Panelli, M.C., et al. 2000 J Immunol 164:4382-4392 (lymphocyte cultures derived from tumor infiltrating lymphocytes (TILs) in tumor biopsies are disclosed), and Dupont, J., et al. 2005 Cancer Res 65:5417-5427, Papanicolaou, G.A., et al. 2003 Blood 102:2498-2505 (the use of artificial antigen presenting cells (AAPCs) or pulsed dendritic cells for the selective in vitro expansion of antigen-specific peripheral blood leukocytes is disclosed). In some embodiments, the immunoreactive cells (e.g., T cells) may be of autologous origin, allologous origin (e.g., allogeneic), or derived in vitro from engineered progenitor cells or stem cells.

[0216] 3. Methods of Use

[0217] The subject of this disclosure further provides methods of using the disclosed antibodies and antibody derivatives. In some embodiments, these methods relate to the therapeutic use of the antibodies or antibody derivatives of this disclosure. In some embodiments, these methods relate to the diagnostic use of the antibodies or antibody derivatives of this disclosure.

[0218] 3.1 Therapeutic Methods

[0219] This disclosure provides methods and uses for using the antibodies or antibody derivatives disclosed herein to treat diseases and disorders or enhance immune responses. In some examples, the antibodies, antibody derivatives, or drug compositions comprising the antibodies or antibody derivatives disclosed herein can be administered to a subject (e.g., a mammal (e.g., human)) to treat diseases and disorders or enhance immune responses. In some examples, these diseases and disorders relate to Treg-mediated immunosuppression and / or abnormal GARP / TGFβ activity. In some examples, diseases and disorders treatable with the antibodies or antibody derivatives disclosed herein include, but are not limited to, tumorigenesis (e.g., cancer).

[0220] In some embodiments, the Disclosure provides antibodies or antibody derivatives (or fragments thereof) as described herein for use in the preparation of drugs. In some embodiments, the Disclosure provides antibodies or antibody derivatives (or fragments thereof) as described herein for use in the preparation of drugs for the treatment of cancer. In some embodiments, the Disclosure provides antibodies or antibody derivatives (or fragments thereof) as described herein for use in the treatment of cancer in a subject. In some embodiments, the Disclosure provides drug compositions comprising antibodies or antibody derivatives (or fragments thereof) as described herein for use in the treatment of cancer in a subject. In some embodiments, cancer may be hematological cancers (e.g., leukemia, leukemia and myeloma), ovarian cancer, breast cancer, bladder cancer, brain cancer, colon cancer, intestinal cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, skin cancer, gastric tumors, glioblastoma, laryngeal cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various other cancers (including prostate cancer and small cell lung cancer). Appropriate cancers further include any known cancers in the field of oncology, which include astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymal cell tumor, medulloblastoma, primary neuroectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small cell and large cell lung adenocarcinoma, chordoma, angiosarcoma, endosarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma, and their liver metastases, lymphangiosarcoma, intralymphatic sarcoma, liver cancer, cholangiocarcinoma, and synovial mammary gland. Mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon cancer, basal cell carcinoma, sweat adenoma, papillary carcinoma, sebaceous gland carcinoma, papillary carcinoma, cystadenocarcinoma, medullary carcinoma, bronchial cancer, renal cell carcinoma, cholangiocarcinoma, choriocarcinoma, seminomas, fetal cancer, Wilms' tumor, medulloblastoma, myopharyngoma, ependymoma, pineal gland tumor, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia This includes, but is not limited to, macroglobulinemia, breast tumors (e.g., ductal adenocarcinoma and lobular adenocarcinoma), cervical squamous cell carcinoma and adenocarcinoma, uterine and ovarian epithelial carcinoma, prostate cancer, bladder transition squamous cell carcinoma, B and T lymphomas (nodular and dispersive), plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma, and leiomyosarcoma.

[0221] In some embodiments, the cancer may be melanoma, NSCLC, head and neck cancer, urothelial carcinoma, breast cancer (e.g., triple-negative breast cancer, TNBC), gastric cancer, cholangiocarcinoma, classical Hodgkin lymphoma (cHL), non-Hodgkin lymphoma primary mediastinal B-cell lymphoma (NHL PMBCL), mesothelioma, ovarian cancer, lung cancer (e.g., small cell lung cancer), esophageal cancer, nasopharyngeal cancer (NPC), biliary tract cancer, colorectal cancer, cervical cancer, or thyroid cancer. In some embodiments, the cancer exhibits high microsatellite instability (MSI-high). In some embodiments, the cancer exhibits low microsatellite instability (MSI-low).

[0222] In some embodiments, the subjects to be treated are mammals (e.g., humans, non-primates, rats, mice, cattle, horses, pigs, sheep, goats, dogs, cats, etc.). In some embodiments, the subjects are human. In some embodiments, the subjects are suspected of having cancer, are at risk of having cancer, or have been diagnosed with cancer or any other disease exhibiting abnormal GARP / TGFβ complex expression or activity.

[0223] Diagnostic methods for many cancers or any other diseases exhibiting abnormal GARP / TGFβ activity, and clinical descriptions of these diseases, are known in this field. Such methods include, but are not limited to, immunohistochemistry, PCR, and fluorescence insight hybridization (FISH). Further details regarding diagnostic methods for abnormal GARP / TGFβ activity or expression are described, for example, in Gupta et al. (2009) Mod Pathol. 22(1): 128-133, Lopez-Rios et al. (2013) J Clin Pathol. 66(5): 381-385, Ellison et al. (2013) J Clin Pathol 66(2): 79-89, and Guha et al. (2013) PLoS ONE 8(6): e67782.

[0224] For example, it may be administered by any suitable route, including intravenous, intramuscular, or subcutaneous. In some examples, the antibodies or antibody derivatives (or fragments thereof) and / or compositions according to this specification may be administered in combination with a second, third, or fourth agent (e.g., an antitumor agent, a growth inhibitor, a cytotoxic agent, or a chemotherapeutic agent) to treat diseases or disorders related to abnormal GARP / TGFβ activity. Such drugs include, for example, anti-PD1 antibodies (e.g., pembrolizumab, nivolumab, serplulimab), docetaxel, gefitinib, FOLFIRI (irinotecan, 5-fluorouracil, and folinic acid), irinotecan, cisplatin, carboplatin, paclitaxel, bevacizumab (anti-VEGF antibody), FOLFOX-4, infused fluorouracil, folinic acid and oxaliplatin, alfartinib, gemcitabine, capecitabine, pemetrexed, tecartinib, everolimus, CpG-ODN, rapamycin, lenalidomide, berofinil, endostatin, lapatinib, PX-866, Imprime PGG, and irinotinib. In some examples, an antibody or antibody derivative (or a fragment thereof) is conjugated with another drug.

[0225] In some examples, the antibodies or antibody derivatives (or fragments thereof) and / or compositions according to this specification are administered in combination with one or more other therapies (e.g., radiotherapy, surgery, chemotherapy and / or targeted therapy). In some examples, the antibodies, antibody derivatives (or fragments thereof) and / or compositions according to this specification are administered in combination with radiotherapy. In some examples, the antibodies, antibody derivatives (or fragments thereof) and / or compositions according to this specification are used in combination with radiotherapy for the treatment of neoplasms or cancers disclosed herein.

[0226] In some examples, the anti-GARP / TGFβ antibody, antibody derivative (or fragment thereof) and / or composition according to this specification are administered in combination with an anti-PD1 antibody (e.g., celpurimab). In some examples, the anti-GARP / TGFβ antibody and the anti-PD1 antibody are administered simultaneously or sequentially. In some examples, the anti-GARP / TGFβ antibody and the anti-PD1 antibody are administered simultaneously. In some examples, one or more anti-PD1 antibodies are administered before the administration of the anti-GARP / TGFβ antibody. In some examples, the subject receives a complete course of anti-PD1 antibody therapy before the administration of the anti-GARP / TGFβ antibody. In some examples, the anti-GARP / TGFβ antibody is administered during a second course of anti-PD1 antibody therapy. In some examples, the subject receives at least one, at least two, at least three, or at least four anti-PD1 antibodies before the administration of the anti-GARP / TGFβ antibody. In some examples, at least one anti-PD1 antibody is administered simultaneously with the anti-GARP inhibitor. In some examples, one or more anti-GARP / TGFβ antibodies are administered before administering the anti-PD1 antibody. In some examples, subjects receive at least two, at least three, at least three, or at least four anti-GARP / TGFβ antibodies before administering the anti-PD1 antibody. In some examples, at least one anti-GARP / TGFβ antibody is administered simultaneously with the anti-PD1 antibody. In some examples, the anti-GARP / TGFβ antibody and the anti-PD1 antibody are administered every 1, 2, 3, 4, or 5 weeks. In some examples, the cancer is recurrent or progressive after a therapy selected from the group consisting of surgery, chemotherapy, radiotherapy, and any combination thereof.

[0227] Depending on the indication to be treated and the administration-related factors well known to those skilled in the art, the antibody or antibody derivative according to this specification is administered in a dose effective to treat the indication while minimizing toxicity and side effects. In the treatment of cancer, a typical dose may be, for example, in the range of 0.001 to 1000 μg, however doses lower or higher than this exemplary range are within the scope of the present invention. The daily dose may be about 0.1 μg / kg to about 100 mg / kg of total body weight, about 0.1 μg / kg to about 100 μg / kg of total body weight, or about 1 μg / kg to about 100 μg / kg of total body weight. As described above, the therapeutic or preventive effect can be monitored by regularly evaluating the treated patient. For repeated administrations of several days or more, treatment should be repeated, depending on the symptoms, until the desired suppression of disease symptoms occurs. However, other dosage schemes may be useful and are within the scope of the present invention. The desired dose may be delivered by a single bolus of the composition, multiple bolus of the composition, or by continuous infusion of the composition.

[0228] Drug compositions comprising antibodies or antibody derivatives disclosed herein may be administered once, twice, three or four times daily. Compositions may also be administered at a lower frequency than daily, for example, six times weekly, five times weekly, four times weekly, three times weekly, twice weekly, once weekly, once every two weeks, once every three weeks, once monthly, once every two months, once every three months, or once every six months. Compositions may also be administered, for example, in the form of a sustained-release formulation via an implant, which gradually releases the composition for use over a period of time and allows for even lower-frequency administration of the composition, for example, once monthly, once every two to six months, once annually, or even as a single dose. Sustained-release devices (e.g., pellets, nanoparticles, microparticles, nanospheres, microspheres, etc.) may be administered by injection or surgically implanted at various locations.

[0229] For example, cancer treatment may be evaluated by, but is not limited to, tumor regression, reduction in tumor weight or size, time of progression, survival time, progression-free survival time, overall response rate, response time, quality of life, protein expression and / or activity. For example, methods for determining therapeutic effect may be used, including measuring the response by radiographic imaging.

[0230] In some examples, the therapeutic effect was measured as a percentage of tumor growth inhibition (% TGI), calculated using the equation 100 - (T / C x 100), where T is the mean relative tumor volume of the treated tumor and C is the mean relative tumor volume of the untreated tumor. In some examples, % TGI may be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, or more than 95%.

[0231] 3.2 Diagnostic and Imaging Methods

[0232] Labeled antibodies or antibody derivatives may be used diagnostically to detect, diagnose, or monitor diseases and / or disorders related to the expression, abnormal expression, and / or activity of the GARP / TGFβ complex. For example, the antibodies and antibody derivatives described herein may be used in in situ, in vivo, ex vivo, and in vitro diagnostic assays or imaging assays. A method for detecting GARP / TGFβ complex expression includes (a) measuring expression in the cells (e.g., tissues) or body fluids of an individual using one or more antibody or antibody derivative assay polypeptides, and (b) comparing the gene expression level with a standard gene expression level, wherein an increase or decrease in the measured gene expression level compared to a standard expression level indicates abnormal expression.

[0233] Another embodiment described herein includes a method for diagnosing a disease or disorder in an animal (e.g., a mammal, e.g., a human) that involves the expression or abnormal expression of the GARP / TGFβ complex. These methods involve detecting the GARP / TGFβ complex in a mammal. In some embodiments, the diagnosis includes (a) administering an effective amount of a labeled antibody or antibody derivative to a mammal; (b) waiting for a period of time after administration to allow the labeled antibody or antibody derivative to preferentially concentrate at the site of GARP / TGFβ complex expression in the subject (and remove unbound labeled molecules to background levels); (c) determining the background level; and (d) detecting labeled molecules in the subject such that the detection of labeled molecules at a high background level indicates that the subject is suffering from a specific disease or disorder that involves the expression or abnormal expression of the GARP / TGFβ complex. The background level may be determined by different methods, which include comparing the amount of detected labeled molecules to a standard value previously determined for one specific system.

[0234] The antibodies and antibody derivatives described herein may be used to measure protein levels in biological samples using classical immunohistochemical methods well known to those skilled in the art (see, for example, Jalkanen, et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096 (1987)). Other antibody-based methods useful for detecting protein gene expression include immunoassays, e.g., enzyme-linked immunosorbent assays (ELISA) and radioimmunoassays (RIA). Suitable antibody assay labels are known in the art and include enzyme labels (e.g., glucose oxidase), radioisotopes, e.g., iodine. 131 I, 125 I, 123 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium (3 H), Indium ( 115m In, 113m In, 112 In, 111 In), and technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti), Gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 PM, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 It contains Ru, luminol, and fluorescent labels (e.g., fluorescein, rhodamine, and biotin).

[0235] Known techniques in this field are applicable to the labeled antibodies (or fragments thereof) according to this specification. Such techniques include, but are not limited to, the use of bifunctional conjugates (see, for example, U.S. Patent Nos. 5,756,065, 5,714,631, 5,696,239, 5,652,361, 5,505,931, 5,489,425, 5,435,990, 5,428,139, 5,342,604, 5,274,119, 4,994,560 and 5,808,003).

[0236] Alternatively or additionally, the level of nucleic acid or mRNA encoding a GARP polypeptide in a cell can be measured, for example, by fluorescence in situ hybridization using a nucleic acid-based probe corresponding to the nucleic acid encoding GARP or its complementary sequence (see FISH, WO 98 / 45479, disclosed in October 1998), DNA blotting, RNA blotting or polymerase chain reaction (PCR) techniques, such as real-time quantitative PCR (RT-PCR). The overexpression of the GARP / TGFβ complex can also be studied by measuring shed antigens in biological fluids (e.g., serum), for example, using an antibody-based assay (see also, for example, U.S. Patent No. 4,933,294, disclosed on June 12, 1990; WO 91 / 05264, disclosed on April 18, 1991; U.S. Patent No. 5,401,638, disclosed on March 28, 1995; and Sias et al., J. Immunol. Methods 132:73-80 (1990)). In addition to the above assays, those skilled in the art can obtain various in vivo and ex vivo assays. For example, cells in a mammalian body are exposed to an antibody, which is optionally labeled with a detectable label (e.g., a radioisotope) and the binding of the antibody to the cells can be evaluated, for example, by external scanning for radioactivity or analysis of a sample (e.g., a biopsy or other biological sample) taken from a mammal previously exposed to the antibody.

[0237] 4. Pharmaceutical Formulations

[0238] The present disclosure further provides a pharmaceutical formulation comprising an antibody or antibody derivative disclosed herein and a pharmaceutically acceptable carrier agent. In some embodiments, the pharmaceutical composition may comprise a combination of multiple (e.g., two or more) antibodies and / or antibody derivatives of the present disclosure.

[0239] In some embodiments, the disclosed drug formulations may be prepared by combining an antibody or antibody derivative of a desired purity with one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), and may be in the form of a lyophilized formulation or an aqueous solution. For example, lyophilized antibody formulations are described in U.S. Patent No. 6,267,958, but are not limited thereto. In some embodiments, aqueous antibody formulations may include those described in U.S. Patent No. 6,171,586 and WO 2006 / 044908, the latter of which comprises a histidine-acetate buffer. In some examples, the antibody or antibody derivative may have a purity greater than about 80%, greater than about 90%, greater than about 91%, greater than about 92%, greater than about 93%, greater than about 94%, greater than 95%, greater than about 96%, greater than about 97%, greater than about 98%, greater than about 99%, greater than about 99.1%, greater than about 99.2%, greater than about 99.3%, greater than about 99.4%, greater than about 99.5%, greater than about 99.6%, greater than about 99.7%, greater than about 99.8%, or greater than about 99.9%.

[0240] Pharmacologically acceptable carriers are non-toxic to the recipient at the dose and concentration used, and include buffers (e.g., phosphates, citrates, and other organic acids), antioxidants including ascorbic acid and methionine, preservatives (e.g., benzyldimethyloctadecylammonium chloride, hexamethonium chloride, benzalkonium chloride, phenethylammonium chloride, phenol, butanol, or benzyl alcohol, alkylparabens (e.g., methyl or propylparaben), catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin, and chelating agents such as EDTA. These include, but are not limited to, sugars such as sucrose, mannitol, trehalose, or sorbitol, chlorided counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or nonionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers as described herein further include mesenchymal drug dispersants, e.g., soluble neutral active hyaluronidase glycoproteins (sHASEGP), e.g., human soluble PH-20 hyaluronidase glycoprotein, e.g., rHuPH20 (HYLENEX®, Baxter International). Several exemplary sHASEGPs containing rHuPH20 and their uses are described in U.S. Patents 2005 / 0260186 and 2006 / 0104968. In some examples, sHASEGP is combined with one or more other glycosaminoglycans (e.g., chondroitinases).

[0241] The carrier agent may be suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound (e.g., anti-GARP / TGFβ antibody) may be coated with a material to protect it from the effects of acids and other natural conditions that may inactivate the compound.

[0242] The drug compositions disclosed herein may be used in combination therapy, i.e., administered in combination with other drugs. In some examples, the drug compositions disclosed herein may further contain one or more active ingredients that are essential for the specific indication being treated, and which, for example, have complementary activity and do not cause adverse effects on each other. In some examples, the drug formulation may contain a second active ingredient for treating the same disease treated by the first therapeutic agent. Such active ingredients are present in appropriate combinations in amounts effective for the desired purpose. For example, the formulations disclosed herein may further contain one or more active ingredients that are essential for the specific indication being treated, and which preferably have complementary activity and do not cause adverse effects on each other. For example, it may be desirable to provide a second therapeutic agent for treating the same disease. Such active ingredients are present in appropriate combinations in amounts effective for the desired purpose.

[0243] The compositions of this disclosure may be administered by various methods known in the art. The route and / or method of administration will depend on the desired outcome. These active compounds can be prepared using controlled-release formulations, including carriers that protect the compounds from rapid release, such as implants, transdermal patches, and microencapsulation delivery systems. Biodegradable and biocompatible polymers, such as ethylene vinyl acetate, polyacid anhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, may also be used. Many methods for preparing such formulations are described, for example, in *Sustained and Controlled Release Drug Delivery Systems*, JR Robinson, ed., Marcel Dekker, Inc., New York, 1978. In some examples, the drug compositions are produced under the Good Manufacturing Practice (GMP) conditions of the U.S. Food and Drug Administration.

[0244] Sustained-release formulations containing antibodies or antibody derivatives disclosed herein may be prepared. A preferred example of a sustained-release formulation is a semipermeable matrix containing a solid hydrophobic polymer of the antibody or antibody derivative, the matrix being in the form of a molded article (e.g., a film or microcapsule). In some examples, the active ingredient can be embedded in microcapsules prepared by, for example, coacervation technology or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or crude emulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0245] To administer the antibodies or antibody derivatives of this disclosure via several routes of administration, it may be necessary to coat the compounds with a material that prevents their inactivation, or to administer them together with the compounds. For example, the compounds may be administered to a subject in a suitable carrier (e.g., liposomes) or diluent. Pharmaceutically acceptable diluents include physiological saline and buffered aqueous solutions. Liposomes include water-in-oil-in-water CGF emulsions and conventional liposomes (Strejan et al. (1984) J Neuroimmunol. 7:27).

[0246] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the temporary preparation of sterile injectable solutions or dispersions. Substances that use such media and agents for their pharmaceutical activity are known in the art.

[0247] Unless any conventional medium or agent is unsuitable for the active compound, it may be considered to use it in the drug composition of this disclosure. Supplemental active compounds may be doped into the composition.

[0248] Therapeutic compositions are typically sterile, substantially isotonic, and stable under production and storage conditions. The composition may be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for high drug concentrations. The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof. Adequate fluidity may be maintained by the use of coatings (e.g., lecithin), by maintaining the desired particle size in the case of dispersions, and by the use of surfactants. Often, it is preferable that the composition contains isotonic agents, such as sugars, polyols (e.g., mannitol, sorbitol), or sodium chloride. Longer absorption of these injectable compositions can be achieved by incorporating absorption-delaying agents, such as monostearates and gelatin, into the composition.

[0249] A sterile injection solution may be prepared by doping a desired amount of one or more antibodies or antibody derivatives disclosed herein with a suitable solvent and, if necessary, a combination of one or more of the components listed above, and then by sterile microfiltration (e.g., filtration through a sterile filtration membrane). Typically, dispersions are prepared by doping the active compound into a sterile medium, the sterile medium comprising a basic dispersion medium and other desired components from those listed above. In the case of sterile powders for the preparation of sterile injection solutions, preferred preparation methods are vacuum drying and freeze-drying, which produce a powder of the active component and any other desired components from the previously sterile filtered solution.

[0250] The therapeutic composition may also be administered using medical devices known in the art. For example, the therapeutic composition of the present invention may be administered with a needleless subcutaneous injection device, such as those disclosed in U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824 or 4,596,556. Examples of implants and modules that can be used in this disclosure include U.S. Patent No. 4,487,603 disclosing an implantable microinfusion pump for dispensing drugs at a controlled rate, U.S. Patent No. 4,486,194 disclosing a therapeutic device for administering drugs through the skin, U.S. Patent No. 4,447,233 disclosing a drug infusion pump for delivering drugs at a precise infusion rate, U.S. Patent No. 4,447,224 disclosing a variable flow rate implantable infusion device for continuous drug delivery, U.S. Patent No. 4,439,196 disclosing a permeable drug delivery system having multiple compartments, and U.S. Patent No. 4,475,196 disclosing a permeable drug delivery system. Many such implants, delivery systems, and modules are known.

[0251] Regarding therapeutic compositions, the formulations of this disclosure include formulations suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. These formulations may conveniently exist in unit dosage forms and may be prepared by any method well known in the pharmaceutical field. The amount of antibody or antibody derivative that can be combined with a carrier material to produce a single dosage form varies depending on the subject being treated and the specific method of administration. The amount of antibody or antibody derivative that can be combined with a carrier material to produce a single dosage form is usually the amount of the composition that produces the therapeutic effect. Typically, this amount is about 0.01% to about 99%, about 0.1% to about 70%, or about 1% to about 30% of the active ingredient as a percentage.

[0252] Dosage forms for topical or transdermal administration of the compositions of this disclosure include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound may be mixed under sterile conditions with a pharmaceutically acceptable carrier and any necessary preservatives, buffers, or propellants.

[0253] The terms "parenteral administration" and "administration by parenteral method" usually refer to administration modes other than enteral and local administration by injection, and include, but are not limited to, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intra-articular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injections and infusions.

[0254] These drug compositions may contain adjuvants, such as preservatives, humectants, emulsifiers, and dispersants. Prevention of the presence of microorganisms can be ensured by the sterilization procedures described above and by including various antimicrobial and antifungal agents, such as parabens, chlorobutanol, and phenolsorbic acid. It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in these compositions. Furthermore, the inclusion of absorption-delaying agents, such as aluminum monostearate and gelatin, can induce sustained absorption of the injectable drug form.

[0255] In some embodiments, when the antibodies or antibody derivatives of the present disclosure are administered to humans and animals as drugs, they can be administered alone or in combination with a pharmaceutically acceptable carrier as a drug composition, which comprises, for example, about 0.01% to about 99.5% (or about 0.1% to about 90%) of the antibody or antibody derivative.

[0256] 5. Products

[0257] The subject of this disclosure further provides products (e.g., kits) that include materials used for the treatment, prevention and / or diagnosis of the above-mentioned disorders.

[0258] In some embodiments, the product / kit includes a container and a label or packaging insert on or associated with the container. Non-limiting examples of suitable containers include bottles, vials, syringes, and IV solution bags. The container may be formed from a variety of materials (e.g., glass or plastic). The container may contain a composition effective in treating, preventing, and / or diagnosing a disease (either by itself or in combination with another composition) and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial with a stopper that can be punctured by a subcutaneous needle).

[0259] In some embodiments, at least one activator in the composition is an antibody or antibody derivative of the present disclosure. A label or packaging insert may indicate that the composition is used for the treatment of a selected medical condition.

[0260] In some embodiments, the product / kit may include (a) a first container containing a composition comprising the antibody or antibody derivative of the Disclosure, and (b) a second container containing a composition comprising other cytotoxic or therapeutic agents. In some embodiments, the product / kit may further include a packaging insert indicating that the composition may be used to treat a particular medical condition.

[0261] Alternatively or additionally, the product / kit may further include another container, for example, a second or third container, which may include, but is not limited to, pharmaceutically acceptable buffers, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The product / kit may also include other materials desirable from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes.

[0262] TIFF0007843284000003.tif250160 TIFF0007843284000004.tif253161 TIFF0007843284000005.tif246156 TIFF0007843284000006.tif232161 TIFF0007843284000007.tif248161 TIFF0007843284000008.tif247155 TIFF0007843284000009.tif215161 TIFF0007843284000010.tif240161 TIFF0007843284000011.tif246155 TIFF0007843284000012.tif203161 TIFF0007843284000013.tif250162 TIFF0007843284000014.tif100161

[0263] The following examples are for illustrative purposes only and should not be considered as limiting in any way. This disclosure relates, for example, to the following: [1] An antibody that binds to the GARP / TGFβ complex, a) (1) SEQ ID NO: A heavy chain variable region CDR-H1 containing one amino acid sequence from among 1, 11, 21, 31, 41, 51, 61 and 105, or a variant of said amino acid sequence containing at most about three amino acid substitutions, (2) SEQ ID NO: Heavy chain variable region CDR-H2 containing one amino acid sequence from among 2, 12, 22, 32, 42, 52, 62 and 106, or a variant of said amino acid sequence containing at most about three amino acid substitutions, (3) SEQ ID NO: A heavy chain variable region CDR-H3 comprising one amino acid sequence from among 3, 13, 23, 33, 43, 53, 63 and 107, or a variant of said amino acid sequence containing at most about three amino acid substitutions, b) (1) SEQ ID NO: Light chain variable region CDR-L1 containing one amino acid sequence from among 4, 14, 24, 34, 44, 54, 64 and 108, or a variant of said amino acid sequence containing at most about three amino acid substitutions, (2) SEQ ID NO: Light chain variable region CDR-L2 containing one amino acid sequence from among 5, 15, 25, 35, 45, 55, 65 and 109, or a variant of said amino acid sequence containing at most about three amino acid substitutions, (3) An antibody that binds to the GARP / TGFβ complex, comprising a light chain variable region CDR-L3 containing one amino acid sequence from among 6, 16, 26, 36, 46, 56, 66, and 110, or a variant of said amino acid sequence containing at most about three amino acid substitutions, and a light chain variable region. [2] This antibody is 1 x 10 -7 The antibody described in [1] above, which binds to the GARP / TGFβ complex with a KD of M or less. [3] This antibody is 1 x 10 -8 The antibody described in [1] or [2] above, which binds to the GARP / TGFβ complex with a KD of M or less. [4] Antibodies are approximately 1 x 10⁶ -11 From M to approximately 1x10 -7 An antibody according to any one of the above [1] to [3], which binds to the GARP / TGFβ complex at M KD. [5] This antibody is approximately 1 x 10⁶ -10 M to approximately 5x10 -8 An antibody according to any one of the above [1] to [4], which binds to the GARP / TGFβ complex at M KD. [6] This antibody cross-competes with the reference anti-GARP / TGFβ antibody, and the reference anti-GARP / TGFβ antibody, a) A heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 6. b) A heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 11, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 12, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 14, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 15, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 16. c) A heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 21, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 22, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 24, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 25, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 26. d) A heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 31, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 32, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 33, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 34, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 35, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 36. e) A heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 41, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 42, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 43, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 44, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 45, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 46. f) A heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 51, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 52, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 53, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 54, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 55, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 56. g) A heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 61, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 62, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 64, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 65, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 66, or h) An antibody according to any one of the above [1] to [5], comprising a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 105, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 106, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 107, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 108, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 109, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 110. [7] The antibody is, a) A heavy chain variable region comprising a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain, wherein the CDR-H1 domain, the CDR-H2 domain, and the CDR-H3 domain each include a CDR-H1 domain, a CDR-H2 domain, and a CDR-H3 domain, respectively, which are included in a reference heavy chain variable region, and the reference heavy chain variable region comprises a heavy chain variable region containing an amino acid sequence selected from the group consisting of SEQ ID NO: 7, 17, 27, 37, 47, 57, 67, 85, 89, 93, 97, 101, and 111, b) The antibody according to any one of the above [1] to [6], comprising a light chain variable region comprising a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, wherein the CDR-L1 domain, the CDR-L2 domain, and the CDR-L3 domain each comprise a CDR-L1 domain, a CDR-L2 domain, and a CDR-L3 domain, respectively, and the reference light chain variable region comprises a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 18, 28, 38, 48, 58, 68, 83, 84, 86, 90, 94, 98, 102, and 112. [8] The antibody is the antibody according to any one of the above [1] to [7], comprising a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 6. [9] The antibody is the antibody according to any one of the above [1] to [7], comprising a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 11, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 12, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 14, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 15, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 16.

[10] The antibody is the antibody according to any one of the above [1] to [7], comprising a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 21, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 22, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 24, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 25, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 26.

[11] The antibody is the antibody according to any one of the above [1] to [7], comprising a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 31, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 32, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 33, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 34, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 35, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 36.

[12] The antibody is the antibody according to any one of the above [1] to [7], comprising a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 41, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 42, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 43, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 44, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 45, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 46.

[13] The antibody is the antibody according to any one of the above [1] to [7], comprising a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 51, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 52, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 53, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 54, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 55, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 56.

[14] The antibody is the antibody according to any one of the above [1] to [7], comprising a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 61, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 62, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 64, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 65, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 66.

[15] The antibody is the antibody according to any one of the above [1] to [7], comprising a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 105, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 106, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 107, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 108, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 109, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 110.

[16] The antibody according to any one of the above [1] to

[15] , comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 8.

[17] The antibody according to any one of the above [1] to

[15] , comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 17 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 18.

[18] The antibody according to any one of the above [1] to

[15] , comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 28.

[19] The antibody according to any one of the above [1] to

[15] , comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 37 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 38.

[20] The antibody according to any one of the above [1] to

[15] , comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 37, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 83.

[21] The antibody according to any one of the above [1] to

[15] , comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 47 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 48.

[22] The antibody according to any one of the above [1] to

[15] , comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 47 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 84.

[23] The antibody according to any one of the above [1] to

[15] , comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 57 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 58.

[24] The antibody according to any one of the above [1] to

[15] , comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 67 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 68.

[25] The antibody according to any one of the above [1] to

[15] , comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 85, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 86.

[26] The antibody according to any one of the above [1] to

[15] , comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 89 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 90.

[27] The antibody according to any one of the above [1] to

[15] , comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 93 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 94.

[28] The antibody according to any one of the above [1] to

[15] , comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 97 and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 98.

[29] The antibody according to any one of the above [1] to

[15] , comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 101, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 102.

[30] The antibody according to any one of the above [1] to

[15] , comprising a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence shown in SEQ ID NO: 111, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO: 112.

[31] The antibody is the antibody according to any one of the above [1] to

[30] , comprising a human framework.

[32] The antibody is a human antibody, as described in any one of the above [1] to

[31] .

[33] The antibody is a humanized antibody, as described in any one of the above [1] to

[31] .

[34] The antibody is the antibody according to any one of the above [1] to

[33] , comprising a full-length immunoglobulin, a single-stranded Fv(scFv) fragment, a Fab fragment, a Fab' fragment, F(ab')2, a Fv fragment, a disulfide-bonded Fv fragment (dsFv), (dsFv)2, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a triabody, a tetrabody, or any combination thereof.

[35] The antibody is the antibody described in any one of the above [1] to

[34] , comprising an Fc region.

[36] The antibody according to any one of the above [1] to

[35] , wherein the Fc region includes a human Fc region.

[37] The antibody according to any one of the above [1] to

[36] , wherein the Fc region includes an Fc region selected from the group consisting of IgG, IgA, IgD, IgE, and IgM Fc regions.

[38] The antibody according to any one of the above [1] to

[37] , wherein the Fc region includes an Fc region selected from the group consisting of the Fc regions of IgG1, IgG2, IgG3, and IgG4.

[39] The antibody according to any one of the above [1] to

[38] , wherein the Fc region includes the IgG1 Fc region.

[40] The antibody according to any one of the above [1] to

[38] , wherein the Fc region includes the IgG4 Fc region.

[41] The antibody is the antibody described in any one of the above [1] to

[40] that binds to the human GARP / TGFβ complex.

[42] The antibody is the antibody described in any one of the above [1] to

[41] that binds to the cynomolgus monkey GARP / TGFβ complex.

[43] The antibody is the antibody described in any one of the above [1] to

[42] , which binds to the human GARP / TGFβ complex, the cynomolgus monkey GARP / TGFβ complex, and the mouse GARP / TGFβ complex.

[44] The Fc region comprises a C-terminal lysine, and is the antibody according to any one of the above [1] to

[43] .

[45] The antibody according to any one of the above [1] to

[43] , wherein the Fc region includes a deletion of the C-terminal lysine.

[46] The antibody is included in a multispecific antibody, for example, a bispecific antibody, wherein the multispecific antibody includes a second antibody moiety that specifically binds to a second antigen, as described in any one of the above [1] to

[45] .

[47] The second antigen is the antibody described in

[46] above, which is a tumor-associated antigen.

[48] The tumor-associated antigens are Her-2, EGFR, PDL1, MSLN, c-Met, B-cell maturation antigen (BCMA), carbonic anhydrase IX (CA1X), carcinoembryonic antigen (CEA), CD5, CD7, CD10, CD19, CD20, CD22, CD30, CD33, CD34, CD38, CD41, CD44, CD47, CD49f, CD56, CD74, CD123, CD133, CD138, CD276 (B7H3), epithelial glycoprotein (EG P2), trophoblast cell surface antigen 2 (TROP-2), epithelial glycoprotein-40 (EGP-40), epithelial cell adhesion molecule (EpCAM), receptor tyrosine kinase erb-B2, 3, 4, folate-binding protein (FBP), fetal acetylcholine receptor (AChR), folate receptor-a, ganglioside G2 (GD2), ganglioside G3 (GD3), human telomerase reverse transcriptase (hTERT), kinase insertion domain receptor (KDR), Lewis An antibody as described in

[47] above, selected from the group consisting of A (CA 1.9.9), Lewis Y (LeY), phosphatidylinositol proteoglycan-3 (GPC3), L1 cell adhesion molecule (L1CAM), mucin 16 (Muc-16), mucin 1 (Muc-1), NG2D ligand, tumor embryonic antigen (h5T4), prostate stem cell antigen (PSCA), prostate-specific membrane antigen (PSMA), tumor-associated glycoprotein 72 (TAG-72), claudin 18.2 (CLDN18.2), vascular endothelial growth factor R2 (VEGF-R2), nephroblastoma protein (WT-1), type 1 tyrosine kinase transmembrane receptor (ROR1), PVR, PVRL2, and any combination thereof.

[49] The second antigen is the antibody described in

[48] above, which is an immune checkpoint modulator.

[50] The immune checkpoint modulator is an antibody selected from the group consisting of TIGIT, PD1, CTLA4, LAG-3, 2B4, BTLA, and any combination thereof, as described in

[49] .

[51] The antibody according to

[48] , wherein the second antigen is an immunocostimulatory molecule or a subunit of a T cell receptor / CD3 complex.

[52] The immunocostimulatory molecule is the antibody described in

[51] , selected from the group consisting of CD28, ICOS, CD27, 4-1BB, OX40, CD40, and any combination thereof.

[53] The antibody according to

[51] , wherein the subunit of the T cell receptor / CD3 complex is selected from the group consisting of CD3γ, CD3δ, CD3ε and any combination thereof.

[54] An immunoconjugate comprising an antibody according to any one of the preceding paragraphs [1] to

[53] , which is linked to a therapeutic agent or label.

[55] The therapeutic agent is an immunoconjugate as described in

[54] , wherein the therapeutic agent is a cytotoxin or a radioisotope.

[56] The label is an immunoconjugate as described in

[54] , selected from the group consisting of radioisotopes, fluorescent dyes, and enzymes.

[57] An antigen-recognition receptor comprising an extracellular antigen-binding domain containing an antibody as described in any one of the above [1] to

[53] .

[58] The antigen-recognition receptor described in

[57] above, which is a chimeric antigen receptor (CAR) or a recombinant T cell receptor.

[59] An antigen-recognition receptor, which is a CAR, as described in

[57] or

[58] .

[60] The antibody is an antigen-recognition receptor according to any one of the above

[57] to

[59] , wherein the antibody is scFv or Fab.

[61] An immune-responsive cell comprising an antigen-recognizing receptor as described in any one of the preceding paragraphs

[57] to

[60] .

[62] The immune-responsive cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, and myeloid cells, as described in

[61] above.

[63] The immune-responsive cell is a T cell, as described in

[62] .

[64] a) an antibody according to any one of the items [1] to

[53] above, an immune conjugate according to any one of the items

[54] to

[56] above, or immune-responsive cells according to any one of the items

[61] to

[63] above, and b) a pharmaceutically acceptable carrier agent.

[65] One or more nucleic acids encoding an antibody as described in any one of the above items [1] to

[53] .

[66] One or more vectors comprising the nucleic acids described in

[65] above.

[67] A host cell comprising the nucleic acid described in

[65] or the vector described in

[66] .

[68] A method for preparing an antibody according to any one of the preceding paragraphs [1] to

[53] , comprising expressing the antibody in a host cell according to the preceding paragraph

[67] and isolating the antibody from the host cell.

[69] A method for reducing the tumor burden of a subject, comprising administering to the subject an effective amount of an antibody described in any one of the preceding [1] to

[53] , an immunoconjugate described in any one of the preceding

[54] to

[56] , or a drug composition described in

[64] .

[70] The method according to

[69] , which reduces the number of tumor cells.

[71] The method is the method according to

[69] or

[70] , which reduces the size of a tumor.

[72] The method is the method according to any one of the preceding

[69] to

[71] , for eradicating a tumor in a subject.

[73] The method according to any one of the claims

[69] to

[72] , wherein the tumor exhibits high microsatellite instability (MSI).

[74] The method according to any one of the above

[69] to

[73] , wherein the tumor is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic carcinoma, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof.

[75] A method for treating and / or preventing cancer, comprising administering to a subject an effective amount of an antibody described in any one of the preceding [1] to

[53] , an immunoconjugate described in any one of the preceding

[53] to

[56] , or a drug composition described in

[64] .

[76] A method for extending the survival period of a subject having cancer, comprising administering to the subject an effective amount of an antibody described in any one of the above [1] to

[63] , an immunoconjugate described in any one of the above

[54] to

[56] , or a drug composition described in the above

[64] .

[77] The method according to

[75] or

[76] , wherein the cancer exhibits high microsatellite instability (MSI).

[78] The cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof, according to any one of the above

[75] to

[77] .

[79] An antibody as described in any one of the above [1] to

[53] , used as a drug.

[80] An antibody used in the treatment of cancer, as described in any one of the above [1] to

[53] .

[81] A drug composition as described in

[64] above, used as a drug.

[82] A drug composition as described in

[64] above, used for the treatment of cancer.

[83] The cancer is the antibody described in

[86] or the drug composition described in

[82] , which exhibits high microsatellite instability (MSI).

[84] The cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof, and is the antibody described in

[80] or the drug composition described in

[82] .

[85] A kit comprising an antibody according to any one of items [1] to

[53] , an immune conjugate according to any one of items

[54] to

[56] , a drug composition according to item

[64] , a nucleic acid according to item

[65] , a vector according to item

[66] , or an immune-responsive cell according to any one of items

[61] to

[63] .

[86] The kit described above

[85] further includes a manual for the treatment and / or prevention of neoplasms.

[87] A method for treating cancer in a subject, comprising administering to the subject an effective amount of anti-GARP / TGFβ antibody and anti-PD1 antibody.

[88] The method according to

[87] , wherein the anti-GARP / TGFβ antibody is the anti-GARP / TGFβ antibody described in any one of the above [1] to

[53] .

[89] The method according to

[87] or

[88] , wherein the cancer exhibits high microsatellite instability (MSI).

[90] The method according to any one of the above

[87] to

[89] , wherein the cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof.

[91] The method according to any one of the above

[87] to

[90] , wherein the anti-GARP / TGFβ antibody and the anti-PD1 antibody are administered simultaneously or sequentially.

[92] The method according to any one of the above

[87] to

[91] , wherein the anti-GARP / TGFβ antibody and the anti-PD1 antibody are administered simultaneously.

[93] The method according to any one of the claims

[87] to

[92] , wherein one or more anti-PD1 antibodies are administered before administering the anti-GARP / TGFβ antibody.

[94] The method according to any one of the claims

[87] to

[93] , wherein the subject receives a complete course of anti-PD1 antibody therapy prior to the administration of the anti-GARP / TGFβ antibody.

[95] The method according to any one of the

[87] to

[94] , wherein the anti-GARP / TGFβ antibody is administered during a second course of treatment of the anti-PD1 antibody therapy.

[96] The method according to any one of the claims

[87] to

[95] , wherein the subject receives at least one, at least two, at least three, or at least four of the anti-PD1 antibodies prior to administration of the anti-GARP / TGFβ antibody.

[97] The method according to any one of the claims

[87] to

[96] , wherein at least one anti-PD1 antibody and the anti-GARP / TGFβ antibody are administered simultaneously.

[98] The method according to any one of the above

[87] to

[97] , wherein one or more anti-GARP / TGFβ antibodies are administered before the administration of the anti-PD1 antibody.

[99] The method according to any one of the claims

[87] to

[98] , wherein the subject receives at least two, at least three, at least three, or at least four anti-GARP / TGFβ antibodies before administration of the anti-PD1 antibody.

[0100] The method according to any one of the claims

[87] to

[99] , wherein at least one anti-GARP / TGFβ antibody is administered simultaneously with the anti-PD1 antibody.

[0101] The method according to any one of the above

[87] to

[0100] , wherein the anti-GARP / TGFβ antibody and the anti-PD1 antibody are administered every 1, 2, 3, 4, or 5 weeks.

[0102] The method according to any one of the above

[87] to

[0101] , wherein the cancer is recurrent or progressive after a therapy selected from the group consisting of surgery, chemotherapy, radiotherapy and any combination thereof.

[0264] example

[0265] Example 1. Screening and testing of the anti-GARP / TGFβ antibody GA1.

[0266] Anti-GARP / TGFβ antibody clones were isolated from an internally synthesized natural human Fab phage library and screened for GARP N-terminal ECD using enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS). A natural human Fab phage library was produced using PBMC samples isolated from eight healthy donors. The VL and VH nucleotide sequences of the obtained clones were then fused with the constant region of human IgG1 using standard assembly PCR techniques, and the resulting clones were used to produce full-length antibodies. Clone GA1 was identified as the top clone.

[0267] The total cell binding capacity of GA1 was tested using transfected CHO-S cells expressing human, cynomolgus monkey, and mouse human GARP / TGFβ1 complexes, as well as activated platelets and Treg cells expressing human GARP / latent TGFβ1 on their cell surface. Treg cells were activated by incubation with anti-CD3 / CD28 Dynabead (Gibco) at a 1:1 cell-to-bead ratio for 24 hours. Platelets were activated by incubation with 1 U / mL thrombin (Sigma) for 1 hour. The total cell binding capacity of each antibody was then tested by incubation of the cells with anti-GARP / TGFβ monoclonal antibodies sequentially diluted in FACS buffer (1x PBS containing 2% FBS) at 4°C for 30 minutes. The cells were washed with FACS buffer, and binding was re-detected at 4°C for 30 minutes using goat anti-human IgG(H+L) FITC Ab. Flow cytometry analysis was performed using the CytoFLEX platform (Beckman Coulter). An isotype control (bevacizumab) was used as a negative control. A GARP reference antibody (an ABBV-151 analog synthesized internally based on the sequence information disclosed in US 2016 / 0251438) was used as a positive control. ABBV-151, also known as LHG10.6, is a clinical-stage anti-GARP / TGFβ1 antibody.

[0268] As shown in Figures 1A-1E, GA1 bound to the human GARP / TGFβ1 complex expressed in CHO-S cells, the cynomolgus monkey GARP / TGFβ1 complex, and the mouse GARP / TGFβ1 complex, as well as the endogenous human GARP / TGFβ1 complex in thrombin-activated human platelets and activated human Treg cells. Conversely, the ABBV-151 analog, as shown in Figure 1C, can bind to the human and cynomolgus monkey GARP / TGFβ1 complex, but cannot bind to the mouse GARP / TGFβ1 complex. In addition to targeting the human GARP / TGFβ1 complex, GA1's ability to target the mouse GARP / TGFβ1 complex gives it an advantage, allowing its therapeutic effects to be tested in various mouse models, thus providing more therapeutic information and guidance before human clinical trials. Furthermore, as shown in Figures 1D and 1E, GA1 exhibited a higher binding ability to activated platelets and Treg cells compared to the ABBV-151 analog, indicating an enhanced ability of GA1 to bind to the human GARP / TGFβ1 complex.

[0269] Next, the ability of GA1 to inhibit the release of mature TGFβ1 from activated platelets was tested. Platelets were prepared as described below. Blood was aspirated into a BD vacutainer glass blood collection tube (BD Biosciences) containing acidic glucose citrate (ACD), and centrifuged at 200x g for 20 min to collect the platelet-rich plasma. The collected platelet-rich plasma was gently mixed with an equal volume of HEP buffer (140 mM NaCl, 2.7 mM KCl, 3.8 mM HEPES, 5 mM EGTA, pH 7.4) containing 1 μM prostaglandin E1 (Sigma-Ace), and centrifuged at 100x g for 20 min to remove RBCs and leukocytes. The supernatant was then transferred to a new tube, and platelets were precipitated by centrifuging at 800x g for 20 min. The precipitate was further washed with washing buffer (10 mM sodium citrate, 150 mM NaCl, 1 mM EDTA, 1% (w / v) dextrose, pH 7.4), and the platelet precipitate was resuspended in Tyrode buffer (134 mM NaCl, 12 mM NaHCO3, 2.9 mM KCl, 0.34 mM Na2HPO4, 1 mM MgCl2, 10 mM HEPES, pH 7.4). Platelets were stimulated for 1 hour with 1 U / mL thrombin (Sigma-A) at 1000 rpm vibration, either in the presence or absence of the specified Ab. After stimulation, the reaction supernatant was harvested and used for the quantification of mature TGFβ1. Mature TGFβ1 quantification was determined without acidification using the TGFβ1 Duoset® ELISA kit (R&D Corporation), as described by the manufacturer. The GARP reference antibody ABBV-151 analog was used as a positive control.

[0270] As shown in Figure 2, compared to a sample containing only platelets, thrombin stimulated the release of mature TGFβ1 from platelets, and GA1 inhibited the release of mature TGFβ1 from thrombin-activated human platelets in a dose-dependent manner.

[0271] Furthermore, the ability of GA1 to reduce platelet-mediated T cell inhibition was tested. Human CD4+ T cells were isolated using the MagniSort Human CD4 T Cell Concentration Kit (eBioscience). Platelets (1 x 10⁶) were enriched with anti-CD3 / CD28 Dynabeads (Gibco) in a bead-to-cell ratio of 1:40, with or without the specified antibody. 7 ) If present or absent, CD4+ T cells (5 x 10 4 The cells were stimulated for 4 days. After incubation, the culture supernatant was collected and used for IFNγ quantification. The amount of IFNγ was measured using the Human IFNγ ELISA MAX Deluxe Kit (Biolegend Biotechnology Co., Ltd.) according to the manufacturer's instructions. An isotype control (bevacizumab) was used as a negative control. A GARP reference antibody ABBV-151 analog was used as a positive control.

[0272] As shown in Figure 3, anti-CD3 / CD28 beads stimulated IFNγ secretion from CD4+ T cells compared to T cells alone, while the addition of platelets inhibited IFNγ secretion. Both GA1 and the ABBV-151 analog reduced platelet-mediated inhibition of IFNγ secretion, but the isotype control antibody did not. Compared to the ABBV-151 analog, GA1 showed a greater reduction in platelet inhibition, particularly at higher dose levels, and induced higher IFNγ secretion from CD4+ T cells. Since IFNγ is an important antitumor cytokine, the results indicate that GA1 has a better antitumor effect.

[0273] Furthermore, the ability of GA1 to reduce Treg-mediated T cell inhibition was tested in a mixed leukocyte reaction assay. Human T cells were isolated using the MagniSort Human T Cell Enrichment Kit (eBioScience). Based on the manufacturer's description, EasySep Human CD4+CD127 was used. 低 Human CD4+CD25+CD127 cells were isolated using the CD25+ Regulatory T Cell Isolation Kit (Stemcell). 低Treg cells were isolated and amplified for 13–15 days in X-VIVO 15 medium (LONZA Group) containing IL-2 (300 U / ml, eBioScience), rapamycin (1 nM, Selleckchem), and 5% human serum (Sigma-A) in the presence of anti-CD3 / CD28 Dynabeads. Treg cells (2.5 × 10⁶) were incubated at 37°C in a 5% CO₂ atmosphere. 3 ) in RPMI-1640 complete medium, dose-titrated T cells (1 × 10⁶) with or without antibody. 5 ) and allogeneic dendritic cells (DCs) (1 × 10⁻¹⁰ 4 The mixture was added to the following. After incubation for 5 days, IFNγ and IL-2 secretion in the culture supernatant were quantified using the Human IFNγ ELISA MAX Deluxe Kit and the Human IL-2 ELISA MAX Deluxe Kit (Baekjin Biotechnology Co., Ltd.), respectively. An isotype control (bevacizumab) was used as a negative control. A GARP reference antibody ABBV-151 analog was used as a positive control.

[0274] As shown in Figures 4A and 4B, dendritic cells (DCs) stimulated IFNγ and IL-2 secretion from human T cells, while the addition of Treg cells inhibited IFNγ and IL-2 secretion. Compared to isotype control antibodies and the no-antibody group, both GA1 and the ABBV-151 analog reduced Treg inhibition and improved IFNγ and IL-2 secretion levels. Compared to the ABBV-151 analog, GA1 improved T cell IFNγ secretion. Since IFNγ and IL-2 are important antitumor cytokines, the results indicate that GA1 has a better antitumor effect.

[0275] Furthermore, the in vivo antitumor effect of GA1 was tested in a similar MC38 mouse model (colon cancer) both alone and in combination with an anti-PD1 antibody. A total of 3 × 10⁶ ions were obtained in 100 μL PBS. 5MC38 cells (mouse colon cancer cells) were mixed with 100 μL of Matrigel (Corning, CA, USA) in a 1:1 ratio and subcutaneously transplanted into both flanks of male C57BL / 6 mice (Biolasco, Taipei, Taiwan). The tumor size was 100-150 mm. 3 When the target was reached, the indicator antibody or control reagent for each group was administered intraperitoneally twice a week for three weeks. The tumor was observed and measured twice a week. Tumor volume was calculated as TV (tumor volume) = (length × width) 2 It was defined as ) / 2. All data points represent the mean ± SEM. Tumor growth inhibition (TGI) was calculated by comparing the tumor volume of each treatment group with that of the medium control group.

[0276] As shown in Figure 5, GA1 alone significantly reduced tumor growth compared to the control group (TGI = 53%). The anti-PD1 antibody (RMP1-14) alone also significantly reduced tumor growth, as expected (TGI = 75%). Furthermore, the combination of GA1 and the anti-PD1 antibody further enhanced tumor growth inhibition (TGI = 95.0%). The results indicate that GA1 itself has an antitumor effect in vivo, and that the combination of GA1 and the anti-PD1 antibody can provide a significantly enhanced antitumor effect compared to monotherapy using either antibody alone. Since anti-PD1 antibodies such as pembrolizumab and nivolumab are widely used to treat various types of cancer, these results suggest that GA1, when used in combination, may further enhance the therapeutic effect of anti-PD1 antibodies.

[0277] Example 2. Screening and testing of GA1 variants

[0278] To further enhance the therapeutic efficacy of antibody clone GA1, affinity maturation based on in vitro phage display was performed on it according to a standard procedure to enhance its affinity for the GARP / TGFβ antigen. In short, one or more CDR residues were mutated, the mutant antibody was displayed on phages, and it was screened based on its higher binding ability to the GARP / TGFβ1 complex using enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS).

[0279] The total cell binding capacity of GA1 mutants was tested using transfected CHO-S cells expressing human, cynomolgus monkey, and mouse human GARP / TGFβ1 complexes, as well as activated platelets and Treg cells expressing human GARP / TGFβ1 complexes on their cell surface. Treg cells were activated by incubation with anti-CD3 / CD28 Dynabead (Gibco) at a 1:1 cell-to-bead ratio for 24 hours. Platelets were activated by incubation with 1 U / mL thrombin (Sigma) for 1 hour. The total cell binding capacity of each antibody was then tested by incubation of the cells with anti-GARP / TGFβ monoclonal antibodies sequentially diluted in FACS buffer (1x PBS containing 2% FBS) at 4°C for 30 minutes. The cells were washed with FACS buffer, and binding was re-detected at 4°C for 30 minutes using goat anti-human IgG(H+L) FITC Ab. Flow cytometry analysis was performed using the CytoFLEX platform (Beckman Coulter). An isotype control (bevacizumab) was used as a negative control. A GARP reference antibody (an ABBV-151 analog synthesized internally based on the sequence information disclosed in US 2016 / 0251438) was used as a positive control.

[0280] As shown in Figures 6A-6E, GA1 and its variants (GA1#4, GA1#6, GA1#7, GA1#8, GA1#9, and GA1#12) bound to the human GARP / TGFβ1 complex expressed in CHO-S cells, the cynomolgus monkey GARP / TGFβ1 complex, and the mouse GARP / TGFβ1 complex, as well as the human GARP / TGFβ1 complex in thrombin-activated human platelets and activated human Treg cells.

[0281] Furthermore, GA1 mutants with modifications in the framework / constant region were tested. For example, compared to GA1#8, GA1#8K includes the addition of a heavy chain C-terminal lysine, and GA1#8K(LC_FS / IT) includes two amino acid substitutions in the light chain framework region (FR1 and FR3) of GA1#8K. Using the above method, the total cell binding ability of these constant region mutants to human GARP / latent TGFβ1-transfected CHO-S cells was tested. Isotype control (bevacizumab) was used as a negative control. As shown in Figure 7, the framework / constant region mutants (GA1#8K and GA1#8K(LC_FS / IT)) can bind to human GARP / latent TGFβ1-transfected CHO-S cells in the same manner as GA1#8. These results demonstrate that modifications in the framework / constant region do not alter the ability of GA1 mutants to bind to the antigen.

[0282] Next, the ability of GA1 mutants to inhibit the release of mature TGFβ1 from activated platelets was tested. Platelets were prepared as described below. Blood was aspirated into BD vacutainer glass blood collection tubes (BD Biosciences) containing acidic glucose citrate (ACD), centrifuged at 200x g for 20 min, and the platelet-rich plasma (PRP) was collected. The collected PRP was gently mixed with an equal volume of HEP buffer (140 mM NaCl, 2.7 mM KCl, 3.8 mM HEPES, 5 mM EGTA, pH 7.4) containing 1 μM prostaglandin E1 (Sigma-Ace), and centrifuged at 100x g for 20 min to remove RBCs and leukocytes. The supernatant was then transferred to a new tube, and platelets were precipitated by centrifuging at 800x g for 20 min. The precipitate was further washed with washing buffer (10 mM sodium citrate, 150 mM NaCl, 1 mM EDTA, 1% (w / v) dextrose, pH 7.4), and the platelet precipitate was resuspended in Tyrode buffer (134 mM NaCl, 12 mM NaHCO3, 2.9 mM KCl, 0.34 mM Na2HPO4, 1 mM MgCl2, 10 mM HEPES, pH 7.4). Platelets were stimulated for 1 hour with 1 U / mL thrombin (Sigma-A) at 1000 rpm vibration, either in the presence or absence of the specified Ab. After stimulation, the reaction supernatant was harvested and used for the quantification of mature TGFβ1. Mature TGFβ1 was quantified without acidification using the TGFβ1 Duoset® ELISA kit (R&D Corporation), as described by the manufacturer. Isotype control (bevacizumab) was used as a negative control. The GARP reference antibody ABBV-151 analog was used as a positive control.

[0283] As shown in Figure 8, compared to a sample containing only platelets, thrombin stimulated the release of mature TGFβ1 from platelets, and the GA1 mutant inhibited the release of mature TGFβ1 from thrombin-activated human platelets in a dose-dependent manner.

[0284] Furthermore, the ability of GA1 mutants to reduce platelet-mediated T cell inhibition was tested. Human CD4+ T cells were isolated using the MagniSort Human CD4 T Cell Enrichment Kit (eBioscience). Platelets (1 x 10⁶) were enriched with anti-CD3 / CD28 Dynabeads (Gibco) in a bead-to-cell ratio of 1:40, with or without the specified antibody. 7 ) or not, CD4+ T cells 5 x 10 4 The cells were stimulated for 4 days. After incubation, the culture supernatant was collected and used for IFNγ quantification. The amount of IFNγ was measured using the Human IFNγ ELISA MAX Deluxe Kit (Baijin Biotechnology) according to the manufacturer's instructions. An isotype control (bevacizumab) was used as a negative control. A GARP reference antibody ABBV-151 analog was used as a positive control.

[0285] As shown in Figure 9, anti-CD3 / CD28 beads stimulated IFNγ secretion from CD4+ T cells compared to T cells alone, while the addition of platelets inhibited IFNγ secretion. Both the GA1 mutant and the ABBV-151 analog reduced platelet-mediated inhibition of IFNγ secretion, but the isotype control antibody did not reduce platelet inhibition. Compared to the ABBV-151 analog, the GA1 mutant showed a greater reduction in platelet inhibition and induced higher IFNγ secretion from CD4+ T cells. Since IFNγ is an important antitumor cytokine, these results indicate that the GA1 mutant has a better antitumor effect.

[0286] Furthermore, the ability of GA1 variants to reduce Treg-mediated T cell inhibition was tested in a mixed leukocyte reaction assay. Human T cells were isolated using the MagniSort Human T Cell Enrichment Kit (eBioScience). Based on the manufacturer's description, EasySep Human CD4+CD127 was used. 低 Human CD4+CD25+CD127 cells were isolated using the CD25+ Regulatory T Cell Isolation Kit (Stemcell). 低Treg cells were isolated and amplified for 13-15 days in X-VIVO 15 medium (Lonza Group) containing IL-2 (300 U / ml, eBioScience), rapamycin (1 nM, Selleckchem), and 5% human serum (Sigma-A) in the presence of anti-CD3 / CD28 Dynabeads. Treg cells (2.5 × 10⁶) were incubated at 37°C in a 5% CO₂ atmosphere. 3 ) in RPMI-1640 complete medium, dose-titrated T cells (1 × 10⁶) with or without antibody. 5 ) and allogeneic dendritic cells (DCs) (1 × 10⁻¹⁰ 4 The mixture was added to the following. After incubation for 5 days, IFNγ and IL-2 secretion in the culture supernatant were quantified using the Human IFNγ ELISA MAX Deluxe Kit and the Human IL-2 ELISA MAX Deluxe Kit (Baijin Biotechnology Co., Ltd.), respectively. The isotype control (bevacizumab) was used as a negative control. The GARP reference antibody ABBV-151 analog was used as a positive control.

[0287] As shown in Figures 10A and 10B, dendritic cells (DCs) stimulated IFNγ and IL-2 secretion from human T cells, while the addition of Treg cells inhibited IFNγ and IL-2 secretion. Compared to isotype control antibodies and the no-antibody group, both the GA1 mutant and the ABBV-151 analog reduced Treg inhibition and improved IFNγ and IL-2 secretion levels. Compared to the ABBV-151 analog, the GA1 mutant induced higher IFNγ secretion from T cells. Since IFNγ and IL-2 are important antitumor cytokines, the results indicate that the GA1 mutant has a better antitumor effect.

[0288] The ability of GA1 mutants to inhibit TGFβ-mediated Smad2 phosphorylation in activated human Treg cells was tested. Based on the manufacturer's description, EasySep human CD4+CD127 低 Human CD4+CD25+CD127 cells were isolated using the CD25+ Regulatory T Cell Isolation Kit (Stemcell). 低Treg cells were isolated and amplified for 13-15 days in X-VIVO™ 15 medium (Lonza Group) containing IL-2 (300 U / ml, eBioScience), rapamycin (1 nM, Selleckchem), and 5% human serum (Sigma-A) in the presence of anti-CD3 / CD28 Dynabeads (Thermo). Amplified Treg cells (1 × 10⁶) were then measured in serum-free X-VIVO 15 medium using anti-CD3 / CD28 Dynabeads, either in the presence or absence of the antibody. 6 Cells were stimulated for 24 hours (10 cells / ml). Recombinant human TGFβ1 (20 ng / mL, PeproTech) stimulation was performed by incubation with the cells for 30 minutes. After stimulation, the cells were lysed and subjected to SDS-polyacrylamide gel electrophoresis under reducing conditions. The gel was blotted on a nitrocellulose film using a Wet / Tank blot system (Bio-Rad). After blocking, the film was incubated with a first antibody against P-Smad2 (Cell Signaling Technologies) or GAPDH (Cell Signaling Technologies), and then incubated with a second HRP coupling antibody, followed by color development with ECL substrate (Septure). The presence of P-Smad2 indicated that stimulated Treg cells produced active TGFβ1. An isotype control (bevacizumab) was used as a negative control. Recombinant human TGFβ (rhTGFβ) and a GARP reference antibody ABBV-151 analog were used as positive controls. Anti-TGFβ (commercially available anti-TGFβ antibody (1D11) from Bio X Cell) was also used as a positive control.

[0289] As shown in Figure 11, antibody-free and negative control samples exhibited similar baseline P-Smad2 levels, and recombinant human TGFβ (rhTGFβ) treatment improved P-Smad2 levels as expected. Furthermore, representative GA1 variants (GA1#8), ABBV-151 analogues, and anti-TGFβ could inhibit Smad2 phosphorylation in Treg cells. Since TGFβ-mediated Smad2 signaling is crucial for Treg cell activation, the results indicate that GA1 variants can inhibit Treg cell activation, further enhancing effector T cell function and improving the subject's immune response to disease and tumors.

[0290] Furthermore, the in vivo antitumor effect of the GA1 mutant was tested in a related MC38 mouse model (colon cancer). A total of 3 × 10⁶ ions were obtained in 100 μL PBS. 5 MC38 cells (mouse colon cancer cells) were mixed with 100 μL of Matrigel (Corning Corporation, California, USA) in a 1:1 ratio and subcutaneously transplanted into both flanks of male C57BL / 6 mice (Less Biotechnology Co., Ltd., Taipei, Taiwan, China). The tumor size was 100-150 mm. 3 When the target was reached, the indicator antibody or control reagent for each group was administered intraperitoneally twice a week for three weeks. The tumor was observed and measured twice a week. Tumor volume was calculated as TV (tumor volume) = (length × width) 2 It was defined as ) / 2. All data points represent the mean ± SEM. Tumor growth inhibition (TGI) was calculated by comparing the tumor volume of each treatment group with that of the medium control group.

[0291] Previous studies on GA1 in the MC38 mouse model demonstrated the antitumor effect of GA1, but compared to the control group, GA1 treatment did not show tumor inhibition until day 12 post-treatment, as shown in Figure 5. Similarly, in this study, tumor inhibition by GA1 treatment was minimized at day 13 post-treatment (TGI = 9%), as shown in Figure 12. However, greater tumor inhibition was observed at day 13 post-treatment from treatment with GA1 mutants GA1#7 (TGI = 57%), GA1#8 (TGI = 54%), and GA1#9 (TGI = 48%). The results indicate that the antitumor effects of GA1 mutants GA1#7, GA1#8, and GA1#9 are superior to those of GA1.

[0292] Based on the same scheme as above, GA1#8 was further tested in the MC38 mouse model at a low dose level (10 mg / kg). As shown in Figure 13, compared to the control group, the GA1 mutant GA1#8 alone significantly reduced tumor growth (TGI = 37.8%), similar to the anti-PD1 antibody (RMP1-14, TGI = 37.3%). Furthermore, the combination of GA1#8 and the anti-PD1 antibody further enhanced tumor growth inhibition (TGI = 98.0%). The results indicate that GA1#8 itself, when combined with the anti-PD1 antibody, has an in vivo antitumor effect at low dose levels.

[0293] Furthermore, GA1#8 was tested at a high dose (25 mg / kg) in a syngeneic mouse model of the CT26 mouse model (mouse colon cancer). Compared to the MC38 model, the CT26 mouse model was reported to be more resistant to PD1 inhibitor treatment and to have a higher level of Treg cells in the tumor microenvironment compared to the MC38 model, which may affect the antitumor effect of anti-GARP / TGFβ antibody treatment. A total of 5 x 10 in 100 μL PBS. 5 CT26 cells (mouse colon cancer) were mixed with 100 μL of Matrigel (Corning Corporation, California, USA) in a 1:1 ratio and subcutaneously transplanted into the bilateral flanks of BALB / c mice (Less Biotechnology Co., Ltd., Taipei, Taiwan, China). The tumor size was 100-150 mm.3 When the target was reached, the indicator antibody or control for each group was administered intraperitoneally twice a week for three weeks. The tumor was observed and measured twice a week. Tumor volume was calculated as TV (tumor volume) = (length × width) 2 It was defined as ) / 2. All data points represent the mean ± SEM. Tumor growth inhibition (TGI) was calculated by comparing the tumor volume of each treatment group with that of the medium control group.

[0294] As shown in Figure 14, compared to the control group, the GA1 mutant GA1#8 alone significantly reduced tumor growth (TGI = 50%), similar to the anti-PD1 antibody (RMP1-14, TGI = 48%). Furthermore, the combination of GA1#8 and the anti-PD1 antibody further enhanced tumor growth inhibition (TGI = 73%). These results are consistent with those from the MC38 model and also indicate that the GA1 mutant GA1#8 itself has an antitumor effect in vivo, and that the combination of GA1#8 and the anti-PD1 antibody can provide a significantly enhanced antitumor effect compared to monotherapy using either antibody. Since anti-PD1 antibodies such as pembrolizumab and nivolumab are widely used to treat various types of cancer, these results suggest that GA1 mutants like GA1#8, when used in combination, may further enhance the therapeutic effect of anti-PD1 antibodies.

[0295] Example 3. Screening and testing of the anti-GARP / TGFβ antibody hGA17.

[0296] Screening of the Fab phage library identified another anti-GARP / TGFβ antibody clone. This phage library was produced from hybridomas, which were constructed from mice immunized with the GARP / TGFβ1 complex or the GARP ECD / TGFβ1 complex. A representative clone, GA17, was selected for use in the humanization of the framework. In short, a database of human germline genes was searched by performing Igblast using the clone sequence. An ideal germline sequence was selected, and mutations were performed on the framework sequence to change it from a mouse sequence to a human sequence, producing the humanized clone hGA17.

[0297] ELISA binding of hGA17 was tested using human GARP / TGFβ1 complexes and human GARP proteins not present in any GARP / TGFβ1 complex. 96-well plates (Costar, 3690) were coated overnight at 4°C with 30 μl / well of 4 μg / ml GARP / TGFβ1 complex or 2 μg / ml GARP in PBS buffer. The coated plates were washed five times with PBST buffer (PBS containing 0.05% Tween 20, pH 7.4) and then SuperBlocked. TMThe cells were blocked with buffer (Saimar, 37516). Repeated titrations of hGA17, GA1#8, and the reference antibody (in the range of 1000 ng / ml to 0.32 ng / ml) were produced, added to washed plates, and incubated at room temperature for 2 hours. GARP reference antibody 1 (an ABBV-151 analog synthesized internally based on the sequence information disclosed in US 2016 / 0251438) and GARP reference antibody 2 (a DS-1005a analog synthesized internally based on the sequence information disclosed in US 2018 / 0258184) were used as positive controls. ABBV-151, also known as LHG10.6, is a clinical-stage anti-GARP / TGFβ1 IgG4 antibody. DS-1005a, also known as H151D-H1L1, is a clinical-stage anti-GARP / TGFβ IgG1 antibody. The plates described above were washed, and 30 μl / well of 1 / 8000 diluted goat anti-human IgG and monkey ads-HRP (SouthernBiotech) were added and incubated at room temperature for 1 hour. After further washing, the conjugated antibodies were detected with 30 μl / well of TMB substrate (SurModics, TMBS-1000-01), and the samples were stopped with ELISA stop solution (Solarbio, C1058-100 ml). Absorbance was measured at 450 nm, and the binding curve of the test antibody was compared with that of the reference antibody. Absorbance was plotted against sample concentration. The binding ability of the antibodies to human GARP and human GARP / TGFβ1 complexes was further tested using Octect.

[0298] As shown in Figure 15A, both GA1#8 and hGA17 bound to the human GARP / potential TGFβ1 complex and showed better binding than the two reference antibodies. As shown in Figure 15B, both hGA17 and GARP reference antibody 2 bound to human GARP individually, with hGA17 showing much stronger binding than GARP reference antibody 2. In comparison, GARP reference antibody 1 and GA1#8 did not bind to human GARP other than the GARP / TGFβ complex.

[0299] The binding ability of antibodies to human GARP and the human GARP / TGFβ1 complex was further tested using Octect, and the results are shown in Table 3. Unlike GA1#8 and ABBV-151, which bind only to the GARP / TGFβ1 complex, hGA17 bound to the GARP / TGFβ1 complex and GARP with considerable affinity (Table 3). The DS-1005a analog could bind to the GARP / TGFβ1 complex and GARP, but the binding was much weaker than that of hGA17.

[0300] [Table 3]

[0301] The total cell binding capacity of hGA17 and GA1#8 was tested using tumor cells Hs 578T expressing the GARP / TGFβ complex, GARP-transfected CHO-S cells expressing human GARP protein, and human platelets and Treg cells expressing human GARP / latent TGFβ1 on their cell surface. The platelets were from Miao Tong Biological Science & Technology. Based on the manufacturer's description, EasySep human CD4+CD127 was used. 低 Human CD4+CD25+CD127 cells were isolated using the CD25+ Regulatory T Cell Isolation Kit (Stemcell). 低Treg cells were isolated from human PBMCs (Naeto Bioscience & Technology Co., Ltd.) and amplified for 13–15 days in X-VIVO 15 medium (Lonza Group) containing IL-2 (300 U / ml, eBioscience), rapamycin (1 nM, Selleckchem), and 5% human serum (Sigma-Ace) in the presence of anti-CD3 / CD28 Dynabeads. Treg cells were activated by incubation with anti-CD3 / CD28 Dynabeads (Gibco, 111.32D) at a 1:1 cell-to-bead ratio for 24 hours. The total cell binding capacity of each antibody was then tested by incubation with sequentially diluted anti-GARP / TGFβ monoclonal antibodies in FACS buffer (1x PBS containing 2% FBS) at 4°C for 1 hour. The cells were then washed with FACS buffer, and binding was re-detected for 30 minutes at 4°C using goat anti-human IgG PE Ab (Baekjin Biotechnology). Flow cytometry analysis was performed using the CytoFLEX platform (Beckman Coulter). IgG isotype control (anti-CLDN18.2 antibody) was used as a negative control. GARP reference antibody 1 (ABBV-151 analog) and GARP reference antibody 2 (DS-1005a analog) were used as positive controls.

[0302] As shown in Figures 16A-16D, hGA17 bound to the human GARP / TGFβ complex expressed in Hs 578T tumor cells (Figure 16A), human GARP expressed in CHO-S cells (Figure 16B), and the human GARP / latent TGFβ1 complex in human platelets (Figure 16C) and activated human Treg cells (Figure 16D). As shown in Figure 16A, hGA17 showed much stronger binding activity to Hs 578T tumor cells than GA1#8, GARP reference antibody 1, and GARP reference antibody 2. Furthermore, as shown in Figure 16B, hGA17 and GARP reference antibody 2 can bind to human GARP in CHO-S cells, and to the human GARP / latent TGFβ1 complex in human platelets and activated human Treg cells. Conversely, as shown in Figure 16B, GA1#8 and GARP reference antibody 1 did not bind to human GARP in CHO cells. The ability to target human GARP other than the GARP / TGFβ complex and the human GARP / TGFβ1 complex conferred superiority to hGA17, and its therapeutic effect mediated a broader ADCC effect because its efficacy was induced by two forms of GARP. Furthermore, as shown in Figures 16C and 16D, hGA17 showed higher binding affinity to human platelets and Treg cells compared to GARP reference antibody 2, and hGA17 had higher binding affinity to the human GARP / potential TGFβ1 complex compared to GARP reference antibody 2.

[0303] Furthermore, the ability of hGA17 to inhibit the release of mature TGFβ1 from activated platelets was tested. Platelets were from Naeto Biotechnology Co., Ltd. Platelets pre-washed in DMEM medium were inoculated into 96-well plates, incubated with the specified antibody at 4°C for 1 hour, and stimulated with 2 U / mL thrombin (Sigma-A) at 1000 rpm vibration for 1 hour in or without the specified Ab. After stimulation, the reaction supernatant was harvested and used for the quantification of mature TGFβ1. Mature TGFβ1 quantification was determined without acidification using the TGFβ1 Duoset® ELISA kit (R&D Co., Ltd.) as described by the manufacturer. GARP reference antibody 1 (ABBV-151 analog) and GARP reference antibody 2 (DS-1055a analog) were used as positive controls.

[0304] As shown in Figure 17, GA1#8 and hGA17 inhibited the release of mature TGFβ1 from thrombin-activated human platelets at a dose of 50 μg / ml, compared to a sample containing only platelets. GARP reference antibody 1 showed a similar pattern of inhibiting the release of TGFβ1 from thrombin-activated human platelets; however, no inhibitory effect of GARP reference antibody 2 was observed in this assay.

[0305] Furthermore, the ability of hGA17 to reduce Treg-mediated T cell inhibition was tested. Human CD3 + The T cells were purchased from Naeto Biotechnology Co., Ltd. Based on the manufacturer's description, they were EasySep Human CD4+CD127. 低 Treg cells were isolated from human PBMCs (Naetsu Biotechnology Co., Ltd.) using the CD25+ Regulatory T Cell Isolation Kit (Stemcell). Treg cells (5 x 10) were isolated using anti-CD3 / CD28 Dynabeads (Gibco) in a 1:10 bead-to-cell ratio, with or without the specified antibody. 4 ) or if there is no CD3 + T cells (1x10 5The cells were stimulated for 3 days. After incubation, the culture supernatant was collected and used for IL-2 quantification. The amount of IL-2 was measured using the Human IL-2 ELISA MAX Deluxe Kit (Baijin Biotechnology) according to the manufacturer's instructions. Human IgG1 (Sino) was used as a negative control. GARP reference antibody 1 (ABBV-151 analog) was used as a positive control.

[0306] As shown in Figure 18, compared to the non-irritating sample, the anti-CD3 / CD28 beads showed a difference in CD3 + While T cell IL-2 secretion was stimulated, the addition of Tregs inhibited IL-2 secretion. GA1#8, hGA17, and GARP reference antibody 1 reduced Treg-mediated inhibition of IL-2 secretion, but isotype control antibodies did not. Since IL-2 is an important cytokine for immune activation, the results suggest that GA1#8 and hGA17 may enhance patients' anti-tumor immunity.

[0307] The ability of hGA17 to promote the lysis of tumor cells expressing the GARP / TGFβ complex, mediated by NK cells, was further investigated. Briefly, Hs 578T tumor cells were used as target cells. PBMCs were used as effector cells. PBMCs were mixed overnight with Hs 578T (10,000 cells / well) at a 20:1 effector-to-target cell (E / T) ratio in the presence of a series of diluted antibodies (10,000 ng / ml to 0.1 ng / ml). GARP reference antibody 1 (ABBV-151 analog) and GARP reference antibody 2 (DS-1055a analog) were used as controls. Cytotoxicity was measured according to the instructions for the Cytotoxicity LDH Assay Kit-WST (Dojindi, CK12). Based on OD490 readings, the percentage of antibody-dependent cell lysis was calculated using the formula [(test - mean background) / (mean maximum - mean background)] × 100. PBMCs isolated from two healthy donors were tested.

[0308] As shown in Figures 19A and 19B, hGA17 induced strong cytotoxicity in a dose-dependent manner, while other anti-GARP / TGFβ antibodies induced only weak cytotoxicity against Hs 578 T cells or no cytotoxicity at all. The superior ADCC effect of hGA17 indicates that hGA17 has a better antitumor effect in tumors that highly express the GARP / TGFβ complex.

[0309] GARP + The ability of hGA17 to deplete Treg cells was tested. Human PBMCs from four healthy donors (Naeto Biotechnology Co., Ltd.) were cultured in RPMI 1640 containing CD3 / CD28 dynabeads (Gibco) in the presence of anti-human GARP / TGFβ antibody or human IgG1 (Sino). GARP reference antibody 2 (DS-1055a analog) was used as a control. After two days of culture, the cells were washed and stained with LIVE / DEAD (Thermo Fisher Scientific), Alexa Flour 700-CD3 (Baekjin Biotechnology), PE / CY7-CD4 (Baekjin Biotechnology), PE / CY5.5-CD25 (Baekjin Biotechnology), Pacific Blue-FOXP3 (Baekjin Biotechnology), and PE-GARP (BD Biosciences). Using the CytoFLEX platform (Beckman Coulter), stained cells were evaluated, and CD3 + CD25 + CD4 + FOXP3 + GARP in T cell populations + The decrease in the Treg cell population was measured.

[0310] As shown in Figure 20, hGA17 is superior to all other anti-GARP / TGFβ antibodies in four different donors compared to GARP. + The Treg population was reduced to its maximum extent. The results indicate that, compared to other anti-GARP / TGFβ antibodies, hGA17 possesses superior Treg depletion activity and can enhance its antitumor effect by reducing the Treg population in the tumor microenvironment.

[0311] GA1#8 could cross-react with the mouse GARP / TGFβ complex, but hGA17, GARP reference antibody 1, and GARP reference antibody 2 could not bind to mouse GARP. To compare the in vivo antitumor effects of GA1#8, hGA17, and the reference antibodies, human GARP knock-in (KI) c57 / BL6 mice were used as an MC38 colon cancer model. GARP reference antibody 1 (ABBV-151 analog) and GARP reference antibody 2 (DS-1055a analog) were used as controls.

[0312] A total of 5 × 10 in 100 μL of PBS 5 MC38 cells were mixed with 100 μL of Matrigel (Corning, California, USA) (in a 1:1 ratio) and subcutaneously transplanted into the forelimbs of mice. The tumor size was 80-100 mm. 3When the target was reached, the designated antibody or medium in each group was administered intraperitoneally at a dose of 25 mg / kg twice a week for 3 weeks. Tumors were observed and measured twice a week. Tumor volume was defined as TV(tumor volume) = (length × width²) / 2. All data points represent the mean ± SEM. Tumor growth inhibition (TGI) was calculated by comparing the tumor volume of each treatment group with that of the medium control group. Mice were killed on day 24, and spleens and blood were harvested. Spleens were prepared as single-cell suspensions by centrifugation at 4°C and 400 g, and pulverization and filtration using a 40 μm cell filter (Falcon®). Each spleen was suspended and precipitated in 5 ml of 1x RBC lysis buffer (Invitrogen) and incubated at room temperature for 4 minutes. Lysis of red blood cells was stopped in 30 ml of PBS buffer. Mouse blood was lysed with 1 ml of 1x RBC lysis buffer per 1 ml of mouse blood for 4 minutes, and erythrocyte lysis was stopped with 30 ml of PBS buffer. Spleen and hematocyte precipitates were collected and stained with surface labels (Live / dead-eflour 506, mCD45-BV605, mCD3-AF700, mCD4-APC-H7, mCD8-Percp-cy5.5, mCD25-PE-cy7, mPD1-APC, hGARP-BV421 / mGARP-BV421). After washing with FACS buffer (PBS containing 2% FBS), the cell precipitates were fixed / permeabilized with Foxp3 and incubated at 4°C for 16 hours. After washing twice with 1x permeabilizing buffer, cells were stained with mFOXP3-PE in the dark at 4°C for 30 minutes. Finally, the cells were washed twice with 1 × permeabilizing buffer, suspended in FACS buffer, and used for flow cytometry analysis.

[0313] As shown in Figure 21A, both hGA17 and GA1#8 showed antitumor effects in the human GARP KI MC38 mouse model. On day 24, compared to the media control group, the tumor growth inhibition (TGI) of hGA17 and GA1#8 was 45.81% and 38.55%, respectively. Conversely, on day 24, GARP reference antibody 1 showed significantly less tumor growth inhibition (TGI = 16.57%) compared to the media control group, while GARP reference antibody 2 treatment showed no tumor inhibition. Flow cytometry was used to analyze Treg cells from the blood and spleen of human GARP KI mice. As shown in Figures 21B and 21C, each antibody treatment group showed GARP in the blood (Figure 21B) and spleen (Figure 21C) of hGARP KI mice. + It reduced Treg cells. These results indicate that GA1#8 and hGA17, compared to the reference antibody, exhibit superior antitumor efficacy and better GARP in vivo. + It was shown to exhibit the ability to deplete Treg cells.

[0314] In addition to the various embodiments illustrated and claimed, the disclosed themes further cover other embodiments having other combinations of the features disclosed and claimed herein. Thus, certain features presented herein may be combined with each other in other ways within the scope of the disclosed themes, such that the disclosed themes include any suitable combination of the features disclosed herein. The above descriptions of specific embodiments of the disclosed themes are provided for illustrative and explanatory purposes only. The above descriptions are not intended to be exhaustive or to limit the disclosed themes to those disclosed embodiments.

[0315] It will be apparent to those skilled in the art that various modifications and changes can be made to the structure and methods of the disclosed theme without departing from the spirit or scope of the disclosed theme. Accordingly, the disclosed theme is intended to include modifications and changes that fall within the scope of the appended claims and their equivalents.

[0316] Various publications, patents, and patent applications are incorporated herein by reference, and their contents are incorporated as a whole.

Claims

1. An antibody that binds to the GARP / TGFβ complex, a) A heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 1, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 2, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 3, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 4, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 5, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO:

6. b) A heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 11, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 12, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 13, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 14, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 15, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO:

16. c) A heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 21, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 22, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 23, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 24, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 25, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO:

26. d) A heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 31, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 32, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 33, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 34, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 35, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO:

36. e) A heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 41, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 42, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 43, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 44, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 45, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO:

46. f) A heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 51, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 52, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 53, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 54, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 55, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO:

56. g) A heavy chain variable domain (VH) sequence comprising (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 61, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 62, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 63, and a light chain variable domain (VL) sequence comprising (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 64, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 65, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO: 66, or h) An antibody comprising a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 105, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 106, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 107, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 108, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 109, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO:

110.

2. The antibody according to claim 1, comprising a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 41, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 42, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 43, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 44, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 45, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO:

46.

3. The antibody according to claim 1, comprising a heavy chain variable domain (VH) sequence including (1) CDR-H1 containing the amino acid sequence shown in SEQ ID NO: 105, (2) CDR-H2 containing the amino acid sequence shown in SEQ ID NO: 106, and (3) CDR-H3 containing the amino acid sequence shown in SEQ ID NO: 107, and a light chain variable domain (VL) sequence including (1) CDR-L1 containing the amino acid sequence shown in SEQ ID NO: 108, (2) CDR-L2 containing the amino acid sequence shown in SEQ ID NO: 109, and (3) CDR-L3 containing the amino acid sequence shown in SEQ ID NO:

110.

4. The antibody comprises a heavy chain variable region and a light chain variable region. a) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 7, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:

8. b) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 17, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:

18. c) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 27, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:

28. d) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 37, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:

38. e) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 37, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:

83. f) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 47, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:

48. g) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 47, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:

84. h) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 57, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:

58. i) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 67, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:

68. j) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 85, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:

86. k) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 89, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:

90. l) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 93, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:

94. m) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 97, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:

98. n) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 101, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO: 102, or o) The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO: 111, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO:

112. The antibody according to any one of claims 1 to 3.

5. The antibody according to any one of claims 1 to 3, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 85, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

86.

6. The antibody according to any one of claims 1 to 3, wherein the antibody comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO: 111, and the light chain variable region comprising the amino acid sequence shown in SEQ ID NO:

112.

7. The antibody according to any one of claims 1 to 3, comprising a full-length immunoglobulin, a single-stranded Fv (scFv) fragment, a Fab fragment, a Fab' fragment, F(ab')2, an Fv fragment, a disulfide-bonded stable Fv fragment (dsFv), (dsFv)2, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a triabody, or a tetrabody.

8. The antibody is the antibody according to any one of claims 1 to 3, comprising an Fc region.

9. The antibody according to claim 8, wherein the Fc region includes an Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions.

10. The antibody according to claim 9, wherein the Fc region includes an IgG1 Fc region or an IgG4 Fc region.

11. The antibody according to claim 8, wherein the Fc region includes C-terminal lysine.

12. The antibody according to claim 8, wherein the Fc region includes a deletion of the C-terminal lysine.

13. An immunoconjugate comprising an antibody according to any one of claims 1 to 12, which is linked to a therapeutic agent or label.

14. The immunoconjugate according to claim 13, wherein the therapeutic agent is a cytotoxin or a radioisotope.

15. The immunoconjugate according to claim 13, wherein the label is selected from the group consisting of radioisotopes, fluorescent dyes, and enzymes.

16. An antigen-recognition receptor comprising an extracellular antigen-binding domain containing an antibody according to any one of claims 1 to 12.

17. The antigen-recognition receptor according to claim 16, which is a chimeric antigen receptor (CAR).

18. The antigen-recognition receptor according to claim 16 or 17, wherein the antibody is scFv.

19. An immune-responsive cell comprising an antigen-recognizing receptor according to any one of claims 16 to 18.

20. The immune-responsive cells according to claim 19, wherein the immune-responsive cells are selected from the group consisting of T cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), regulatory T cells, natural killer T (NKT) cells, and myeloid cells.

21. The immune-responsive cell according to claim 20, wherein the immune-responsive cell is a T cell.

22. a) an antibody according to any one of claims 1 to 12, an immune conjugate according to any one of claims 13 to 15, or an immune-responsive cell according to any one of claims 19 to 21, and b) a pharmaceutically acceptable carrier agent.

23. One or more nucleic acids encoding the antibody according to any one of claims 1 to 12.

24. One or more vectors comprising the nucleic acid described in claim 23.

25. A host cell comprising the nucleic acid described in claim 23 or the vector described in claim 24.

26. A method for preparing an antibody according to any one of claims 1 to 12, comprising expressing the antibody in a host cell according to claim 25 and isolating the antibody from the host cell.

27. A drug composition according to claim 22, which is used as a drug.

28. A drug composition according to claim 22, used for the treatment of cancer.

29. The drug composition according to claim 28, wherein the cancer exhibits high microsatellite instability (MSI).

30. The drug composition according to claim 28, wherein the cancer is selected from the group consisting of mesothelioma, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, colon cancer, pleural tumor, glioblastoma, esophageal cancer, gastric cancer, synovial sarcoma, thymic cancer, endometrial cancer, gastric tumor, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof.

31. A kit comprising an antibody according to any one of claims 1 to 12, an immune conjugate according to any one of claims 13 to 15, a drug composition according to claim 22, a nucleic acid according to claim 23, a vector according to claim 24, or an immune-responsive cell according to any one of claims 19 to 21.

32. The kit according to claim 31, further comprising a manual for the treatment and / or prevention of neoplasms.

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