Anti-PDL1 Antibodies, Multispecific Antibodies, and Methods of Use

Abstract

The present disclosure relates to antibodies and antibody derivatives that bind to PDL1 and methods of using them. In certain embodiments, the antibodies or antibody derivatives disclosed herein comprise single domain antibodies that bind to PDL1. In certain embodiments, the antibody derivatives are multispecific antibodies that bind to PDL1 and an additional antigen, such as CD47.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to International Patent Application No. PCT / CN2022 / 143518, filed December 29, 2022, the entire contents of which are incorporated by reference and priority to which is claimed.

[0002] The present disclosure relates to antibodies and antibody derivatives that bind to PDL1 and methods of using them. In certain embodiments, the antibody derivatives are multispecific antibodies that bind to PDL1 and an additional antigen, such as CD47. [Background technology]

[0003] PDL1 (also known as CD274) is an immunoinhibitory receptor ligand expressed by immune cells and various types of tumor cells. Interaction of this ligand with its receptor PD1 inhibits T cell activation and cytokine secretion. In the tumor microenvironment, this interaction can provide immune evasion for tumor cells through the inactivation of cytotoxic T cells, and inhibition of the PD1 / PDL1 interaction mediates potent antitumor activity in preclinical models. Although the use of antibody inhibitors of the PD1 / PDL1 interaction to treat cancer has entered clinical trials, there remains a need in the art for the development of molecules and methods that target PDL1 for cancer therapy.

[0004] CD47 (also known as IAP, MER6, and OA3) is a membrane receptor with an extracellular N-terminal domain, five transmembrane domains, and a C-terminal intracellular tail. It binds to various membrane integrins and two soluble ligands, thrombospondin-1 (TSP-1) and signal-regulatory protein alpha (SIRPα). CD47 is involved in various cellular processes, including apoptosis, proliferation, adhesion, and migration. Furthermore, it plays an important role in immune and angiogenic responses. In particular, CD47 functions as a "do not eat me" signal to macrophages, helping non-malignant cells maintain immune tolerance under physiological conditions. Many types of tumor cells overexpress this immunosuppressive signaling molecule, thereby contributing to their survival. Furthermore, CD47 is expressed on normal red blood cells. Because CD47 plays an important role in immune regulation, there is a need in the art for the development of therapeutic molecules and methods that target CD47 for immunotherapy and cancer treatment, as well as for CD47-targeting molecules with reduced binding to red blood cells. Summary of the Invention

[0005] The present disclosure provides isolated monoclonal antibodies and antibody derivatives that specifically bind to PDL1 with high affinity, including monospecific anti-PDL1 antibodies and multispecific antibodies that bind to PDL1 and one or more additional targets. In certain embodiments, the one or more additional targets are CD47. In certain embodiments, the antibodies or antibody derivatives disclosed herein include single-domain antibodies that bind to PDL1. The present disclosure further provides methods of making and using the antibodies and antibody derivatives disclosed herein, as well as pharmaceutical compositions comprising them, for example, to treat diseases and disorders, such as cancer. The present invention is based, in part, on the discovery of novel single-domain antibodies that bind to PDL1, novel anti-CD47 antibodies with reduced binding to erythrocytes, and novel multispecific antibodies that bind to PDL1 and CD47, each of which can target tumor cells and / or increase the immune response against tumor cells, thereby resulting in improved anti-tumor effects.

[0006] The present disclosure provides a multispecific antibody that binds to PDL1 and CD47, comprising: i) a first antigen-binding portion comprising a single-domain anti-PDL1 antibody that binds to PDL1; and ii) a second antigen-binding portion comprising an anti-CD47 antibody that binds to CD47. In certain embodiments, the single-domain antibody comprises a VHH. In certain embodiments, the single-domain antibody or the VHH comprises a heavy chain variable region (VH).

[0007] In certain embodiments, the single domain antibody is administered at a concentration of 1×10 -7 In certain embodiments, the single domain antibody binds to PDL1 with a KD of 5×10 -8 In certain embodiments, the single domain antibody binds to PDL1 with a KD of 1×10 or less. -8 In certain embodiments, the single domain antibody binds to PDL1 with a KD of about 1 x 10 -10 M ~ approx. 5×10 -8 In certain embodiments, the single domain antibody comprises a VHH. In certain embodiments, the single domain antibody or the VHH comprises a heavy chain variable region (VH).

[0008] In certain embodiments, the single domain antibody has, with respect to binding to PDL1, a) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3; b) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8; c) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11. a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 12, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 13, a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 17, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 18, a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 23, f) a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 26, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 27, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 28; g) a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 31, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 32, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 33; h) a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 36, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 37, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 39; a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 41, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 42, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 43; j) a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 46, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 47, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 48;k) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53; l) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 58; m) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 61, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 62, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63; or n) a heavy chain variable region comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68.

[0009] In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising: a) a heavy chain variable region CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, or 66, or a variant thereof comprising up to about three amino acid substitutions; b) a heavy chain variable region CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, or 67, or a variant thereof comprising up to about three amino acid substitutions; and c) a heavy chain variable region CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 3, 8, 13, 18, 23, 28, 33, 38, 43, 48, 53, 58, 63, or 68, or a variant thereof comprising up to about three amino acid substitutions.

[0010] In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising a CDR1 domain, a CDR2 domain, and a CDR3 domain, wherein the CDR1 domain, the CDR2 domain, and the CDR3 domain respectively comprise the CDR1 domain, the CDR2 domain, and the CDR3 domain contained in a reference heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, or 69.

[0011] In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 2, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 3. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 6, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 7, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 8. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 11, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 12, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 13. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 16, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 17, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 18. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38.In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 43. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 46, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 47, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 58. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 61, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 62, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68.

[0012] In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, or 69. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 4. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 9. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 14. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 19. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 24. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 29. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 34. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 39. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 44. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 49. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 54. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 59. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 64. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 69. In certain embodiments, the single domain antibody comprises a humanized framework.

[0013] In certain embodiments, the second antigen-binding portion comprises a heavy chain variable region (VH) comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 71; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 72; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 73; and a light chain variable region (VL) comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 74; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 75; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 76. In certain embodiments, the second antigen-binding portion comprises a heavy chain variable region (VH) comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 81; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 82; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 83; and a light chain variable region (VL) comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 84; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 85; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 86.

[0014] In certain embodiments, the second antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 77 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 78. In certain embodiments, the second antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 87 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 88.

[0015] In certain embodiments, the anti-CD47 antibody comprises a human antibody. In certain embodiments, the second antigen-binding portion comprises an anti-CD47 antibody comprising two antibody heavy chains and two antibody light chains. In certain embodiments, the first antigen-binding portion comprises one or more anti-PDL1 antibodies. In certain embodiments, the first antigen-binding portion comprises two anti-PDL1 antibodies.

[0016] In certain embodiments, the C-terminus of at least one of the two anti-CD47 light chains is linked to the anti-PDL1 antibody of the first antigen-binding moiety. In certain embodiments, the C-terminus of each of the two anti-CD47 light chains is linked to the anti-PDL1 antibody of the first antigen-binding moiety. In certain embodiments, the N-terminus of each of the two anti-CD47 light chains is linked to the anti-PDL1 antibody of the first antigen-binding moiety. In certain embodiments, the N-terminus of each of the two anti-CD47 light chains is linked to the anti-PDL1 antibody of the first antigen-binding moiety. In certain embodiments, the C-terminus of at least one of the two anti-CD47 heavy chains is linked to the anti-PDL1 antibody of the first antigen-binding moiety. In certain embodiments, the C-terminus of each of the two anti-CD47 heavy chains is linked to the anti-PDL1 antibody of the first antigen-binding moiety. In certain embodiments, the N-terminus of at least one of the two anti-CD47 heavy chains is linked to the anti-PDL1 antibody of the first antigen-binding portion. In certain embodiments, the N-terminus of each of the two anti-CD47 heavy chains is linked to the anti-PDL1 antibody of the first antigen-binding portion.

[0017] In certain embodiments, the first antigen-binding moiety is linked to the second antigen-binding moiety via a linker, hi certain embodiments, the linker is a peptide linker.

[0018] In certain embodiments, the peptide linker comprises about 4 to about 30 amino acids. In certain embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 93-135.

[0019] In certain embodiments, the anti-CD47 antibody of the second antigen-binding portion comprises an Fc region. In certain embodiments, the Fc region comprises a human Fc region. In certain embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG, IgA, IgD, IgE, and IgM Fc regions. In certain embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions. In certain embodiments, the Fc region comprises an IgG4 Fc region. In certain embodiments, the Fc region comprises an IgG1 Fc region. In certain embodiments, the IgG1 Fc region comprises one or more mutations that enhance antibody-dependent cellular cytotoxicity (ADCC). In certain embodiments, the IgG1 Fc region comprises the following mutations: L235V, F243L, R292P, and Y300L. In certain embodiments, the IgG1 Fc region comprises the following mutations: S239D, A330L, and I332E.

[0020] In certain embodiments, the multispecific antibody comprises a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab'), an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv), a VHH, a VHH-Fc fusion, an Fv-Fc fusion, an scFv-Fc fusion, an scFv-Fv fusion, a diabody, a tribody, a tetrabody, or any combination thereof.

[0021] In certain embodiments, the multispecific antibody is a bispecific antibody.

[0022] In certain embodiments, the multispecific antibody comprises: i) a first antigen-binding portion comprising a single-domain anti-PDL1 antibody comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53; and ii) a second antigen-binding portion comprising an anti-CD47 antibody comprising a heavy chain variable region (VH) comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 71, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 72, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 73, and a light chain variable region (VL) comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 74, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 75, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 76. In certain embodiments, the multispecific antibody comprises: i) a first antigen-binding portion comprising a single-domain anti-PDL1 antibody comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53; and ii) a second antigen-binding portion comprising an anti-CD47 antibody comprising a heavy chain variable region (VH) comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 81, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 82, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 83, and a light chain variable region (VL) comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 84, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 85, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 86.

[0023] In certain embodiments, the multispecific antibody comprises i) a first antigen-binding portion comprising a single-domain anti-PDL1 antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 54, and ii) a second antigen-binding portion comprising an anti-CD47 antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 77 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 78.

[0024] In certain embodiments, the multispecific antibody comprises i) a first antigen-binding portion comprising a single-domain anti-PDL1 antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 54, and ii) a second antigen-binding portion comprising an anti-CD47 antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 87 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 88.

[0025] In certain embodiments, the multispecific antibody comprises a first chain comprising the amino acid sequence set forth in SEQ ID NO: 136 and a second chain comprising the amino acid sequence set forth in SEQ ID NO: 137. In certain embodiments, the multispecific antibody comprises a first chain comprising the amino acid sequence set forth in SEQ ID NO: 138 and a second chain comprising the amino acid sequence set forth in SEQ ID NO: 139. In certain embodiments, the multispecific antibody comprises a first chain comprising the amino acid sequence set forth in SEQ ID NO: 140 and a second chain comprising the amino acid sequence set forth in SEQ ID NO: 141.

[0026] The present disclosure provides immunoconjugates comprising any of the antibodies disclosed herein linked to a therapeutic agent or label. In certain embodiments, the therapeutic agent is a cytotoxin or a radioisotope. In certain embodiments, the label is selected from the group consisting of a radioisotope, a fluorescent dye, and an enzyme.

[0027] The present disclosure provides an antigen-recognizing receptor comprising an extracellular antigen-binding domain comprising an antibody disclosed herein. In certain embodiments, the antigen-recognizing receptor is a chimeric antigen receptor (CAR) or a recombinant T cell receptor. In certain embodiments, the antigen-recognizing receptor is a CAR. In certain embodiments, the antibody comprised in the extracellular antigen-binding domain comprises a VHH.

[0028] The present disclosure provides an immune response cell comprising an antigen-recognizing receptor disclosed herein. In certain embodiments, the immune response cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT) cell, and a myeloid cell. In certain embodiments, the immune response cell is a T cell.

[0029] The present disclosure provides a pharmaceutical composition comprising: a) any antibody disclosed herein, any immunoconjugate disclosed herein, or any immune response cell disclosed herein, and b) a pharmaceutically acceptable carrier.

[0030] The present disclosure further provides one or more nucleic acids encoding any of the antibodies disclosed herein, one or more vectors comprising any of the nucleic acids disclosed herein, and host cells comprising any of the nucleic acids or vectors disclosed herein.

[0031] The present disclosure provides a method of preparing an antibody disclosed herein, comprising expressing the antibody in a host cell disclosed herein and isolating the antibody from the host cell.

[0032] The present disclosure further provides methods for reducing tumor burden in a subject. In certain embodiments, the method comprises administering to the subject an effective amount of an antibody disclosed herein, an immunoconjugate disclosed herein, or a pharmaceutical composition disclosed herein. In certain embodiments, the method reduces the number of tumor cells. In certain embodiments, the method reduces tumor size. In certain embodiments, the method eradicates the tumor in the subject. In certain embodiments, the tumor exhibits high microsatellite instability (MSI). In certain 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 cancer, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof.

[0033] The present disclosure provides methods for treating and / or preventing cancer or prolonging survival of a subject with cancer. In certain embodiments, the method comprises administering to a subject an effective amount of an antibody disclosed herein, an immunoconjugate disclosed herein, or a pharmaceutical composition disclosed herein. In certain embodiments, the cancer exhibits high microsatellite instability (MSI). In certain embodiments, 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 cancer, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof.

[0034] The present disclosure further provides any antibody and / or pharmaceutical composition disclosed herein for use as a medicament. The present disclosure further provides any antibody and / or pharmaceutical composition disclosed herein for use in treating cancer. In certain embodiments, the cancer exhibits high microsatellite instability (MSI). In certain embodiments, 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 cancer, bile duct cancer, head and neck cancer, hematological cancer, and combinations thereof.

[0035] The present disclosure provides kits comprising an antibody disclosed herein, an immunoconjugate disclosed herein, a pharmaceutical composition disclosed herein, a nucleic acid disclosed herein, a vector disclosed herein, or an immune response cell disclosed herein. In certain embodiments, the kit further comprises written instructions for treating and / or preventing a neoplasm. [Brief explanation of the drawings]

[0036] [Figure 1] 1 shows the ability of anti-PDL1 antibodies to block human PDL1 / PD1 interaction, as tested in a whole cell blocking assay. [Figure 2] Figure 1 shows the ability of various humanized anti-PDL1 VHH antibodies to block human PDL1 / PD1 interaction, tested in a whole cell blocking assay. [Figure 3] Figure 1 shows the ability of humanized anti-PDL1 VHH antibodies to block human PDL1 / PD1 interaction compared to two reference antibodies. [Figure 4] 1 shows the antitumor effect of a humanized anti-PDL1 VHH antibody in a xenograft mouse model bearing H292 human lung cancer. [Figure 5] Figure 1 shows the ability of various affinity matured anti-PDL1 VHH antibodies to block human PDL1 / PD1 interaction, as tested in a whole cell blocking assay. [Figure 6A] Figure 6 shows the anti-tumor effect of anti-PDL1 clone 2H4 in a syngeneic mouse model bearing B16F10 mouse melanoma cells. Figure 6A shows the tumor growth curves of various treatment groups. [Figure 6B] Figure 6B shows the anti-tumor effect of anti-PDL1 clone 2H4 in a syngeneic mouse model bearing B16F10 mouse melanoma cells. Figure 6B shows the individual tumor volumes 20 days after tumor inoculation. [Figure 6C] Figure 6 shows the anti-tumor effect of anti-PDL1 clone 2H4 in a syngeneic mouse model bearing B16F10 mouse melanoma cells. Figure 6C shows the weight changes of mice in various treatment groups. [Figure 7A]Figure 7 shows the anti-tumor effect of anti-PDL1 clone 3D5 in a syngeneic mouse model bearing B16F10 mouse melanoma cells. Figure 7A shows the tumor growth curves of various treatment groups. [Figure 7B] Figure 7B shows the anti-tumor effect of anti-PDL1 clone 3D5 in a syngeneic mouse model bearing B16F10 mouse melanoma cells. Figure 7B shows the individual tumor volumes 20 days after tumor inoculation. [Figure 7C] Figure 7 shows the anti-tumor effect of anti-PDL1 clone 3D5 in a syngeneic mouse model bearing B16F10 mouse melanoma cells. Figure 7C shows the weight changes of mice in various treatment groups. [Figure 8] 1 shows a schematic diagram of an exemplary anti-PDL1 / CD47 bispecific antibody. [Figure 9A] Shown is the total cell binding of anti-PDL1 / CD47 antibodies to PDL1-CHO-K1 cells expressing hPDL1. [Figure 9B] 1 shows the total cell binding of anti-PDL1 / CD47 antibodies to Jurkat cells (leukemia cell line) expressing CD47. [Figure 10] 1 shows the whole cell binding of anti-PDL1 / CD47 antibodies to human primary red blood cells (RBCs). [Figure 11] Binding of anti-PDL1 / CD47 antibodies to primary human platelets is shown. [Figure 12A] 1 shows total cell binding of anti-PDL1 / CD47 antibodies to normal tissues containing human CD3+ T cells. [Figure 12B] 1 shows total cell binding of anti-PDL1 / CD47 antibodies to normal tissues, including human CD56+ NK cells. [Figure 12C] 1 shows total cell binding of anti-PDL1 / CD47 antibodies to normal tissues containing human CD14+ monocytic cells. [Figure 12D] 1 shows total cell binding of anti-PDL1 / CD47 antibodies to normal tissues containing human CD19+ B cells. [Figure 13A] Figure 13A shows the ability of anti-PDL1 / CD47 antibodies to induce cytokine release by immune cells. [Figure 13B]Figure 13B shows the ability of anti-PDL1 / CD47 antibodies to induce cytokine release by immune cells. Figure 13B shows antibody-induced IL2 release by immune cells. [Figure 14A] Figure 14 shows the anti-tumor effect of anti-PDL1 / CD47 antibodies in the A375 melanoma model. Figure 14A shows the tumor growth curves of the various treatment groups. [Figure 14B] Figure 4B shows the anti-tumor effect of anti-PDL1 / CD47 antibodies in an A375 melanoma model. Figure 4B shows the weight changes of mice in various treatment groups. [Figure 15A] Figure 15 shows the anti-tumor effect of anti-PDL1 / CD47 antibodies in the MDA-MB-231 triple-negative breast cancer model. Figure 15A shows tumor growth curves for various treatment groups. [Figure 15B] Figure 15B shows the anti-tumor effect of anti-PDL1 / CD47 antibodies in an MDA-MB-231 triple-negative breast cancer model. Figure 15B shows the weight changes of mice in various treatment groups. [Figure 16A] Figure 16 shows drug resistance testing of anti-PDL1 / CD47 antibodies in a mouse model. Figure 16A shows RBC counts in mice on specific days after antibody re-treatment. [Figure 16B] Figure 16B shows drug resistance testing of anti-PDL1 / CD47 antibodies in a mouse model. Figure 16B shows hemoglobin levels of mice at specific days after antibody re-treatment. [Figure 17] 1 shows changes in body weight in a drug resistance test of an anti-PDL1 / CD47 antibody in a cynomolgus monkey model. [Figure 18] 1 shows changes in red blood cells (RBC), white blood cells (WBC), hematocrit (HCT), hemoglobin (HGB), reticulocytes (ABRETIC), and platelets (PLT) in a drug resistance test of an anti-PDL1 / CD47 antibody in a cynomolgus monkey model. DETAILED DESCRIPTION OF THE INVENTION

[0037] The present disclosure provides isolated monoclonal antibodies and antibody derivatives that specifically bind to PDL1 with high affinity, including monospecific anti-PDL1 antibodies and multispecific antibodies that bind to PDL1 and one or more additional targets. In certain embodiments, the antibodies or antibody derivatives disclosed herein include single-domain antibodies that bind to PDL1. The present disclosure also provides isolated monoclonal antibodies and antibody derivatives that specifically bind to CD47 with high affinity. The present disclosure also provides methods of making and using the antibodies and antibody derivatives disclosed herein, as well as pharmaceutical compositions comprising them, for example, to treat diseases and disorders, such as cancer. The present invention is based, in part, on the discovery of novel single-domain antibodies that bind to PDL1, novel anti-CD47 antibodies with reduced binding to erythrocytes, and novel multispecific antibodies that bind to PDL1 and CD47, each of which can target tumor cells and / or increase the immune response against tumor cells, thereby resulting in improved anti-tumor effects.

[0038] For clarity, and not by way of limitation, the detailed description of the subject matter disclosed herein is divided into the following subsections: 1. Definition, 2. Antibodies and antibody derivatives, 3.How to use, 4. Pharmaceutical preparations, and 5. Manufactured products.

[0039] 1. Definition

[0040] As used herein, the term "antibody" includes full-length antibodies and any antigen-binding fragments thereof (i.e., antibody fragments). An "antibody" can be a free-standing molecule or part of an antibody derivative. Exemplary antibody derivatives include, but are not limited to, multispecific antibodies (e.g., bispecific antibodies), antigen-recognizing receptors (e.g., chimeric antigen receptors), antibody conjugates comprising additional proteinaceous or non-proteinaceous moieties (e.g., antibody-drug conjugates or polymer-coated antibodies), and other multifunctional molecules comprising antibodies.

[0041] "Full-length antibody," "intact antibody," and "whole antibody" refer to antibodies having heavy chains that resemble native antibody structures or contain an Fc region as defined herein. In certain embodiments, a full-length antibody comprises two heavy chains and two light chains. In certain embodiments, the variable regions of the light and heavy chains are responsible for antigen binding. The variable regions of the heavy and light chains may be referred to as "VH" and "VL," respectively. The variable regions of both chains generally contain three hypervariable loops called complementarity-determining regions (CDRs) (light chain (LC) CDRs comprising LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC) CDRs comprising HC-CDR1, HC-CDR2, and HC-CDR3). CDR boundaries for the antibodies and antigen-binding fragments disclosed herein may be defined or identified by well-known conventions, such as the conventions of Kabat, Chothia, MacCallum, IMGT, and AHo, as described below. The three CDRs of a heavy or light chain are interposed between flanking stretches known as framework regions (FRs), which are more conserved than the CDRs and form a scaffold to support the hypervariable loops. The constant regions of the heavy and light chains are not involved in antigen binding but exhibit various effector functions. Antibodies are assigned to classes based on the amino acid sequence of the constant region of their heavy chains. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, which are characterized by the presence of α, δ, ε, γ, and μ heavy chains, respectively. Some of the major antibody classes are divided into subclasses, such as 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 certain embodiments, full-length antibodies are glycosylated. In certain embodiments, full-length antibodies contain glycans linked to their Fc region. In certain embodiments, the full-length antibody comprises branched glycans.

[0042] As used herein, the terms "antigen-binding portion," "antibody fragment," and "antibody portion" of an antibody refer to one or more fragments of an antibody that retain 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, nanobodies, domain antibodies, bivalent domain antibodies, or any other fragment of an antibody that binds to an antigen, or a combination thereof. "VHH" refers to a single-domain antibody isolated from a camelid. In certain embodiments, the VHH comprises the variable region of the heavy chain of a camelid heavy-chain antibody. In certain embodiments, the VHH has a size not exceeding about 25 kDa. In certain embodiments, the VHH has a size not exceeding about 20 kDa. In certain embodiments, the VHH has a size not exceeding about 15 kDa.

[0043] An "antibody that cross-competes for binding" with a reference antibody refers to an antibody that blocks the reference antibody from binding to its antigen by 50% or more in a competition assay, and conversely, a reference antibody that blocks the antibody from binding to its antigen by 50% or more in a competition assay. Exemplary competition assays are described in Antibodies, Harlow and Lane (Cold Spring Harbor Press, Cold Spring Harbor, NY).

[0044] An "Fv" is the minimum antibody fragment containing a complete antigen-recognition and antigen-binding site. This fragment consists of a dimer of one heavy- and one light-chain variable region in tight, non-covalent association. The folding of these two domains results in six hypervariable loops (three loops per heavy and light chain) that contribute amino acid residues to antigen binding and confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three antigen-specific CDRs) can recognize and bind antigen, although in some cases with lower affinity than the entire binding site.

[0045] "Single-chain Fv," also abbreviated as "sFv" or "scFv," is a VFv consisting of a single Fv fragment connected to a single polypeptide chain. H and V L In some embodiments, the scFv polypeptide is an antibody fragment comprising a V domain that enables the scFv to form the desired structure for antigen binding. H and V L It further comprises a polypeptide linker between the domains. For a review of scFvs, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0046] For purposes herein, an "acceptor human framework," or "human framework," is a framework comprising the amino acid sequence of a light chain variable region (VL) framework or a heavy chain variable region (VH) framework derived from a human immunoglobulin framework or a human consensus framework. An acceptor human framework "derived from" a human immunoglobulin framework or a human consensus framework may comprise the same amino acid sequence or may contain amino acid sequence changes. In certain embodiments, the number of amino acid changes is 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 certain embodiments, the VL acceptor human framework is identical in sequence to the VL human immunoglobulin framework sequence or the human consensus framework sequence.

[0047] "Affinity" refers to the strength of the sum of 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 indicated, as used herein, "binding affinity" refers to the intrinsic binding affinity, which reflects a 1:1 interaction between members of a binding pair (e.g., an antibody and an antigen). The affinity of a molecule X for its partner Y can generally be expressed as a dissociation constant (KD). Affinity can be measured by methods well known in the art, including those described herein. Specific descriptions and exemplary embodiments for measuring binding affinity are provided below.

[0048] An "affinity matured" antibody refers to an antibody that has one or more alterations in one or more CDRs or hypervariable regions (HVRs) compared to a parent antibody that does not possess such alterations, which alterations result in improved affinity of the antibody for antigen.

[0049] The terms "anti-PDL1 antibody" and "antibody that binds to PDL1" refer to an antibody that is capable of binding to PDL1 with sufficient affinity such that the antibody is useful as a diagnostic and / or therapeutic agent for targeting PDL1. In one embodiment, the extent of binding of an anti-PDL1 antibody to an unrelated, non-PDL1 protein can be measured using, for example, BIACORE (登録商標) The antibody binds to PDL1 at a binding affinity of less than about 10% as measured by surface plasmon resonance assay. In certain embodiments, the antibody binds to PDL1 at a binding affinity of less than about 1 μM, less than about 100 nM, less than about 10 nM, less than about 1 nM, less than about 0.1 nM, less than about 0.01 nM, or less than about 0.001 nM (e.g., 10 -8 M or less, e.g., 10 -8 M~10 -12 M, e.g., 10 -9 M~10 -10 In certain embodiments, the anti-PDL1 antibody binds to an epitope on PDL1 that is conserved among PDL1 from different species. In certain embodiments, the anti-PDL1 antibody binds to an epitope on PDL1 that is in the ECD of the protein.

[0050] The term "chimeric" antibody refers to an antibody in which a portion of the heavy and / or light chain is derived from a particular source or species, while the remainder of the heavy and / or light chain is derived from a different source or species. In certain embodiments, a chimeric antibody disclosed herein comprises a murine heavy chain variable region and a human Fc region. In certain embodiments, a chimeric antibody disclosed herein comprises a camelid heavy chain variable region and a human Fc region.

[0051] As used herein, the term "CDR" or "complementarity determining region" is intended to mean non-contiguous antigen-binding sites within the variable regions of the heavy and / or light chains. These specific regions have been described by 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 Marin, Mol. Immunol., 45:3832-3839 (2008), Lefranc The definitions are described by MP et al., Dev. Comp. Immunol., 27:55-77 (2003), and Honegger and Pluckthun, J. Mol. Biol., 309:657-670 (2001), and these definitions include overlapping or subsets of amino acid residues when compared with each other. Nevertheless, application of any one of the definitions to refer to the CDRs of an antibody or grafted antibody, or variants thereof, is intended to be within the scope of the term as defined and used herein. The amino acid residues encompassing the CDRs as defined in each of the above-cited references are set forth in Table 1 below for comparison. CDR prediction algorithms and interfaces are well known in the art, for example, Abhinandan and Marin, 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 references cited in this section are incorporated herein by reference in their entirety for use in this application and as may be included in one or more claims herein.

[0052] [Table 1] 1 Residue numbering follows the nomenclature of Kabat et al., supra. 2 Residue numbering follows the nomenclature of Chothia et al., supra. 3 Residue numbering follows the nomenclature of MacCallum et al., supra. 4 Residue numbering follows the nomenclature of Lefranc et al., supra. 5 Residue numbering follows the nomenclature of Honegger and Pluckthun et al., supra.

[0053] The phrases "Kabat variable region residue numbering" or "Kabat amino acid position numbering," and variations thereof, refer to the numbering system used for the heavy or light chain variable regions of the antibody compilation in Kabat et al., supra. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FRs or CDRs of the variable region. For example, a heavy chain variable region may contain a single amino acid insertion after residue 52 of H2 (residue 52a according to Kabat) and inserted residues after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by aligning the homologous regions of the antibody sequence with the "standard" Kabat numbering sequence.

[0054] In certain embodiments, the amino acid residues encompassing the CDRs of a single domain antibody are defined according to the IMGT nomenclature in Lefranc et al., supra. In certain embodiments, the amino acid residues encompassing the CDRs of a full-length antibody are defined according to the Kabat nomenclature in Kabat et al., supra. In certain embodiments, the numbering of residues in an immunoglobulin heavy chain, e.g., in the Fc region, is that of the EU index as in Kabat et al., supra. "EU index as in Kabat" refers to the residue numbering of a human IgG1 EU antibody.

[0055] "Framework" or "FR" refers to those variable domain residues other than the CDR residues as herein defined.

[0056] A "humanized" antibody refers to a chimeric antibody comprising amino acid residues derived from non-human CDRs / HVRs and human FRs. In certain embodiments, a humanized antibody comprises at least one, and typically two, variable domains, in which all or substantially all of the HVRs / CDRs correspond to those of a non-human antibody and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody may optionally comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0057] A "human antibody" is an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or an antibody produced using any of the techniques for producing human antibodies disclosed herein. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques well 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). Methods described by 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 to prepare human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomouse, which have been modified to produce such antibodies in response to antigen challenge, but whose endogenous gene loci have been disabled (e.g., XENOMOUSE). TM (See U.S. Patent Nos. 6,075,181 and 6,150,584 regarding this technology.) See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), regarding human antibodies produced by human B cell hybridoma technology.

[0058] With respect to the polypeptide and antibody sequences identified herein, "percent amino acid sequence identity," or "homology," is defined as the percent of amino acid residues in a candidate sequence that are identical to those in a compared polypeptide, after aligning the sequences and taking into account any conservative substitutions as part of the sequence identity. Alignment to determine percent amino acid sequence identity can be accomplished in a variety of ways within the skill of the art, 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 for measuring alignment, including any algorithms required to achieve maximal alignment over the entire length of the sequences being compared. However, for purposes of this specification, percent amino acid sequence identity values ​​are generated using the sequence comparison computer program MUSCLE (Edgar, R.C., Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, R.C., BMC Bioinformatics 5(1):113, 2004).

[0059] "Homologous" refers to sequence similarity or sequence identity between two polypeptides or two nucleic acid molecules. If a position in both compared sequences is occupied by the same base or amino acid monomer subunit, for example, if each position in two DNA molecules is occupied by adenine, the molecules are homologous at that position. The percent 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 compared positions, multiplied by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, the two sequences are 60% homologous. For example, the DNA sequences ATTGCC and TATGGC share 50% homology. Generally, comparisons are performed by aligning two sequences to obtain maximum homology.

[0060] The "light chains" of antibodies (e.g., immunoglobulins) from any mammalian species can be assigned to one of two clearly distinct types, called kappa ("κ") and lambda ("λ"), based on the amino acid sequences of their constant domains.

[0061] The term "constant domain" refers to the portion of an immunoglobulin molecule that has a more conserved amino acid sequence compared to the other portion of the immunoglobulin, i.e., the variable domain, which contains the antigen-binding site. The constant domain is the C H 1. C H 2, and C H 3 domains (collectively, C H ), and light chain C L Includes the domain.

[0062] In certain embodiments, the "CH1 domain" (also called "C1" for "H1" domain) extends from about amino acid 118 to about amino acid 215 (EU numbering system).

[0063] In certain embodiments, the "hinge region" is generally defined as the region in IgG corresponding to Glu216 to Pro230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain S—S bonds in the same positions.

[0064] In certain embodiments, the "CH2 domain" (also called the "C2" domain) of the human IgG Fc region typically extends from about amino acid 231 to about amino acid 340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains in an intact, native IgG molecule. It has been speculated that the carbohydrates may provide a substitute for domain pairing and help stabilize the CH2 domain. Burton, Molec Immunol. 22:161-206 (1985).

[0065] In certain embodiments, the "CH3 domain" (also referred to as the "C2" domain) comprises residues between the CH2 domain and the C-terminus of the Fc region (i.e., from about amino acid residue 341 to the C-terminus of the antibody sequence, typically amino acid residue 446 or 447 of an IgG).

[0066] The terms "Fc region" or "fragment crystallizable region" herein are used to define the C-terminal region of an immunoglobulin heavy chain, including native-sequence Fc regions and variant Fc regions, or dimers thereof. In certain embodiments, a human IgG Fc region extends from Cys226 to its carboxyl terminus. In certain embodiments, a human IgG Fc region extends from Pro231 to its carboxyl terminus. In certain embodiments, a human IgG Fc region comprises a CH2 domain and a CH3 domain. In certain embodiments, the C-terminal lysine of the Fc region (residue 447 according to the EU numbering system) can be removed, for example, during antibody production or purification, or by genetically modifying a nucleic acid encoding the antibody heavy chain. In certain embodiments, a composition of intact antibodies can include an antibody population having all of the K447 residues removed, an antibody population in which the K447 residue is not removed, or a mixture of antibodies with and without the K447 residue. Suitable native sequence Fc regions for use in the antibodies described herein include those of human IgG1, IgG2 (IgG2A, IgG2B), IgG3, and IgG4.

[0067] "Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native human FcR. Furthermore, a preferred FcR is one that binds IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed 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). Other FcRs, including FcRs identified in the future, are encompassed by the term "FcR" herein.

[0068] As used herein, the term "epitope" refers to a specific group of atoms or amino acids on an antigen to which an antibody or antibody derivative binds. Two antibodies or antigen-binding portions can bind to the same epitope in an antigen if they exhibit competitive binding to the antigen.

[0069] As used herein, the terms "specifically bind," "specifically recognize," and "specific for" refer to a measurable and reproducible interaction, such as binding between a target and an antibody or antibody molecule, that determines the presence of the target in the presence of a heterogeneous population of molecules, including biomolecules. For example, an antibody or antibody portion that specifically recognizes a target (which can be an epitope) is an antibody or antibody portion that binds to this target with higher affinity, higher affinity, more rapid response, and / or longer duration than its binding to other targets. In some embodiments, the extent of binding of an antibody to an unrelated target is less than about 10% of the binding of the antibody to the target, as measured, for example, by radioimmunoassay (RIA). In some embodiments, an antibody that specifically binds to a target has a binding affinity of ≦10 -5 M, ≤10 -6 M, ≤10 -7 M, ≤10 -8 M, ≤10 -9 M, ≤10 -10 M, ≤10 -11 M, or ≦10 -12 Dissociation constant of M (K D ) In some embodiments, the antibody specifically binds to an epitope on a protein that is conserved among proteins from different species. In some embodiments, specific binding can include, but is not necessarily, exclusive binding. The binding specificity of an antibody or antigen-binding domain can be determined experimentally by methods well known in the art. Such methods include Western blot, ELISA test, RIA test, ECL test, IRMA test, EIA test, BIACORE test, and the like. TM These include, but are not limited to, peptide scans and peptide tests.

[0070] An "isolated" antibody (or construct) is one that has been identified, separated, and / or recovered from a component of its production environment (e.g., natural or recombinant). In certain embodiments, an isolated polypeptide is free from association or substantially free from association with all other components from its production environment.

[0071] An "isolated" nucleic acid molecule encoding a construct, antibody, or antigen-binding fragment thereof described herein is a nucleic acid molecule that has been identified and separated from at least one contaminant nucleic acid molecule with which it is normally associated in the environment in which it is produced. In certain embodiments, an isolated nucleic acid is free or substantially free from association with all components associated with the production environment. Isolated nucleic acid molecules encoding the polypeptides and antibodies described herein are in a form other than the form or setting in which they are found in nature. Isolated nucleic acid molecules are therefore distinguished from nucleic acids encoding the polypeptides and antibodies described herein that are naturally present in cells. Isolated nucleic acids include nucleic acid molecules that are contained in cells that normally contain the nucleic acid molecule, but where the nucleic acid molecule is present extrachromosomally or at a chromosomal location that is different from its natural chromosomal location.

[0072] The term "regulatory sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Regulatory sequences that are suitable for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0073] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to promote translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading frame. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0074] As used herein, the term "vector" refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes vectors that act as self-replicating nucleic acid structures as well as vectors that integrate into the genome of a host cell into which they are introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors."

[0075] As used herein, the terms "transfected" or "transformed" or "transduced" refer to the process by which exogenous nucleic acid is transferred or introduced into a host cell. A "transfected" or "transformed" or "transduced" cell is one that has been transfected, transformed, or transduced with exogenous nucleic acid, and this cell includes the primary subject cell and its progeny.

[0076] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and its progeny without regard to the number of passages. The progeny may not be completely identical in nucleic acid content to the parent cell and may contain mutations. Included herein are mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell.

[0077] The terms "subject," "individual," and "patient" are used interchangeably herein and refer to a mammal, including, but not limited to, a human, bovine, equine, feline, canine, rodent, or primate. In some embodiments, the subject is a human.

[0078] An "effective amount" of an agent refers to an amount effective to achieve a desired therapeutic or prophylactic result, at the dosage and for the period of time necessary. The specific dosage may vary depending on one or more of the particular agent selected, the administration regimen to be followed, whether it is administered in combination with other compounds, the timing of administration, the tissue to be imaged, and the physical delivery system in which it is delivered.

[0079] A "therapeutically effective amount" of a substance / molecule, agonist, or antagonist herein can vary depending on factors such as the individual's condition, age, sex, and weight, and the ability of the substance / molecule, agonist, or antagonist to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the substance / molecule, agonist, or antagonist are outweighed by the therapeutically beneficial effects. A therapeutically effective amount can be delivered in one or more administrations.

[0080] A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or preventive result. Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0081] As used herein, "treatment" or "treating" is an approach to obtaining beneficial or desired results, including clinical results. For purposes of this application, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or slowing the progression of the disease), preventing or slowing the spread of the disease (e.g., metastasis), preventing or slowing the recurrence of the disease, slowing or attenuating the progression of the disease, improving the condition, providing remission (partial or complete) of the disease, reducing the dose of one or more other drugs required to treat the disease, slowing the progression of the disease, improving or improving quality of life, increasing weight gain, and / or extending survival. "Treatment" also includes reducing the pathological consequences of cancer (e.g., tumor volume, etc.). The methods of this application contemplate any one or more of these aspects of treatment. "Treatment" does not necessarily mean that the condition being treated is cured.

[0082] It is understood that embodiments of the present application described herein include "consisting of" and / or "consisting essentially of" embodiments.

[0083] As used herein, the term "about" or "approximately" means that a particular value, as determined by one of ordinary skill in the art, is within an acceptable error range, which depends in part on how the value is measured or determined, i.e., is limited by the measurement system. In certain embodiments, "about" can mean within 3 standard deviations, or more than 3 standard deviations, in accordance with the practice in the art. In certain embodiments, "about" can mean a range of up to 20%, e.g., up to 10%, up to 5%, or up to 1% of a given value. In certain embodiments, particularly with respect to biological systems or processes, the term can mean within an order of magnitude of a value, e.g., within 5-fold or within 2-fold.

[0084] As used herein, the term "modulation" means to alter in a positive or negative direction. Exemplary modulations include a change of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

[0085] As used herein, the term "increase" means a positive modification of at least about 5%. The modification can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, about 100%, or more.

[0086] As used herein, the term "reduce" means to alter in a negative direction by at least about 5%. The alteration can be about 5%, about 10%, about 25%, about 30%, about 50%, about 75%, or even about 100%.

[0087] As used herein, the term "about X to Y" has the same meaning as "about X to about Y."

[0088] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise.

[0089] "Effector function" refers to a biological activity attributable to the Fc region of an antibody, 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 cellular cytotoxicity (ADCC), phagocytosis, down-regulation of cell surface receptors (e.g., B cell receptors), and B cell activation.

[0090] An "immunoconjugate" refers to an antibody conjugated to one or more heterologous molecules, including, but not limited to, cytotoxic agents.

[0091] The term "pharmaceutical formulation" refers to a formulation that is in a form such that the biological activity of the active ingredient contained therein is effective and that does not contain additional ingredients that are unacceptably toxic to a subject to which the formulation may be administered.

[0092] As used herein, "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, that is non-toxic to a subject. Pharmaceutically acceptable carriers include, but are not limited to, buffers, excipients, stabilizers, or preservatives.

[0093] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. In certain embodiments, the heavy and light chain variable domains (VH and VL, respectively) of natural antibodies generally have a similar structure, with each domain containing four conserved framework regions (FR) and three CDRs. (See, e.g., Kindt et al., Kuby Immunology, 61 ed., W.H. Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind to a specific antigen can be isolated from an antibody that binds that antigen using the VH or VL domain to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0094] As used herein, the term "antigen-recognizing receptor" refers to a receptor that can activate an immune response cell (e.g., a T cell) in response to its binding to an antigen. Non-limiting examples of antigen-recognizing receptors include natural and modified T cell receptors ("TCRs") and chimeric antigen receptors ("CARs").

[0095] As used herein, the term "chimeric antigen receptor" or "CAR" refers to a molecule comprising an extracellular antigen-binding domain fused to an intracellular signaling domain capable of activating or stimulating an immune response cell, and a transmembrane domain. In certain embodiments, the extracellular antigen-binding domain of the CAR comprises an antibody or antibody fragment, e.g., a VHH or scFv. In certain embodiments, an antibody (e.g., a VHH or scFv) is fused to a transmembrane domain, which is fused to an intracellular signaling domain. In certain embodiments, the CAR is selected to have high binding affinity or avidity for the antigen.

[0096] "Immune response cell" means a cell that functions in an immune response, or a precursor or progeny thereof.

[0097] As used herein, "PDL1," "PDL1 protein," or "PDL1 polypeptide" refers to any PDL1 polypeptide from any vertebrate source, including mammals such as primates (e.g., humans and cynomolgus monkeys), or any fragment thereof, and can optionally contain up to 1, up to 2, up to 3, up to 4, up to 5, up to 6, up to 7, up to 8, up to 9, or up to 10 amino acid substitutions, additions, and / or deletions. The term encompasses full-length, unprocessed PDL1 as well as any form of PDL1 resulting from processing in cells. The term also encompasses naturally occurring variants of PDL1, such as splice variants or allelic variants. In certain embodiments, the PDL1 polypeptide comprises or has an amino acid sequence that is 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% homologous or identical to a sequence having NCBI reference number NP_001254635.1, NP_001300958.1, or NP_054862.1 (homology herein can be determined using standard software such as BLAST or FASTA). In certain embodiments, the PDL1 polypeptide comprises or has an amino acid sequence that is the entire or a contiguous portion of SEQ ID NO: 91. The term "ECD of PDL1" refers to the extracellular domain of PDL1. In certain embodiments, the ECD of an exemplary PDL1 polypeptide can comprise the amino acid sequence set forth in SEQ ID NO: 92.

[0098] 2. Antibodies and antibody derivatives

[0099] The present disclosure provides antibodies and antibody derivatives.

[0100] In certain embodiments, the antibodies of the present disclosure can be or comprise monoclonal antibodies, including chimeric, humanized, or human antibodies. In certain embodiments, the antibodies disclosed herein comprise humanized antibodies. In certain embodiments, the antibodies comprise an acceptor human framework, such as a human immunoglobulin framework or a human consensus framework.

[0101] In certain embodiments, an antibody of the present disclosure can be an antibody fragment, e.g., an Fv, Fab, Fab', scFv, diabody, or F(ab')2 fragment. In certain embodiments, the antibody is a full-length antibody, e.g., an intact IgG1 antibody, or other antibody class or isotype as defined herein. In certain embodiments, an antibody or antibody derivative of the present disclosure can incorporate, alone or in combination, any of the features described herein, e.g., those detailed in Sections 2.1-2.12 herein.

[0102] The antibodies and antibody derivatives of the present disclosure are useful, for example, for the diagnosis or treatment of neoplasms or cancers. In certain embodiments, neoplasms and cancers whose growth can be inhibited using the antibodies of the present disclosure include neoplasms and cancers that typically respond to immunotherapy. In certain embodiments, such neoplasms and cancers include breast cancer (e.g., breast cell carcinoma), ovarian cancer (e.g., ovarian cell carcinoma), and renal cell carcinoma (RCC). Other examples of cancers that may be treated using the methods of the present disclosure include melanoma (e.g., metastatic malignant melanoma), prostate cancer, colon cancer, lung cancer, bone cancer, pancreatic cancer, skin cancer, brain cancer, chronic or acute leukemia, including acute myeloid leukemia, chronic myeloid leukemia, acute lymphocytic leukemia, and chronic lymphocytic leukemia, lymphoma (e.g., Hodgkin's lymphoma and non-Hodgkin's lymphoma, lymphocytic lymphoma, primary CNS lymphoma, T-cell lymphoma), nasopharyngeal cancer, head or neck cancer, cutaneous or intraocular malignant melanoma, uterine cancer, rectal cancer, and anal region cancer. , gastric cancer, testicular cancer, uterine cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, thyroid cancer, parathyroid cancer, breast cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, bladder cancer, kidney or ureter cancer, breast and pelvic cancer, central nervous system (CNS) neoplasms, tumor angiogenesis, spinal axis tumors, brain stem glioma, pituitary adenoma, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, environmentally induced cancers, e.g., mesothelioma, including those induced by asbestos, and combinations of the above cancers.

[0103] 2.1 Exemplary Monospecific and Multispecific Antibodies

[0104] 2.1.1 Exemplary Anti-PDL1 Antibodies

[0105] The present disclosure provides isolated antibodies that bind to PDL1 protein. In certain embodiments, the present disclosure is based, in part, on the discovery of single-domain antibodies that bind to PDL1, which can be used in anti-tumor therapy, where the antibodies selectively inhibit PDL1-mediated signaling pathways and thereby induce beneficial anti-tumor effects of immune cells against tumor cells. In certain embodiments, the antibodies disclosed herein are antagonistic antibodies that inhibit PDL1-mediated signaling pathways. In certain embodiments, the anti-PDL1 antibodies can increase the anti-tumor immune response of immune cells expressing PDL1 protein. In certain embodiments, the anti-PDL1 antibodies comprise single-domain antibodies, such as camelid antibodies or VHH antibodies. In certain embodiments, the anti-PDL1 antibodies have improved tissue penetration capabilities due to their smaller size compared to conventional antibodies with the same valency in the form of IgG, Fab, and / or scFv. In certain embodiments, the anti-PDL1 antibodies of the present disclosure bind to the ECD of PDL1. In certain embodiments, the anti-PDL1 antibody binds to the same epitope as an anti-PDL1 antibody described herein, such as 10A4.

[0106] In certain embodiments, the anti-PDL1 antibodies disclosed herein can function as antagonists of PDL1-based signaling pathways. In certain embodiments, the anti-PDL1 antibodies can block or reduce the interaction between the PDL1 protein and its receptor. In certain embodiments, the anti-PDL1 antibodies can reduce the interaction between the PDL1 protein and its receptor 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 certain embodiments, the anti-PDL1 antibodies can block downstream immune inhibitory signaling of the PDL1 protein or its receptor. In certain embodiments, treatment with the anti-PDL1 antibodies exhibits an anti-tumor effect in a subject, thereby reducing tumor growth and / or prolonging the subject's survival. In certain embodiments, the anti-PDL1 antibodies enhance the immune response and / or anti-tumor effect of immune cells, such as T cells and / or NK cells. In certain embodiments, anti-PDL1 antibodies including single-domain antibodies (e.g., VHHs) have smaller molecular sizes than full-length antibodies due to the smaller size of single-domain antibodies compared to the Fab domain of full-length antibodies, which can result in superior tissue penetration, for example, at tumor sites, compared to full-length antibodies. In certain embodiments, treatment with such anti-PDL1 antibodies exhibits superior anti-tumor effects compared to treatment with analogs of full-length anti-PDL1 antibodies, such as MPDL3280A or PL3#7-43 deglyco2, the sequences of which are disclosed in WO2016007235A1 and WO2018080812A1, respectively.

[0107] In certain embodiments, the anti-PDL1 antibody comprises a single domain antibody that binds to PDL1. In certain embodiments, the single domain antibody comprises a VHH. In certain embodiments, the single domain antibody comprises a heavy chain variable region (VH). In certain embodiments, the single domain antibody is linked to an Fc region. In certain embodiments, the single domain antibody is not linked to an Fc region.

[0108] In certain embodiments, the single domain antibody is about 1 x 10 -7 In certain embodiments, the single domain antibody binds to PDL1 with a KD of about 1 x 10 -8 In certain embodiments, the single domain antibody binds to PDL1 with a KD of about 5×10 -9 In certain embodiments, the single domain antibody binds to PDL1 with a KD of about 1 x 10 -9 In certain embodiments, the single domain antibody binds to PDL1 with a KD of about 1 x 10 -10 In certain embodiments, the single domain antibody binds to PDL1 with a KD of about 1 x 10 -11 M ~ approx. 1×10 -7 In certain embodiments, the single domain antibody binds to PDL1 with a KD of about 1 x 10 -10 M ~ approx. 1×10 -7 In certain embodiments, the single domain antibody binds to PDL1 with a KD of about 1 x 10 -10 M ~ approx. 1×10 -8 In certain embodiments, the single domain antibody binds to PDL1 with a KD of about 1 x 10 -11 M ~ approx. 1×10 -9 In certain embodiments, the single domain antibody binds to PDL1 with a KD of about 2×10 -10 M ~ approx. 5×10 -9 In certain embodiments, the single domain antibody binds to PDL1 with a KD of about 1 x 10 -9 M ~ approx. 5×10 -8 In certain embodiments, the single domain antibody binds to PDL1 with a KD of about 1 x 10 -10 M ~ approx. 1×10 -9 Binds to PDL1 at the KD of M.

[0109] In certain embodiments, the anti-PDL1 antibody is bivalent, trivalent, tetravalent, pentavalent, hexavalent, heptavalent, or octavalent. In certain embodiments, the anti-PDL1 antibody is bivalent. In certain embodiments, the anti-PDL1 antibody is tetravalent. In certain embodiments, the anti-PDL1 antibody is hexavalent. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising a VHH domain and an Fc region. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising a VHH domain, a CH1 domain, and an Fc region. In certain embodiments, the anti-PDL1 antibody comprises a light chain comprising a VHH domain and a CL domain.

[0110] In certain embodiments, the single domain antibody cross-competes for binding to PDL1 with a reference anti-PDL1 single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 2, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 3. In certain embodiments, the single domain antibody cross-competes for binding to PDL1 with a reference anti-PDL1 single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 6, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 7, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 8. In certain embodiments, the single domain antibody cross-competes for binding to PDL1 with a reference anti-PDL1 single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 11, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 12, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 13. In certain embodiments, the single domain antibody cross-competes for binding to PDL1 with a reference anti-PDL1 single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 16, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 17, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 18. In certain embodiments, the single domain antibody cross-competes for binding to PDL1 with a reference anti-PDL1 single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 23.In certain embodiments, the single domain antibody cross-competes for binding to PDL1 with a reference anti-PDL1 single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 26, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 27, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 28. In certain embodiments, the single domain antibody cross-competes for binding to PDL1 with a reference anti-PDL1 single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 31, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 32, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 33. In certain embodiments, the single domain antibody cross-competes for binding to PDL1 with a reference anti-PDL1 single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 36, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 37, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 38. In certain embodiments, the single domain antibody cross-competes for binding to PDL1 with a reference anti-PDL1 single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 41, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 42, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 43. In certain embodiments, the single domain antibody cross-competes for binding to PDL1 with a reference anti-PDL1 single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 46, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 47, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 48.In certain embodiments, the single domain antibody cross-competes for binding to PDL1 with a reference anti-PDL1 single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 51, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 52, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 53. In certain embodiments, the single domain antibody cross-competes for binding to PDL1 with a reference anti-PDL1 single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 56, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 57, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 58. In certain embodiments, the single domain antibody cross-competes for binding to PDL1 with a reference anti-PDL1 single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 61, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 62, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 63. In certain embodiments, the single domain antibody cross-competes for binding to PDL1 with a reference anti-PDL1 single domain antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 66, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 67, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 68.

[0111] In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising: a) a heavy chain variable region CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, or 66, or a variant thereof comprising up to about three amino acid substitutions; b) a heavy chain variable region CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, or 67, or a variant thereof comprising up to about three amino acid substitutions; and c) a heavy chain variable region CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 3, 8, 13, 18, 23, 28, 33, 38, 43, 48, 53, 58, 63, or 68, or a variant thereof comprising up to about three amino acid substitutions.

[0112] In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising a CDR1 domain, a CDR2 domain, and a CDR3 domain, wherein the CDR1 domain, the CDR2 domain, and the CDR3 domain respectively comprise the CDR1 domain, the CDR2 domain, and the CDR3 domain contained in a reference heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, or 69.

[0113] In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 2, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 3. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 6, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 7, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 8. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 11, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 12, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 13. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 16, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 17, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 18. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 23. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 26, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 27, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 28. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 31, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 32, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 33.In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 36, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 37, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 38. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 41, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 42, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 43. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 46, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 47, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 48. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 51, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 52, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 53. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 56, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 57, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 58. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 61, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 62, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 63. In certain embodiments, the single domain antibody comprises a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 66, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 67, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 68.

[0114] In certain embodiments, the single domain antibody comprises a 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, or 69. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, or 69.

[0115] In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:4. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:9. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:14. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:19. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:24. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:29. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:34. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:39. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:44. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:49. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 54. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 59. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 64. In certain embodiments, the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 69.

[0116] In certain embodiments, the anti-PDL1 antibody comprises a heavy chain 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 to an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, and 70. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 5. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 10. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 15. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 20. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 25. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 30. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 35. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 40. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 45. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 50. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 55. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 60. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 65. In certain embodiments, the anti-PDL1 antibody comprises a heavy chain comprising the amino acid sequence set forth in SEQ ID NO: 70.

[0117] In certain embodiments, any one of the amino acid sequences included in the heavy chain variable region can contain up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions. In certain embodiments, the amino acid substitutions are conservative substitutions.

[0118] In certain embodiments, the single domain antibody comprises a humanized framework, hi certain embodiments, the humanized framework comprises the framework sequence of the heavy chain variable region sequence set forth in SEQ ID NO: 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64, or 69.

[0119] 2.1.2 Exemplary Anti-CD47 Antibodies

[0120] The present disclosure further provides anti-CD47 antibodies. In certain embodiments, the anti-CD47 antibodies disclosed herein bind to the CD47 protein with high affinity. In certain embodiments, the antibodies disclosed herein are antagonist antibodies that inhibit the function of the CD47 receptor. In certain embodiments, the anti-CD47 antibodies inhibit the interaction between the CD47 receptor and one or more of its ligands. In certain embodiments, the anti-CD47 antibodies block or reduce immune inhibitory signals triggered by the CD47 receptor. In certain embodiments, the anti-CD47 antibodies have reduced or decreased binding and / or toxicity to normal cells, e.g., erythrocytes, compared to a reference antibody, e.g., a magrolimab analog. In certain embodiments, the anti-CD47 antibodies can activate immune cells, e.g., T cells and / or NK cells.

[0121] In certain embodiments, the anti-CD47 antibody cross-competes with a reference anti-CD47 antibody comprising: a) a heavy chain variable region (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 71; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 72; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 73; and a light chain variable region (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 74; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 75; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 76. In certain embodiments, the anti-CD47 antibody cross-competes with a reference anti-CD47 antibody comprising: a) a heavy chain variable region (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 81; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 82; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 83; and a light chain variable region (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 84; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 85; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 86.

[0122] In certain embodiments, the anti-CD47 antibody comprises a heavy chain variable region (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 71; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 72; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 73; and a light chain variable region (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 74; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 75; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 76. In certain embodiments, the anti-CD47 antibody comprises a heavy chain variable region (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 81; (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 82; and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 83; and a light chain variable region (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 84; (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 85; and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 86.

[0123] In certain embodiments, the anti-CD47 antibody comprises a 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 to the amino acid sequence of SEQ ID NO: 77, and a light 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 to the amino acid sequence of SEQ ID NO: 78. In certain embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 77 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 78. In certain embodiments, the anti-CD47 antibody comprises a 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 to the amino acid sequence of SEQ ID NO: 87, and a light 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 to the amino acid sequence of SEQ ID NO: 88. In certain embodiments, the anti-CD47 antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 87 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:88.

[0124] In certain embodiments, any one of the amino acid sequences contained in the heavy chain variable region and / or the light chain variable region can contain up to about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, deletions, and / or additions. In certain embodiments, the amino acid substitutions are conservative substitutions.

[0125] In certain embodiments, the anti-CD47 antibody does not comprise an Fc region. In certain embodiments, the anti-CD47 antibody further comprises an Fc region. In certain embodiments, the Fc region comprises a human Fc region. In certain embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG, IgA, IgD, IgE, and IgM Fc regions. In certain embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions. In certain embodiments, the Fc region comprises an IgG1 Fc region. In certain embodiments, the Fc region comprises an IgG2 Fc region. In certain embodiments, the Fc region comprises an IgG4 Fc region. In certain embodiments, the Fc region comprises one or more amino acid modifications, substitutions, or mutations described in Section 2.7.3.

[0126] In certain embodiments, the anti-CD47 antibody comprises a human antibody. In certain embodiments, the anti-CD47 antibody comprises a humanized antibody comprising a humanized framework.

[0127] 2.1.3 Exemplary Multispecific Antibodies

[0128] The present disclosure further provides multispecific antibodies, e.g., bispecific antibodies. Multispecific antibodies are antibody derivatives that have binding specificities for at least two different antigens or antigen epitopes. In certain embodiments, one of the binding specificities is for an epitope present on PDL1, and the other binding specificity is for an epitope present on a different antigen. In certain embodiments, one of the binding specificities is for an epitope present on CD47, and the other binding specificity is for an epitope present on a different antigen. In certain embodiments, the multispecific antibodies of the present disclosure can bind to an epitope on PDL1 and an epitope on CD47. In certain embodiments, the multispecific antibodies of the present disclosure can comprise a full-length antibody, an antibody fragment, and / or any combination thereof.

[0129] In certain embodiments, the multispecific antibodies disclosed herein bind to PDL1 and CD47. In certain embodiments, the multispecific antibodies are bispecific antibodies. In certain embodiments, the multispecific antibodies have at least two different binding specificities; see, e.g., U.S. Pat. Nos. 5,922,845 and 5,837,243; Zeilder (1999) J. Immunol. 163:1246-1252; Somasundaram (1999) Hum. Antibodies 9:47-54; Keler (1997) Cancer Res. 57:4008-401 4. For example, but not limited to, the presently disclosed subject matter provides multispecific antibodies comprising one antigen-binding moiety directed against a first epitope present on PDL1 and a second antigen-binding moiety directed against a second epitope present on CD47. In certain embodiments, the multispecific antibody comprises a first antigen-binding portion comprising an anti-PDL1 antibody disclosed herein and a second antigen-binding portion comprising an anti-CD47 antibody disclosed herein.

[0130] In certain embodiments, the anti-PDL1 / anti-CD47 antibodies disclosed herein can function as antagonists of PDL1 signaling and / or antagonists of CD47 signaling. In certain embodiments, without being bound by any theory, the anti-PDL1 / anti-CD47 antibodies can enhance the anti-tumor function of immune cells. In certain embodiments, treatment with the anti-PDL1 / anti-CD47 antibodies exhibits superior anti-tumor effects compared to treatment with a monospecific anti-PDL1 antibody or a monospecific anti-CD47 antibody. In certain embodiments, treatment with the anti-PDL1 / anti-CD47 antibodies exhibits superior anti-tumor effects compared to treatment with a combination of a monospecific anti-PDL1 antibody and a monospecific anti-CD47 antibody.

[0131] In certain embodiments, the anti-PDL1 / anti-CD47 multispecific antibody comprises a first antigen-binding portion comprising an anti-PDL1 antibody comprising a single-domain antibody that binds to PDL1, and a second antigen-binding portion comprising an anti-CD47 antibody that binds to CD47. In certain embodiments, the first antigen-binding portion comprises an anti-PDL1 antibody disclosed herein. In certain embodiments, the second antigen-binding portion comprises an anti-CD47 antibody disclosed herein.

[0132] In certain embodiments, the anti-PDL1 / anti-CD47 multispecific antibody may be a multivalent antibody. In certain embodiments, the multispecific antibody may be bivalent, trivalent, tetravalent, pentavalent, hexavalent, heptavalent, or octavalent. In certain embodiments, each of the first and second antigen-binding moieties of the anti-PDL1 / anti-CD47 antibody may be monovalent, bivalent, trivalent, tetravalent, pentavalent, hexavalent, heptavalent, or octavalent. In certain embodiments, each of the first and second antigen-binding moieties is monovalent. In certain embodiments, each of the first and second antigen-binding moieties is bivalent. In certain embodiments, the multispecific antibody is bivalent. In certain embodiments, the multispecific antibody is tetravalent. In certain embodiments, the multispecific antibody is hexavalent. In certain embodiments, the multispecific antibody is octavalent.

[0133] In certain embodiments, the second antigen-binding portion comprises an anti-CD47 antibody comprising two antibody heavy chains and two antibody light chains. In certain embodiments, the first antigen-binding portion comprises one or more anti-PDL1 antibodies. In certain embodiments, the first antigen-binding portion comprises two anti-PDL1 antibodies. In certain embodiments, the first antigen-binding portion comprises four anti-PDL1 antibodies. In certain embodiments, the C-terminus of at least one of the two anti-CD47 light chains is linked to the anti-PDL1 antibody of the first antigen-binding portion. In certain embodiments, the C-terminus of each of the two anti-CD47 light chains is linked to the anti-PDL1 antibody of the first antigen-binding portion. In certain embodiments, the N-terminus of at least one of the two anti-CD47 light chains is linked to the anti-PDL1 antibody of the first antigen-binding portion. In certain embodiments, the N-terminus of each of the two anti-CD47 light chains is linked to the anti-PDL1 antibody of the first antigen-binding portion. In certain embodiments, the C-terminus of at least one of the two anti-CD47 heavy chains is linked to the anti-PDL1 antibody of the first antigen-binding moiety. In certain embodiments, the C-terminus of each of the two anti-CD47 heavy chains is linked to the anti-PDL1 antibody of the first antigen-binding moiety. In certain embodiments, the N-terminus of at least one of the two anti-CD47 heavy chains is linked to the anti-PDL1 antibody of the first antigen-binding moiety. In certain embodiments, the N-terminus of each of the two anti-CD47 heavy chains is linked to the anti-PDL1 antibody of the first antigen-binding moiety.

[0134] In certain embodiments, the multispecific antibody comprises: i) a first antigen-binding portion comprising a single-domain anti-PDL1 antibody comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53; and ii) a second antigen-binding portion comprising an anti-CD47 antibody comprising a heavy chain variable region (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 71, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 72, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 73; and a light chain variable region (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 74, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 75, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 76.

[0135] In certain embodiments, the multispecific antibody comprises: i) a first antigen-binding portion comprising a single-domain anti-PDL1 antibody comprising a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53; and ii) a second antigen-binding portion comprising an anti-CD47 antibody comprising a heavy chain variable region (VH) sequence comprising: (1) a CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 81, (2) a CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 82, and (3) a CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 83; and a light chain variable region (VL) sequence comprising: (1) a CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 84, (2) a CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 85, and (3) a CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 86.

[0136] In certain embodiments, the multispecific antibody comprises i) a first antigen-binding portion comprising a single-domain anti-PDL1 antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 54, and ii) a second antigen-binding portion comprising an anti-CD47 antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 77 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 78.

[0137] In certain embodiments, the multispecific antibody comprises i) a first antigen-binding portion comprising a single-domain anti-PDL1 antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 54, and ii) a second antigen-binding portion comprising an anti-CD47 antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 87 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 88.

[0138] In certain embodiments, the first antigen-binding portion is linked to the second antigen-binding portion via a linker. In certain embodiments, the linker is a peptide linker. In certain embodiments, the peptide linker comprises about 4 to about 30 amino acids. In certain embodiments, the peptide linker comprises about 4 to about 15 amino acids. In certain embodiments, the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 93-135.

[0139] In certain embodiments, the anti-CD47 antibody of the second antigen-binding portion comprises an Fc region. In certain embodiments, the Fc region comprises a human Fc region. In certain embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG, IgA, IgD, IgE, and IgM Fc regions. In certain embodiments, the Fc region comprises an Fc region selected from the group consisting of IgG1, IgG2, IgG3, and IgG4 Fc regions. In certain embodiments, the Fc region comprises an IgG1 Fc region. In certain embodiments, the Fc region comprises an IgG2 Fc region. In certain embodiments, the Fc region comprises an IgG4 Fc region. In certain embodiments, the Fc region comprises one or more amino acid modifications, substitutions, or mutations described in Section 2.7.3.

[0140] In certain embodiments, the anti-PDL1 antibody of the first antigen-binding portion comprises a humanized framework. In certain embodiments, the humanized framework comprises the framework sequence of the heavy chain variable region sequence of SEQ ID NO: 9 or 14.

[0141] In certain embodiments, the anti-CD47 antibody of the second antigen-binding portion comprises a human antibody. In certain embodiments, the anti-CD47 antibody of the second antigen-binding portion comprises a humanized antibody comprising a humanized framework.

[0142] In certain embodiments, the multispecific antibody comprises a first chain comprising the amino acid sequence set forth in SEQ ID NO: 136 and a second chain comprising the amino acid sequence set forth in SEQ ID NO: 137. In certain embodiments, the multispecific antibody comprises a first chain comprising the amino acid sequence set forth in SEQ ID NO: 138 and a second chain comprising the amino acid sequence set forth in SEQ ID NO: 139. In certain embodiments, the multispecific antibody comprises a first chain comprising the amino acid sequence set forth in SEQ ID NO: 140 and a second chain comprising the amino acid sequence set forth in SEQ ID NO: 141.

[0143] 2.2 Antibody affinity

[0144] In certain embodiments, the antibodies or antibody derivatives disclosed herein have high binding affinity for their target antigens. In certain embodiments, the antibodies or antibody derivatives have a binding affinity of about 1×10 -7 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -8 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 5×10 -9 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -9 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -10 It binds to the target with a KD of less than or equal to M.

[0145] In certain embodiments, the antibody or antibody derivative is about 1×10 -11 M ~ approx. 1×10 -7 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -10 M ~ approx. 1×10 -7 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -10 M ~ approx. 1×10 -8 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -11 M ~ approx. 1×10 -9 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 2×10 -10 M ~ approx. 5×10 -9 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -9 M ~ approx. 5×10 -8 In certain embodiments, the antibody or antibody derivative binds to the target with a KD of about 1×10 -10 M ~ approx. 1×10 -9 It binds to the target with a KD of M.

[0146] The KD of the antibody or antibody derivative may be determined by methods well known in the art, including, but not limited to, Western blot, ELISA test, RIA test, ECL test, IRMA test, EIA test, Octet-BIACORE® test, and peptide scan.

[0147] In certain embodiments, KD can be measured using a BIACORE® surface plasmon resonance assay. For example, but not limited to, assays using a BIACORE®-2000 or BIACORE® 3000 (Biacore, Inc., Piscataway, NJ) are performed at 25°C using an immobilized antigen CMS chip at approximately 10 response units (RU). In certain embodiments, a carboxymethylated dextran biosensor chip (CMS, Biacore, Inc.) is activated with N-ethyl-N'-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's instructions. The antigen is diluted to 5 μg / mL (approximately 0.2 μM) with 10 mM sodium acetate, pH 4.8, and then injected at a flow rate of 5 μL / min, yielding approximately 10 response units (RU) of coupled protein. After injection of the antigen, 1 M ethanolamine is injected to block any unreacted groups. For kinetic measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS containing 0.05% polysorbate 20 (TWEEN®-20™) surfactant (PBST) at 25°C at a flow rate of approximately 25 μL / min. The association rate (k on ) and dissociation rate (k off) is calculated by simultaneously fitting the association and dissociation sensorgrams using a simple one-to-one Langmuir binding model (BIACORE® Evaluation Software Version 3.2). The equilibrium dissociation constant (KD) can be calculated as the ratio of koff / kon. See, e.g., Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate by the surface plasmon resonance assay described above is 10 6 M -l s -1 If the on-rate exceeds 0.05, the on-rate may be determined by a fluorescence quenching technique, which measures the increase or decrease in fluorescence emission intensity (excitation = 295 nm, emission = 340 nm, 16 nm bandpass) of 20 nM anti-antigen antibody (Fab form) in PBS (pH 7.2) at 25°C in the presence of increasing antigen concentrations, using, for example, a spectrophotometer (Aviv Instruments) equipped with stopped flow or an 8000 series SLM-AMINCO with a stirred cuvette. TM It is measured using a spectrometer such as a ThermoSpectronic spectrophotometer.

[0148] 2.3 Antibody fragments

[0149] In certain embodiments, antibodies of the present disclosure include antigen-binding fragments or antibody fragments. Antibody fragments include, but are not limited to, Fab, Fab', Fab'-SH, F(ab'), VHH, Fv, and scFv fragments, as well as other fragments described herein. For a review of certain antibody fragments, see Hudson et al., Nat. Med. 9:129-134 (2003). For a review of scFv fragments, see, e.g., Pluckthin in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., (Springer-Verlag, New York), pp. 269-315 (1994); see also WO 93 / 16185 and U.S. Pat. Nos. 5,571,894 and 5,587,458. See US Pat. No. 5,869,046 for a discussion of Fab and F(ab)2 fragments that contain salvage receptor binding epitope residues and have increased in vivo half-lives.

[0150] In certain embodiments, the antibodies of the present disclosure can be diabodies. Diabodies are antibody fragments with two antigen-binding sites, which may be bivalent or bispecific. See, e.g., 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).

[0151] In certain embodiments, an antibody of the present disclosure may comprise a single-domain antibody. A single-domain antibody is an antibody fragment comprising all or part of the heavy chain variable region or all or part of the light chain variable region of an antibody. In certain embodiments, the single-domain antibody is a human single-domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516 B1). In certain embodiments, the single-domain antibody is a camelid single-domain antibody. In certain embodiments, the single-domain antibody is a VHH. In certain embodiments, the single-domain antibody is humanized.

[0152] Antibody fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies as well as production by recombinant host cells (e.g., E. coli or phage), as described herein.

[0153] 2.4 Chimeric and humanized antibodies

[0154] In certain embodiments, the antibody of the present disclosure is a chimeric antibody. Certain 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 certain embodiments, a chimeric antibody comprises a non-human variable region (e.g., a variable region derived from a mouse) and a human constant region. In certain embodiments, a chimeric antibody is a "class-switched" antibody in which the class or subclass has been changed from that of the parent antibody. Chimeric antibodies include antigen-binding fragments thereof.

[0155] In certain embodiments, an antibody of the present disclosure can be a humanized antibody. Typically, a non-human antibody is humanized to reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody. Generally, a humanized antibody comprises one or more variable domains in which the HVRs, e.g., CDRs (or portions thereof), are derived from a non-human antibody and one or more framework regions (FRs) (or any portion thereof) are derived from a human antibody sequence. A humanized antibody can optionally also comprise at least a portion of a human constant region. In certain embodiments, certain FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (e.g., the antibody from which the HVR residues are derived), e.g., to restore or improve antibody specificity or affinity.

[0156] Humanized antibodies and methods for producing them are described, for example, in Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and further described, 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) (describing SDR (a-CDR) grafting), Padlan, Mol. Immunol. 28:489-498 (1991) (describing "resurfacing"), Dall'Acqua et al., Methods 36:43-60 (2005) (describing "FR shuffling"), and Osbourn et al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer, 83:252-260 (2000) (describing a "guided selection" approach for FR shuffling).

[0157] Human framework regions that can be used for humanization include framework regions selected using the "best-fit" method (see, e.g., Sims et al., J. Immunol. 151:2296 (1993)), framework regions derived from consensus sequences of human antibodies of a particular subgroup of light 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)), and human framework regions derived from consensus sequences of human antibodies of a particular subgroup of light 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)). Examples of framework regions that may be used include, but are not limited to, human mature (somatically mutated) 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 (1997) and Rosok et al., J. Biol. Chem. 271:22611-22618 (1996)).

[0158] 2.5 Human antibodies

[0159] In certain embodiments, an antibody of the present disclosure can be a human antibody (e.g., a human domain antibody, or human DAb). Human antibodies can be produced using a variety of techniques well 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 well known in the art. See, for example, US20090307787A1, U.S. Patent No. 8,754,287, US20150289489A1, US20100122358A1, and WO2004049794.

[0160] Human antibodies (e.g., human DAbs) can be prepared by administering an immunogen to a transgenic animal modified to produce intact human antibodies or intact antibodies with human variable regions in response to antigen challenge. Such animals typically contain all or part of human immunoglobulin loci that replace endogenous immunoglobulin loci or that are present extrachromosomally or randomly integrated into the animal's chromosomes. In such transgenic mice, the endogenous immunoglobulin loci are typically inactivated. For a review of methods for obtaining human antibodies from transgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). See, e.g., XENOMOUSE. TM US Patent Nos. 6,075,181 and 6,150,584, which describe the technology, and HuMab (登録商標) The technology is described in U.S. Patent No. 5,770,429, KM MOUSE (登録商標) U.S. Patent No. 7,041,870, which describes the technology, and VelociMouse (登録商標) See also U.S. Patent Publication No. US2007 / 0061900, which describes the technology. The human variable regions from intact antibodies produced by such animals can be further modified, for example, by combining with different human constant regions.

[0161] Human antibodies (e.g., human DAbs) can also be produced by hybridoma-based methods. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described (see, 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 generated via human B cell hybridoma technology are also described in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006). Further methods include those described, for example, in U.S. Patent No. 7,189,826 (describing the production of monoclonal human IgM antibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue, 26(4):265-268 (2006) (describing 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).

[0162] Human antibodies (e.g., human DAbs) can also be generated by isolating Fv clone variable domain sequences selected from human-derived phage display libraries. Such variable domain sequences can then be combined with desired human constant domains. Techniques for selecting human antibodies from antibody libraries are described below.

[0163] 2.6 Library-derived antibodies

[0164] Antibodies of the present disclosure can be isolated by screening combinatorial libraries for antibodies with the desired activity. For example, various methods are well known in the art for generating phage display libraries and screening such libraries for antibodies with the desired binding characteristics. Such methods are described, for example, by Hoogenboom et al., in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and further described, for example, by 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, in 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. 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 have been described, see, for example, U.S. Patent No. 7,371,849.

[0165] In certain phage display methods, V H and V LGene repertoires can be separately cloned by polymerase chain reaction (PCR), randomly recombined into phage libraries, and then screened for antigen-binding phage, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phage typically display antibody fragments as either scFv or Fab fragments. Libraries from immunized sources provide high-affinity antibodies to immunogens without the need for hybridoma construction. Alternatively, naive repertoires can be cloned (e.g., from humans) to provide single-source antibodies to a wide range of non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J., 12:725-734 (1993). Finally, naive libraries can also be synthetically generated by cloning unrearranged V gene segments from stem cells and using PCR primers containing random sequences to encode hypervariable CDR3 regions and perform rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, U.S. Patent No. 5,750,373, and U.S. Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0166] Antibodies or antibody fragments isolated from a human antibody library are considered human antibodies or human antibody fragments herein.

[0167] 2.7 Antibody variants

[0168] The present disclosure further provides amino acid sequence variants of the disclosed antibodies. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody, or by peptide synthesis. Such modifications include, but are not limited to, deletions from, and / or insertions into, and / or substitutions of, residues within the amino acid sequence of the antibody. Any combination of deletion, insertion, and substitution can be used to arrive at the final construct, provided that the final antibody, i.e., the modified antibody, possesses the desired properties, e.g., antigen binding.

[0169] 2.7.1 Substitution, Insertion, and Deletion Mutants

[0170] In certain embodiments, antibody variants are provided that have one or more amino acid substitutions. Sites of interest for substitutional mutagenesis include HVRs (or CDRs) and FRs. Conservative substitutions are shown in Table 2 under the heading of "Preferred Substitutions." More substantial changes are provided in Table 2 under the heading of "Exemplary Substitutions," and as further described below with reference to amino acid side chain classes. Amino acid substitutions can be introduced into the antibody of interest, and the products screened for the desired activity, e.g., retained / improved antigen binding, reduced immunogenicity, or improved ADCC or CDC.

[0171] [Table 2]

[0172] Amino acids can be grouped according to common side 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 that affect chain orientation: Gly, Pro, and (6) aromatic: Trp, Tyr, Phe. In certain embodiments, non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0173] In certain embodiments, one type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variant selected for further study will have a modification (e.g., an improvement) in a particular biological property (e.g., increased affinity, decreased immunogenicity) compared to the parent antibody and / or will have substantially retained a particular biological property of the parent antibody. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using, for example, phage-display-based affinity maturation techniques as described herein. Briefly, one or more HVR (or CDR) residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0174] Modifications (e.g., substitutions) can be made in HVRs (or CDRs), for example, to improve antibody affinity. Such modifications can be made in HVR (or CDR) "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or SDRs (a-CDRs), and the resulting variant VH or VL are tested for binding affinity. Affinity maturation by constructing and reselecting from secondary libraries is 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)). In certain embodiments of affinity maturation, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then constructed. This library is then screened to identify any antibody variants with the desired affinity. Another method for introducing diversity involves an HVR (or CDR)-directed approach, in which several HVR (or CDR) residues (e.g., 4 to 6 residues at a time) are randomized. HVR (or CDR) residues involved in antigen binding can be specifically identified, for example, using alanine scanning mutagenesis or modeling. CDR-H3 and CDR-L3, in particular, are often targeted.

[0175] In certain embodiments, substitutions, insertions, or deletions may be made within one or more HVRs (or CDRs) so long as such modifications do not substantially reduce the ability of the antibody to bind to the antigen. For example, conservative modifications (e.g., conservative substitutions provided herein) that do not substantially reduce binding affinity can be made in an HVR (or CDR). Such modifications may be in HVR (or CDR) "hot spots" or outside of the CDRs. In certain embodiments of the above-described variant VHH sequences, each HVR (or CDR) is either unaltered or has no more than one, two, or three amino acid substitutions.

[0176] A useful method for identifying antibody residues or regions that can be targeted for mutagenesis is called "alanine scanning mutagenesis," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, a residue or group of target residues (e.g., charged residues, e.g., Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (e.g., alanine or polyalanine) to determine whether the interaction of the antibody with the antigen is affected. Further substitutions may be introduced at amino acid positions that demonstrate functional sensitivity to the initial substitution. Alternatively, or in addition, a crystal structure of an antigen-antibody complex may be used to identify contact points between the antibody and the antigen. Such contact and adjacent residues may be targeted as candidates for substitution or removed. Mutants may be screened to determine whether they possess desired properties.

[0177] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. An example of a terminal insertion is an antibody with an N-terminal methionyl residue. Other insertional variants of the antibody molecule include the N- or C-terminal fusion of the antibody to an enzyme (e.g., for ADEPT) or a polypeptide which increases the serum half-life of the antibody.

[0178] 2.7.2 Glycosylation variants

[0179] In certain embodiments, antibodies are modified to increase or decrease the degree of glycosylation of the construct. Addition or deletion of glycosylation sites to an antibody can be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed.

[0180] If the antibody comprises an Fc region (e.g., scFv-Fc), the carbohydrate attached thereto can be modified. Natural antibodies produced by mammalian cells typically have the C of the Fc region attached by an N-linkage. H The antibody comprises a branched, biantennary oligosaccharide, typically attached to Asn297 of the 2 domain. See, e.g., Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide may include various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, and sialic acid, as well as a fucose attached to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In certain embodiments, modifications of the oligosaccharides in the antibody can be made to generate antibody variants with certain improved properties.

[0181] In certain embodiments, the antibody has a carbohydrate structure lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such an antibody can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose is determined by calculating the average amount of fucose in the glycan at Asn297 relative to the sum of all glycan structures (e.g., complex, hybrid, and high-mannose structures) attached to Asn297, as measured, for example, by MALDI-TOF mass spectrometry as described in WO 2008 / 077546. Asn297 refers to an asparagine residue located at approximately position 297 (EU numbering of Fc region residues) within the Fc region; however, Asn297 may also be located approximately ±3 amino acids upstream or downstream from position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function. See, e.g., U.S. Patent Publication Nos. US2003 / 0157108 (Presta, L.), US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications relating 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, WO2005 / 053742, WO2002 / 031140, Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004), Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004).Examples of cell lines capable of producing defucosylated antibodies include protein fucosylation-deficient Lec13 CHO cells (Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); U.S. Patent Application No. US 2003 / 0157108 A1, Presta, L; and WO 2004 / 056312 A1, Adams et al.), and knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g., Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO 2003 / 085107).

[0182] In certain embodiments, the antibody has a bisected oligosaccharide, e.g., a biantennary 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. Patent No. 6,602,684 (Umana et al.), and US 2005 / 0123546 (Umana et al.). Antibody variants having at least one galactose residue in the oligosaccharide attached to the Fc region are also provided. 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.).

[0183] 2.7.3 Fc Region Variants

[0184] In certain embodiments, the Fc region of an antibody or antibody derivative disclosed herein can comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) comprising an amino acid modification (e.g., a substitution) at one or more amino acid positions. In certain embodiments, one or more amino acid modifications can be introduced into the Fc region of an antibody portion (e.g., an IgG, scFv-Fc, or VHH-Fc), thereby generating an Fc region variant.

[0185] In certain embodiments, the Fc region possesses some, but not all, effector functions, making it a desirable candidate for uses in which the half-life of the antibody in vivo is important, but certain effector functions (e.g., complement and ADCC) are unnecessary or deleterious. In vitro and / or in vivo cytotoxicity assays can be performed to confirm reduced / depleted CDC and / or ADCC activity. For example, Fc receptor (FcR) binding assays can be performed to ensure that the antibody lacks FcR binding (and thus likely lacks ADCC activity) but retains FcRn binding ability. NK cells, the primary cells for mediating ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest are described in U.S. Patent No. 5,500,362 (see, e.g., 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), 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). TMNon-radioactive cytotoxicity assay (CellTechnology, Inc. Mountain View, CA), and CytoTox 96 (登録商標) (See non-radioactive cytotoxicity assays (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest can be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays can be performed to confirm that the antibody is unable to bind C1q and therefore lacks CDC activity. See, e.g., C1q and C3c binding ELISAs described in WO 2006 / 029879 and WO 2005 / 100402. To assess complement activation, a CDC assay may be performed (see, e.g., 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 measurements can also be performed using methods well known in the art (see, e.g., Petkova, SB et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0186] Antibodies with reduced effector function include those with substitutions at one or more of Fc region residues 238, 265, 269, 270, 297, 327, and 329 (U.S. Patent No. 6,737,056). Such Fc variants include Fc variants with substitutions at two or more of amino acid positions 265, 269, 270, 297, and 327, including the so-called "DANA" Fc variant in which residues 265 and 297 are substituted with alanine (U.S. Patent No. 7,332,581).

[0187] Certain antibody variants have been described that have improved or diminished binding to FcRs (see, e.g., U.S. Pat. No. 6,737,056, WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2):6591-6604 (2001)).

[0188] In certain embodiments, the Fc region comprises one or more mutations according to EU numbering of residues. In certain embodiments, the Fc region is an IgG1 Fc region. In certain embodiments, the Fc region is an IgG2 or IgG4 Fc region.

[0189] In certain embodiments, the IgG1 or IgG4 Fc region comprises one or more mutations that modify effector function. In certain embodiments, the IgG1 Fc region comprises an L234A mutation and / or an L235A mutation. In certain embodiments, the IgG4 Fc region comprises an F234A mutation and / or an L235A mutation. In certain embodiments, the Fc region comprises a substitution at position 297, e.g., N297A, N297Q, or N297G.

[0190] In certain embodiments, the IgG1 Fc region comprises one or more mutations that modify antibody-dependent cellular cytotoxicity (ADCC). In certain embodiments, the IgG1 Fc region comprises one or more mutations that reduce antibody-dependent cellular cytotoxicity (ADCC). In certain embodiments, the IgG1 Fc region comprises one or more mutations that enhance antibody-dependent cellular cytotoxicity (ADCC). In certain embodiments, the IgG1 Fc region comprises the following mutations: L235V, F243L, R292P, Y300L, and P396L. In certain embodiments, the IgG1 Fc region comprises the following mutations: S239D, A330L, and I332E. In certain embodiments, the IgG1 Fc region comprises the following mutations: L235V, F243L, R292P, and Y300L. In certain embodiments, the IgG1 Fc region comprises substitutions at positions 298, 333, and / or 334 of the Fc region, for example, S298A, E333A, and K334A.

[0191] In certain embodiments, the Fc region comprises one or more mutations that modify coupling with an Fc receptor, e.g., FcγRIIa and / or FcγRIIb. In certain embodiments, the Fc region comprises one or more mutations that enhance coupling with an Fc receptor. In certain embodiments, the Fc region comprises one or more mutations that enhance coupling with FcγRIIa, FcγRIIb, or a combination thereof. In certain embodiments, the Fc region comprises the S267E and L328F mutations. In certain embodiments, the Fc region comprises the N325S and L328F mutations.

[0192] In certain embodiments, the Fc region comprises an IgG4 Fc region comprising an S228P mutation.

[0193] In certain embodiments, the Fc region comprises knob-in-hole mutations, which allow two different antibody chains to form heterodimers, resulting in a multispecific antibody. In certain embodiments, the multispecific antibody disclosed herein comprises knob-in-hole mutations selected from the group consisting of T366S, L368A, T366W, Y349C, Y407V, S354C, and any combination thereof. In certain embodiments, the multispecific antibody comprises knob-in-hole mutations T366S and L368A in the hole chain and a knob-in-hole mutation T366W in the knob chain. In certain embodiments, the multispecific antibody comprises knob-in-hole mutations T366S, L368A, and Y407V in the hole chain and a knob-in-hole mutation T366W in the knob chain. In certain embodiments, said multispecific antibody comprises knob-in-hole mutations Y349C, T366S, L368A and Y407V in the hole chain, and knob-in-hole mutations S354C and T366W in the knob chain.

[0194] In certain embodiments, modifications are made in the Fc region that result in altered (i.e., either improved or reduced) C1q binding and / or complement dependent cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 1999 / 51642, and Idusogie et al., J. Immunol. 164:4178-4184 (2000).

[0195] In certain embodiments, antibody (e.g., scFv-Fc or VHH-Fc) variants comprise a variant Fc region containing one or more amino acid substitutions that alter half-life and / or binding to fetal Fc receptor (FcRn). Antibodies with extended half-life and improved binding to fetal Fc receptor (FcRn), which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)), are described in US2005 / 0014934A1 (Hinton et al.). These antibodies comprise an Fc region with one or more substitutions that alter binding of the Fc region to FcRn. Such Fc variants include those with substitutions at one or more Fc region residues, for example, substitutions at Fc region residue 434 (U.S. Patent No. 7,371,826). In certain embodiments, the Fc region comprises the following mutations: M428L and N434S. In certain embodiments, the Fc region comprises the following mutations: M252Y, S254T, and T256E.

[0196] In certain embodiments, the Fc region comprises an Fc region variant described in Duncan & Winter, Nature 322:738-40 (1988), Wang et al., Protein Cell 2018,9(1):63-73, U.S. Patent No. 5,648,260, U.S. Patent No. 5,624,821, and WO 1994 / 29351.

[0197] 2.7.4 Cysteine ​​Engineered Antibody Variants

[0198] In certain embodiments, it is desirable to generate cysteine ​​engineered antibody moieties, e.g., "thioMAbs," in which one or more residues of an antibody are substituted with cysteine ​​residues. In certain embodiments, the substituted residues occur at accessible sites of the antibody. By replacing these residues with cysteine, reactive thiol groups are positioned at accessible sites of the antibody, which may be used to conjugate the antibody to other moieties, such as drug moieties or linker-drug moieties, to generate immunoconjugates, as further described herein. In certain embodiments, any one or more of the following residues can be substituted with cysteine: A118 (EU numbering) of the heavy chain, and S400 (EU numbering) of the heavy chain Fc region. Cysteine ​​engineered antibody moieties can be generated, for example, as described in U.S. Pat. No. 7,521,541.

[0199] 2.8 Antibody derivatives

[0200] In certain embodiments, the antibodies described herein may be further modified into antibody derivatives containing other protein or non-protein moieties that are well known and readily available in the art. Non-protein moieties suitable for derivatizing antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, propylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may be advantageous during manufacturing due to its stability in water. The polymers may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody may vary, and if more than one polymer is attached, they may be the same or different molecules. In general, the number and / or type of polymers used for derivatization may be determined based on considerations including, but not limited to, the particular property or function of the antibody to be improved, whether the antibody derivative will be used in diagnosis under limited conditions, etc.

[0201] In certain embodiments, antibodies may be further modified into antibody derivatives that contain one or more biologically active proteins, polypeptides, or fragments thereof. As used interchangeably herein, "biological activity" or "biologically active" refers to exhibiting biological activity to perform a specific function in the body. For example, it can refer to binding to a specific biomolecule, such as a protein or DNA, and then promoting or inhibiting the activity of such a biomolecule. In certain embodiments, biologically active proteins or fragments thereof include proteins and polypeptides administered to a patient as active drug substances for the prevention or treatment of a disease or condition, proteins and polypeptides used for diagnostic purposes, such as enzymes used in diagnostic tests or in vitro assays, and proteins and polypeptides administered to a patient to prevent a disease, such as vaccines.

[0202] 2.9 Production Method

[0203] The antibodies and antibody derivatives disclosed herein can be produced using any technique available or known in the art. For example, but not limited to, the antibodies and antibody derivatives can be produced using recombinant methods and compositions, such as those described in U.S. Patent No. 4,816,567. Detailed procedures for producing the antibodies and antibody derivatives are described in the Examples below.

[0204] The presently disclosed subject matter further provides an isolated nucleic acid encoding an antibody or antibody derivative disclosed herein. For example, the isolated nucleic acid can encode an amino acid sequence comprising the VL and / or the VH of the antibody, e.g., the light chain and / or the heavy chain of the antibody.

[0205] In certain embodiments, the nucleic acid can 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 has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop into which another DNA segment can be ligated. Another type of vector is a viral vector, in which another DNA segment can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of the host cell after introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors, expression vectors, are capable of directing the expression of genes to which they are operably linked. In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids (vectors). However, the disclosed subject matter is intended to include other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0206] Different portions of the antibodies or antibody derivatives disclosed herein can be assembled into a single multicistronic expression cassette, multiple expression cassettes in a single vector, or multiple vectors. Examples of elements that create polycistronic expression cassettes include, but are not limited to, various viral and non-viral internal ribosome entry sites (IRESs, e.g., FGF-1 IRES, FGF-2 IRES, VEGF IRES, IGF-II IRES, NF-kB IRES, RUNX1 IRES, p53 IRES, Hepatitis A IRES, Hepatitis C IRES, Pestivirus IRES, Aphthovirus IRES, Picornavirus IRES, Poliovirus IRES, and Encephalomyocarditis virus IRES) and cleavable linkers (e.g., 2A peptides, e.g., P2A, T2A, E2A, and F2A peptides). Also suitable are retroviral vectors in combination with appropriate packaging lines, where the capsid protein is functional for infecting human cells. A variety of amphotropic virus-producing cell lines are known, including, but 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). Nonamphotropic particles are also suitable, such as pseudotyped particles with VSVG, RD114, or GALV envelopes, and any others known in the art.

[0207] In certain embodiments, a nucleic acid encoding an antibody or antibody derivative of the present disclosure and / or one or more vectors comprising such a nucleic acid can be introduced into a host cell. In certain embodiments, introduction of a nucleic acid into a cell can be carried out by any method known in the art, including, but not limited to, transfection, electroporation, microinjection, infection with a nucleic acid sequence-containing viral or bacteriophage vector, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. In certain embodiments, a host cell can comprise, for example, a host cell transformed with a vector comprising a nucleic acid encoding a single domain antibody and / or an amino acid sequence comprising the VH of a single domain antibody. In certain embodiments, a host cell can comprise, for example, a host cell that has been transformed with (1) a vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and an amino acid sequence comprising the VH of an antibody, or (2) a first vector comprising a nucleic acid encoding an amino acid sequence comprising the VL of an antibody and a second vector comprising a nucleic acid encoding an amino acid sequence comprising the VH of an antibody. In certain embodiments, the host cell is a eukaryotic cell, such as a Chinese hamster ovary (CHO) cell, or a lymphoid cell (eg, YO, NSO, Sp20 cell).

[0208] In certain embodiments, methods for producing an antibody or antibody derivative disclosed herein include culturing a host cell into which nucleic acid encoding the antibody or antibody derivative has been introduced under conditions suitable for expression of the antibody or antibody derivative, and optionally recovering the antibody or antibody derivative from the host cell and / or host cell culture medium. In certain embodiments, the antibody or antibody derivative is recovered from the host cell by chromatographic techniques.

[0209] For recombinant production of the antibodies or antibody derivatives of the present disclosure, for example, nucleic acids encoding the antibodies or antibody derivatives described above can be isolated and inserted into one or more vectors for further cloning and / or expression in host cells. Such nucleic acids can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody or antibody derivative). Suitable host cells for cloning or expressing antibody-encoding vectors include the prokaryotic and eukaryotic cells described herein. For example, antibodies or antibody derivatives can be produced in bacteria, particularly when glycosylation and Fc effector functions are not required. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Patent Nos. 5,648,237, 5,789,199, and 5,840,523. (See also Charlton, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ, 2003), pp. 245-254, which describes the expression of antibody fragments in E. coli.) After expression, the antibody or antibody derivative can be isolated from the bacterial cell paste in a soluble fraction and further purified.

[0210] In addition to prokaryotes, eukaryotic microbes, such as filamentous fungi or yeast, are suitable cloning or expression hosts for antibody-encoding vectors, including fungal and yeast strains in which the glycosylation pathway has been "humanized" to produce antibodies or antibody derivatives with partial or fully human glycosylation patterns. See Gemgross, Nat. Biotech. 22:1409-1414 (2004) and Li et al., Nat. Biotech. 24:210-215 (2006). Suitable host cells for the expression of glycosylated antibodies can also be derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains have been identified and can be used in combination with insect cells, particularly for transfection of Spodoptera frugiperda cells. In certain embodiments, plant cell cultures can be used as host cells. See, e.g., 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). TM (which describes the technology).

[0211] In certain embodiments, vertebrate cells can also be used as hosts, for example, but not limited to, mammalian cell lines adapted to suspension growth may be useful. Non-limiting examples of useful mammalian host cell lines include monkey kidney CV1 cells transformed with SY40 (COS-7), human embryonic kidney cells (e.g., 293 or 293 cells as described in Graham et al., J Gen Viral. 36:59 (1977)), baby hamster kidney cells (BHK), mouse Sertoli cells (e.g., TM4 cells as described in Mather, Biol. Reprod. 23:243-251 (1980)), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep 02), mouse mammary tumor (MMT 060562), e.g., Mather et al., Annals Examples of useful mammalian host cell lines include TRI cells, MRC 5 cells, and FS4 cells, as described in NYAcad. Sci. 383:44-68 (1982). Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFK CHO cells (Urlaub et al., Proc. Natl. Acad. Sci. USA 77:42 I6 (1980)), and myeloma cell lines such as YO, NSO, and Sp2 / 0. For a review of certain mammalian host cells suitable for the production of antibodies or antibody derivatives, see, e.g., Yazaki and Wu, Methods in Molecular Biology, Vol. 248 (BKC Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003).

[0212] In certain embodiments, techniques for generating bispecific and / or multispecific antibodies include, but are not limited to, recombinant expression of two immunoglobulin heavy-light chain pairs with the same specificity, where one or two of the heavy or light chains are fused to antigen-binding moieties with different specificities (e.g., single-domain antibodies, e.g., VHHs), recombinant coexpression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein and Cuello, Nature 305:537 (1983)), PCT Patent Application No. WO 93 / 08829, and Traunecker et al., EMBO J 10:3655 (1991)), and "knobs-in-holes" engineering (see, e.g., U.S. Pat. No. 5,731,168). Bispecific antibodies can also be generated by engineering electrostatic steering effects (WO 93 / 08829) to generate antibody Fc-heterodimeric molecules. 2009 / 089004A1), cross-linking two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al., Science, 229:81 (1985)), using leucine zippers to produce bispecific antibodies (see, e.g., Kostelny et al., J. Immunol., 148(5):1547-1553 (1992)), use of "diabody" technology to make bispecific antibody fragments (see, e.g., Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)), and the use of single-chain Fv (sFv) dimers (see, e.g., Gruber et al., J. Immunol., 152:5368 (1994)), and, for example, Tutt et al., J. Immunol., 152:5368 (1994)). It can also be produced by the preparation of trispecific antibodies as described in Immunol. 147:60 (1991).

[0213] Bispecific and multispecific molecules of the present disclosure can also be produced using chemical techniques (see, e.g., Kranz (1981) Proc. Natl. Acad. Sci. USA 78:5807), "polydoma" technology (see, e.g., U.S. Pat. No. 4,474,893), or recombinant DNA technology. Bispecific and multispecific molecules of the presently disclosed subject matter can also be prepared by conjugating the constituent binding specificities, e.g., the binding specificities of a first epitope and a second epitope, using methods well known in the art and described herein. For example, but not limited to, each binding specificity of the bispecific and multispecific molecules can be produced together by recombinant fusion protein technology or can be produced separately and then conjugated to one another. When the binding specificities are proteins or peptides, a variety of coupling or cross-linking agents can be used for covalent conjugation. Non-limiting examples of cross-linking 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, e.g., Karpovsky (1984) J. Exp. Med. 160:1686; Liu (1985) Proc. Natl. Acad. Sci. USA 82:8648). Other methods include those described by Paulus (Behring Ins. Mitt. (1985) No. 78, 118-132; Brennan (1985) Science 229:81-83; Glennie (1987) J Immunol. 139:2367-2375). When the binding specificities are antibodies (e.g., two humanized antibodies), they can be conjugated via sulfhydryl bonds in the C-terminal hinge regions of the two heavy chains. In certain embodiments, the hinge region can be modified to contain an odd number of sulfhydryl residues, e.g., one, prior to conjugation.

[0214] In certain embodiments, both binding specificities of a bispecific antibody can be encoded in the same vector and expressed and assembled in the same host cell. This 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 certain embodiments, the bispecific antibodies of the present disclosure can be single-chain molecules, such as single-chain bispecific antibodies, single-chain bispecific molecules comprising one single-chain antibody and a binding-determining cluster, or single-chain bispecific molecules comprising two binding-determining clusters. Bispecific and multispecific molecules can also be single-chain molecules or comprise at least two single-chain molecules. Methods for preparing bispecific and multispecific molecules are described, for example, in U.S. Patent No. 5,260,203, U.S. Patent No. 5,455,030, U.S. Patent No. 4,881,175, U.S. Patent No. 5,132,405, U.S. Patent No. 5,091,513, U.S. Patent No. 5,476,786, U.S. Patent No. 5,013,653, U.S. Patent No. 5,258,498, and U.S. Patent No. 5,482,858. Engineered antibodies with three or more functional antigen-binding sites (e.g., epitope-binding sites), including "octopus antibodies," are also included herein (see, e.g., US 2006 / 0025576A1).

[0215] In certain embodiments, animal systems can be used to produce the antibodies or antibody derivatives of the present disclosure. One animal system for preparing hybridomas is the murine system.

[0216] Hybridoma production in mice is a very well-established procedure. Immunization protocols and techniques for isolating immunized splenocytes are well known in the art. 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).

[0217] 2.10 Assay

[0218] The antibodies and antibody derivatives of the present disclosure provided herein can be identified, screened, or characterized for their physical / chemical properties and / or biological activity by various assays well known in the art and provided herein.

[0219] In certain embodiments, the antigen-binding activity of an antibody or antibody derivative of the present disclosure can be tested by well-known methods, such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or Western blot assay. Each of these assays typically detects the presence of a particular protein-antibody complex of interest by using a labeled reagent (e.g., an antibody) specific for the complex of interest. For example, the antibody or antibody derivative can be detected using, for example, an enzyme-linked antibody or antibody fragment that recognizes and specifically binds to the antibody or antibody derivative. Alternatively, the antibody or antibody derivative can be detected using any of a variety of other immunoassays. For example, the antibody or antibody derivative can be radioactively labeled and used in a radioimmunoassay (RIA) (see, e.g., Weintraub, B., Principles of Radioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, The Endocrine Society, March 1986, incorporated herein by reference). The radioactive isotope can be detected by such means as the use of a Geiger counter or a scintillation counter or by autoradiography.

[0220] In certain embodiments, a competition assay can be used to identify antibodies or antibody derivatives that compete with the antibodies of the present disclosure for binding to PDL1. In certain embodiments, such competing antibodies bind to the same epitope (e.g., a linear or conformational epitope) as the antibodies disclosed herein. Detailed exemplary methods for mapping antibody-binding epitopes are provided in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology, vol. 66 (Humana Press, Totowa, NJ).

[0221] In a non-limiting example of a competitive assay, immobilized PDL1 can be incubated with a solution containing a first labeled antibody or antibody derivative that binds to PDL1 and a second unlabeled antibody being tested for its ability to compete with the first antibody for binding to PDL1. The second antibody may be present in hybridoma supernatant. As a control, immobilized PDL1 is incubated with a solution containing the first labeled antibody but not the second unlabeled antibody. After incubation under conditions that allow the first antibody to bind to PDL1, excess unbound antibody is removed and the amount of label associated with immobilized PDL1 is measured. A significant decrease in the amount of label associated with immobilized PDL1 in the test sample compared to the control sample indicates that the second antibody competes with the first antibody for binding to PDL1. See Harlow and Lane (1988) Antibodies: A Laboratory Manual, ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY).

[0222] The present disclosure provides assays for identifying antibodies, or antibody derivatives thereof, that have biological activity. Biological activity can include, for example, activating immune cells or immune activation reporters, such as NFAT reporters or NF-κB reporters. Antibodies that have such biological activity in vivo and / or in vitro are also provided.

[0223] 2.11 Immune complexes

[0224] The presently disclosed subject matter further provides immunoconjugates comprising an antibody or antibody derivative disclosed herein conjugated to one or more detection probes and / or cytotoxic agents, e.g., chemotherapeutic agents or drugs, growth inhibitory agents, toxins (e.g., protein toxins, enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), or radioisotopes. For example, an antibody or antigen-binding portion of the disclosed subject matter can be functionally linked (e.g., by chemical coupling, genetic fusion, noncovalent bonding, or otherwise) to one or more other binding molecules, e.g., another antibody, antibody fragment, peptide, or binding mimetic.

[0225] In certain embodiments, the immunoconjugate is an antibody that binds to a maytansinoid (see U.S. Pat. Nos. 5,208,020, 5,416,064, and EP 0 425 106). 235), auristatins such as the monomethyl auristatin drug moieties DE and DF (MMAE and MMAF) (see U.S. Pat. Nos. 5,635,483, 5,780,588, and 7,498,298), dolastatins, calicheamicin or derivatives thereof (see U.S. Pat. Nos. 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. Res. 58:2925-2928 (1998)), anthracyclines such as daunomycin 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 (2002), and U.S. Pat. No. 6,630,579), methotrexate, vindesine, taxanes such as docetaxel, paclitaxel, larotaxel, tesetaxel, and ortataxel, trichothecenes, and antibody-drug conjugates (ADCs) conjugated to one or more drugs, including, but not limited to, CC1065.

[0226] In certain embodiments, the immunoconjugate comprises an antibody described herein conjugated to an enzymatically active toxin or fragment thereof, including, but not limited to, diphtheria A chain, a nonbinding active fragment of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Jatropha curcas protein, dianthin protein, pokeweed protein (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, soapwort inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and a trichothecene.

[0227] In certain embodiments, the immunoconjugate comprises an antibody described herein conjugated to a radioactive atom to form a radioconjugate. A variety of radioisotopes are available for the production of radioconjugates. Non-limiting examples include At 211 , I 131 , I 125 , Y 90 ,Re 186 ,Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 When used for detection, the radioconjugate can contain a radioactive atom, such as tc-99m or tc-1123, for scintigraphy studies, or a spin label, such as iodine-123, iodine-131, indium-11, fluorine-19, carbon-13, nitrogen-15, oxygen-17, gadolinium, manganese, or iron, for nuclear magnetic resonance (NMR) imaging (also called magnetic resonance imaging, MRI).

[0228] Conjugates of antibodies and cytotoxic agents can be prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), activated esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., toluene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin 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 radionucleotides to antibodies. See WO 94 / 11026. The linker can be a "cleavable linker" that facilitates release of the cytotoxic drug inside the cell. For example, acid-labile linkers, peptidase-sensitive linkers, photolabile linkers, dimethyl linkers, or disulfide-containing linkers (Chari et al., Cancer Res. 52:127-131 (1992); U.S. Pat. No. 5,208,020) can be used.

[0229] The immunoconjugates or ADCs herein expressly contemplate, but are not limited to, such conjugates prepared with crosslinker reagents, including, but 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, and sulfo-SMPB, and SVSB (succinimidyl-(4-vinylsulfone)benzoate) (e.g., from Pierce Biotechnology, Inc., Rockford, IL, USA).

[0230] 2.12 Antigen Recognition Receptors

[0231] The presently disclosed subject matter further provides an antigen-recognizing receptor comprising the antibody or antibody fragment disclosed herein. An antigen-recognizing receptor is a receptor that can activate, stimulate, or inhibit an immune response cell (e.g., a T cell) in response to binding to an antigen. Non-limiting examples of antigen-recognizing receptors include natural and recombinant T cell receptors (TCRs), chimeric costimulatory receptors (CCRs), chimeric antigen receptors (CARs), or inhibitory CARs (iCARs). The design and use of antigen-recognizing receptors is well known in the art and has been described in the literature, for example, in 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.

[0232] In certain embodiments, the presently disclosed subject matter provides a chimeric antigen receptor (CAR) comprising an antibody, antibody fragment, or multispecific antibody disclosed herein. CARs are engineered receptors that can graft or confer a specificity of interest onto immune effector cells. In certain embodiments, CARs can be used to graft the specificity of a monoclonal antibody onto T cells, with the transfer of the coding sequence facilitated by a vector. In certain embodiments, the CAR is a "first-generation" CAR, which typically consists of an extracellular antigen-binding domain (e.g., scFv, Fab, or VHH) fused to a transmembrane domain, which in turn is fused to a cytoplasmic / intracellular signaling domain. "First-generation" CARs provide de novo antigen recognition and can trigger activation of immune response cells, e.g., CD4+ and CD8+ T cells, via the signaling domain of the CD3z chain in a single fusion molecule, without relying on HLA-mediated antigen presentation. In certain embodiments, the CAR is a "second generation" CAR, which further comprises an intracellular signaling domain from various costimulatory molecules (e.g., CD28, 4-1BB, ICOS, OX40, CD27, CD40 / My88, and NKGD2) in the cytoplasmic tail of the CAR to provide an additional signal to the immune response cell, thereby "second generation" CARs include those that provide both costimulation (e.g., CD28 or 4-1BB) and activating (CD3z) domains. In certain embodiments, the CAR is a "third generation" CAR, which comprises multiple costimulatory domains (e.g., CD28 and 4-1BB) and activating (CD3z) domains. In certain embodiments, the CAR is a second generation CAR. In certain embodiments, the CAR comprises an extracellular antigen-binding domain that binds to an antigen, a transmembrane domain, and an intracellular signaling domain, wherein the intracellular signaling domain comprises a costimulatory signaling domain. In certain embodiments, the CAR further comprises a hinge / spacer region between the extracellular antigen-binding domain and the transmembrane domain, hi certain embodiments, the extracellular antigen-binding domain comprises an antibody, antibody fragment, or multispecific antibody disclosed herein.In certain embodiments, the antibody, antibody fragment, or multispecific antibody comprises a VHH, Fab, or scFv. In certain embodiments, the CAR comprises a multispecific antibody disclosed herein.

[0233] In certain embodiments, the presently disclosed subject matter provides a recombinant TCR comprising an antibody or antibody fragment disclosed herein. Native TCRs are protein complexes comprising a disulfide-linked heterodimeric protein composed of two variable chains expressed as part of a complex with a CD3 chain molecule. Native TCRs are found on the surface of T cells and are responsible for recognizing antigens as peptides bound to major histocompatibility complex (MHC) molecules. In certain embodiments, native TCRs comprise an α chain and a β chain (encoded by the TRA and TRB genes, respectively). In certain embodiments, TCRs comprise a γ chain and a δ chain (encoded by the TRG and TRD genes, respectively). Each of the α, β, γ, and δ chains comprises two extracellular domains: a variable (V) region and a constant (C) region. The constant region is located near the cell membrane and is followed by a transmembrane region and a short cytoplasmic tail. The variable region binds to the peptide / MHC complex. Each variable region has three complementarity-determining regions (CDRs). In certain embodiments, the TCR comprises a receptor complex with CD3δ, CD3γ, CD3ε, and CD3ζ. Upon binding of the TCR complex to its antigen and MHC (peptide / MHC), a T cell expressing the TCR complex is activated.

[0234] In certain embodiments, the recombinant TCR is a non-naturally occurring TCR. In certain embodiments, the recombinant TCR comprises a recombinant α chain and / or a recombinant b chain, wherein part or all of the variable regions of the recombinant α chain and / or the recombinant b chain are replaced by an antibody or antibody fragment disclosed herein. In certain embodiments, the antibody or antibody fragment comprises a VHH, VH, VL, Fab, or scFv. In certain embodiments, the antibody or antibody fragment comprises a VHH. In certain embodiments, the recombinant TCR binds to an antigen of interest in an MHC / HLA-independent manner. In certain non-limiting embodiments, antigen binding can activate immune response cells comprising the recombinant TCR.

[0235] The presently disclosed subject matter provides immune response cells comprising an antigen-recognition receptor (e.g., a CAR or TCR) disclosed herein. In certain embodiments, the antigen-recognition receptor can activate the immune response cell. The immune response cells of the presently disclosed subject matter can be cells of the lymphoid lineage. The lymphoid lineage, including B cells, T cells, and natural killer (NK) cells, provides antibody production, regulation of the cellular immune system, detection of foreign agents in the blood, detection of cells foreign to the host, etc. Non-limiting examples of immune response cells of the lymphoid lineage include T cells, natural killer (NK) cells, embryonic stem cells, and multipotent stem cells (e.g., from which lymphoid cells can differentiate). T cells can be lymphocytes that mature in the thymus and are primarily responsible for cell-mediated immunity. T cells participate in the adaptive immune system. The T cells of the presently disclosed subject matter can be any type of T cell, including, but not limited to, helper T cells, cytotoxic T cells, memory T cells (including central memory T cells, stem cell-like memory T cells (or stem-like memory T cells), and two types of effector memory T cells, e.g., TEM cells and TEMRA cells), regulatory T cells (also called suppressor T cells), natural killer T cells, mucosal-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 cells or tumor cells. A patient's own T cells can be genetically modified to target specific antigens by introduction of an antigen-recognition receptor, e.g., a CAR or TCR. In certain embodiments, the immune response cells are T cells. The T cells may be CD4+ T cells or CD8+ T cells. In certain embodiments, the T cells are CD4+ T cells. In certain embodiments, the T cells are CD8+ T cells. Natural killer (NK) cells can be lymphocytes that are part of cell-mediated immunity and act during the innate immune response. NK cells do not require prior activation to exert a cytotoxic effect on target cells.The types of human lymphocytes of the presently disclosed subject matter include, but are not limited to, peripheral donor lymphocytes, such as Sadelain, M., et al., 2003 Nat Rev Cancer 3:35-45 (disclosing peripheral donor lymphocytes genetically modified to express CARs), Morgan, RA, et al., 2006 Science 314:126-129 (disclosing peripheral donor lymphocytes genetically modified to express full-length tumor antigen-recognizing T cell receptor complexes, including a and b heterodimers), Panelli, MC, et al., 2000 J Immunol 164:495-504, Panelli, MC, et al., 2000 J Immunol 164:4382-4392 (disclosing lymphocyte cultures derived from tumor-infiltrating lymphocytes (TILs) of tumor biopsies), and Dupont, J., et al., 2005 Cancer Res 65:5417-5427; Papanicolaou, GA, et al., 2003 Blood 102:2498-2505 (disclosing selective in vitro expanded antigen-specific peripheral blood leukocytes using artificial antigen presenting cells (AAPCs) or pulsed dendritic cells). In certain embodiments, the immune response cells (e.g., T cells) can be autologous, non-autologous (e.g., allogeneic), or derived in vitro from engineered progenitor or stem cells.

[0236] 3. How to use

[0237] The presently disclosed subject matter further provides methods of using the disclosed antibodies and antibody derivatives. In certain embodiments, the methods relate to therapeutic uses of the antibodies or antibody derivatives disclosed herein. In certain embodiments, the methods relate to diagnostic uses of the antibodies or antibody derivatives disclosed herein.

[0238] 3.1 Treatment method

[0239] The present disclosure provides methods and uses of the antibodies or antibody derivatives disclosed herein for treating diseases and disorders or for increasing immune responses. In certain embodiments, the antibodies, antibody derivatives, or pharmaceutical compositions comprising them disclosed herein can be administered to a subject (e.g., a mammal, such as a human) to treat the disease or disorder or increase the immune response. In certain embodiments, the disease or disorder involves immune checkpoint inhibition and / or aberrant PDL1 activity. In certain embodiments, diseases and disorders treatable with the antibodies or antibody derivatives disclosed herein include, but are not limited to, neoplasms, e.g., cancer.

[0240] In certain embodiments, the present disclosure provides an antibody or antibody derivative (or fragment thereof) described herein for use in the manufacture of a medicament. In certain embodiments, the present disclosure provides an antibody or antibody derivative (or fragment thereof) described herein for use in the manufacture of a medicament for treating cancer. In certain embodiments, the present disclosure provides an antibody or antibody derivative (or fragment thereof) described herein for use in the treatment of cancer in a subject. In certain embodiments, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody derivative (or fragment thereof) provided herein for use in the treatment of cancer in a subject. In certain embodiments, the cancer can be a blood cancer (e.g., leukemia, lymphoma, 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 cancer, glioblastoma, throat cancer, melanoma, neuroblastoma, adenocarcinoma, glioma, soft tissue sarcoma, and various carcinomas (including prostate cancer and small cell lung cancer). Suitable carcinomas further include, but are not limited to, astrocytoma, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, primitive neuroectodermal tumor (PNET), chondrosarcoma, osteogenic sarcoma, pancreatic ductal adenocarcinoma, small cell lung adenocarcinoma and large cell lung adenocarcinoma, chordoma, angiosarcoma, endothelial sarcoma, squamous cell carcinoma, bronchoalveolar carcinoma, epithelial adenocarcinoma and its liver metastases, lymphangiosarcoma, lymphangioendothelial sarcoma, hepatocarcinoma, cholangiocarcinoma, synovium, mesothelioma, Ewing's tumor, rhabdomyosarcoma, colon carcinoma, basal cell carcinoma, sweat gland carcinoma, papillary carcinoma, sebaceous gland carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, cholangiocarcinoma, choriocarcinoma, and seminoma. The cancers include any well-known carcinoma in the field of oncology, including embryonal carcinoma, Wilms' tumor, testicular tumor, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, Waldenstrom's macroglobulinemia, tumors of the breast such as ductal carcinoma and lobular adenocarcinoma, cervical squamous cell carcinoma and adenocarcinoma, uterine epithelial carcinoma and ovarian epithelial carcinoma, prostate cancer, transitional squamous cell carcinoma of the bladder, B- and T-cell lymphoma (nodular and diffuse), plasmacytoma, acute and chronic leukemia, malignant melanoma, soft tissue sarcoma, and leiomyosarcoma.

[0241] In certain embodiments, the cancer can be melanoma, NSCLC, head and neck cancer, urothelial cancer, breast cancer (e.g., triple-negative breast cancer, TNBC), gastric cancer, cholangiocarcinoma, classical Hodgkin's lymphoma (cHL), non-Hodgkin's lymphoma primary mediastinal B-cell lymphoma (NHL PMBCL), mesothelioma, ovarian cancer, lung cancer (e.g., small cell lung cancer), esophageal cancer, nasopharyngeal carcinoma (NPC), biliary tract cancer, colorectal cancer, cervical cancer, or thyroid cancer.

[0242] In certain embodiments, the subject to be treated is a mammal (e.g., a human, a non-human primate, a rat, a mouse, a cow, a horse, a pig, a sheep, a goat, a dog, a cat, etc.). In certain embodiments, the subject is a human. In certain embodiments, the subject is suspected of having cancer, is at risk of having cancer, or has been diagnosed with cancer or any other disease with abnormal PDL1 expression or activity.

[0243] Many diagnostic methods for cancer or any other disease that exhibits abnormal PDL1 activity and the clinical description of these diseases are well known in the art. Such methods include, but are not limited to, immunohistochemistry, PCR, and fluorescence in situ hybridization (FISH). Further details regarding diagnostic methods for abnormal PDL1 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.

[0244] Administration can be by any suitable route, including, for example, intravenous, intramuscular, or subcutaneous. In some embodiments, an antibody or antibody derivative (or fragment thereof), and / or composition provided herein is administered in combination with a second, third, or fourth agent (e.g., an anti-tumor agent, a growth inhibitory agent, a cytotoxic agent, or a chemotherapeutic agent) to treat a disease or disorder associated with aberrant PDL1 activity. Such agents include, for example, docetaxel, gefitinib, FOLFIRI (irinotecan, 5-fluorouracil, and leucovorin), irinotecan, cisplatin, carboplatin, paclitaxel, bevacizumab (anti-VEGF antibody), FOLFOX-4, injectable fluorouracil, leucovorin, and oxaliplatin, afatinib, gemcitabine, capecitabine, pemetrexed, tivantinib, everolimus, CpG-ODN, rapamycin, lenalidomide, vemurafenib, endostatin, lapatinib, PX-866, Imprime PGG, and erlotinib. In some embodiments, the antibody or antibody derivative (or fragment thereof) is conjugated to another agent.

[0245] In certain embodiments, the antibodies or antibody derivatives (or fragments thereof), and / or compositions provided herein are administered in combination with one or more additional therapies, such as radiation therapy, surgery, chemotherapy, and / or targeted therapy. In certain embodiments, the antibodies, antibody derivatives (or fragments thereof), and / or compositions provided herein are administered in combination with radiation therapy. In certain embodiments, the combination of the antibodies, antibody derivatives (or fragments thereof), and / or compositions provided herein with radiation therapy is used to treat neoplasms or cancers as disclosed herein.

[0246] Depending on the indication being treated and dosing-related factors well known to those of skill in the art, the antibodies or antibody derivatives provided herein are administered in dosages effective to treat the indication while minimizing toxicity and side effects. For the treatment of cancer, a typical dosage can range, for example, from 0.001 to 1000 μg, although dosages below or above this exemplary range are within the scope of the present invention. Daily dosages can range from 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 noted above, therapeutic or prophylactic efficacy can be monitored by periodic evaluation of treated patients. For repeated administration over several days or longer, treatment is repeated, depending on the circumstances, until a desired suppression of disease symptoms is achieved. However, other dosage regimens may also be useful and are within the scope of the present invention. The desired dose can be delivered by administration of a single bolus of the composition, by administration of multiple boluses of the composition, or by administration of a continuous infusion of the composition.

[0247] Pharmaceutical compositions containing the antibodies or antibody derivatives disclosed herein can be administered once, twice, three times, or four times daily. The compositions can also be administered less frequently than daily, for example, six times a week, five times a week, four times a week, three times a week, twice a week, once a week, once every two weeks, once every three weeks, once a month, once every two months, once every three months, or once every six months. The compositions can also be administered in sustained-release formulations, such as in implants that gradually release the composition for use over a period of time, allowing for less frequent administration of the administered composition, for example, once a month, once every two to six months, once a year, or even once a single dose. Sustained-release devices (e.g., pellets, nanoparticles, microparticles, nanospheres, microparticles, etc.) can be administered by injection or surgically implanted at various locations.

[0248] Cancer treatment can be evaluated by, for example, but not limited to, tumor regression, shrinkage of tumor weight or size, time to progression, survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression, and / or activity. Approaches for determining the efficacy of therapy can be used, including, for example, measuring response by radiological imaging.

[0249] In certain embodiments, the efficacy of treatment is measured by percent tumor growth inhibition (% TGI), calculated using the equation 100-(T / C x 100), where T is the mean relative tumor volume of treated tumors and C is the mean relative tumor volume of untreated tumors. In certain embodiments, the % TGI is 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 greater than 95%.

[0250] 3.2 Diagnostic and Imaging Methods

[0251] Labeled antibodies or antibody derivatives can be used for diagnostic purposes to detect, diagnose, or monitor diseases and / or disorders associated with PDL1 expression, abnormal expression, and / or activity. For example, the antibodies and antibody derivatives provided herein can be used in in situ, in vivo, ex vivo, and in vitro diagnostic or imaging assays. Methods for detecting PDL1 polypeptide expression include (a) assaying polypeptide expression in an individual's cells (e.g., tissues) or body fluids using one or more antibodies or antibody derivatives, and (b) comparing the gene expression level with a standard gene expression level, wherein an increase or decrease in the assayed gene expression level compared to the standard expression level indicates abnormal expression.

[0252] Another embodiment provided herein includes a method for diagnosing a disease or disorder associated with expression or aberrant expression of PDL1 in an animal (e.g., a mammal, such as a human). The method includes detecting a PDL1 molecule in the mammal. In certain embodiments, the diagnosis includes (a) administering an effective amount of a labeled antibody or antibody derivative to the mammal; (b) allowing a time interval after administration to allow the labeled antibody or antibody derivative to preferentially concentrate at sites in the subject where the PDL1 molecule is expressed (and allowing unbound labeled molecule to be cleared to background levels); (c) determining the background level; and (d) detecting the labeled molecule in the subject, such that detection of the labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with expression or aberrant expression of PDL1. The background level can be determined by various methods, including comparing the amount of detected labeled molecule to a standard value previously determined for a particular system.

[0253] The antibodies and antibody derivatives provided herein can be used to assay protein levels in biological samples using classical immunohistological methods well known to those skilled in the art (see, e.g., 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, such as enzyme-linked immunosorbent assays (ELISAs) and radioimmunoassays (RIAs). Suitable antibody assay labels are well known in the art and include 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 These include Ru, luminol, and fluorescent labels such as fluorescein and rhodamine, and enzyme labels such as biotin.

[0254] Techniques well known in the art can be applied to labeled antibodies (or fragments thereof) provided herein, including, but not limited to, the use of bifunctional conjugation agents (see, e.g., 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).

[0255] Alternatively, or in addition, the level of nucleic acid or mRNA encoding a PDL1 polypeptide in cells can be measured by, for example, fluorescent in situ hybridization (FISH, see WO1998 / 45479 published October 1998), Southern blotting, Northern blotting, or polymerase chain reaction (PCR) techniques such as real-time quantitative PCR (RT-PCR) using a nucleic acid-based probe corresponding to the nucleic acid encoding PDL1 or its complement. For example, it is also possible to study PDL1 overexpression by measuring shed antigens in biological fluids such as serum using antibody-based assays (see, e.g., U.S. Pat. No. 4,933,294, published June 12, 1990; WO 91 / 05264, published April 18, 1991; U.S. Pat. No. 5,401,638, published March 28, 1995; and Sias et al., J. Immunol. Methods 132:73-80 (1990)). In addition to the above assays, various in vivo and ex vivo assays are available to skilled practitioners. For example, cells within a mammal can be exposed to an antibody optionally labeled with a detectable label, e.g., a radioisotope, and binding of the antibody to the somatic cells can be assessed, for example, by external scanning for radioactivity or by analyzing a sample (e.g., a biopsy or other biological sample) taken from a mammal previously exposed to the antibody.

[0256] 4. Pharmaceutical Preparations

[0257] The presently disclosed subject matter further provides pharmaceutical formulations containing an antibody or antibody derivative disclosed herein and a pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical compositions can include a combination of multiple (e.g., two or more) antibodies and / or antibody derivatives of the presently disclosed subject matter.

[0258] In certain embodiments, the disclosed pharmaceutical formulations can be prepared by combining an antibody or antibody derivative having a desired degree of purity in the form of a lyophilized formulation or aqueous solution with any one or more pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)). For example, but not limited to, lyophilized antibody formulations are described in U.S. Pat. No. 6,267,958. In certain embodiments, aqueous antibody formulations can include those described in U.S. Pat. No. 6,171,586 and WO2006 / 044908, the latter formulations including histidine acetate buffers. In certain embodiments, the antibody or antibody derivative can be 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 about 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% pure.

[0259] Pharmaceutically acceptable carriers are generally nontoxic to recipients at the dosages and concentrations employed and include buffers of phosphate, citrate, and other organic acid salts, antioxidants including ascorbic acid and methionine, preservatives (e.g., octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, serum alcohols, and the like. Examples of suitable pharmaceutically acceptable carriers include, but are not limited to, proteins such as lysine, glutamine, 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 dextran, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose, or sorbitol, salt-forming counterions such as sodium, metal complexes (e.g., Zn-protein complexes), and / or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersing agents, such as soluble neutral active hyaluronidase glycoproteins (sHASEGPs), e.g., human soluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publication Nos. 2005 / 0260186 and 2006 / 0104968. In certain embodiments, the sHASEGP is combined with one or more additional glycosaminoglycanases, such as chondroitinases.

[0260] The carrier can 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., an anti-PDL1 antibody or multispecific antibody disclosed herein, can be coated with a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0261] The pharmaceutical compositions of the present disclosure can also be administered in combination therapy, i.e., combined with other drugs. In certain embodiments, the pharmaceutical compositions disclosed herein can optionally contain more than one active ingredient for the particular indication being treated, e.g., those with complementary activities that do not adversely affect each other. In certain embodiments, the pharmaceutical formulation can include a second active ingredient for treating the same disease as that being treated with the first therapeutic agent. Such active ingredients are suitably present in combination in amounts effective for the intended purpose. For example, but not limited to, the formulations of the present disclosure can optionally contain more than one active ingredient for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it may be desirable to further provide a second therapeutic agent useful in treating the same disease. Such active ingredients are suitably present in combination in amounts effective for the intended purpose.

[0262] The compositions of the present disclosure can be administered by various methods known in the art. The route and / or method of administration varies depending on the desired results. The active compounds can be formulated with carriers that will protect the compound against rapid release, such as controlled-release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable, biocompatible polymers, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid, can be used. Many methods for preparing such formulations are described, for example, in *Sustained and Controlled Release Drug Delivery Systems*, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. In certain embodiments, the pharmaceutical compositions are manufactured under Good Manufacturing Practice (GMP) conditions of the U.S. Food and Drug Administration.

[0263] Sustained-release preparations containing the antibodies or antibody derivatives disclosed herein can also be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibodies or antibody derivatives, which matrices are in the form of shaped articles such as films or microcapsules. In certain embodiments, the active ingredient can also be embedded in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in microemulsions, for example, by coacervation techniques or by interfacial polymerization, e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0264] To administer the antibody or antibody derivative of the present disclosure by certain routes of administration, it may be necessary to coat the compound with a material to prevent its inactivation or to co-administer the compound with a material to prevent its inactivation. For example, the compound can be administered to a subject in an appropriate carrier, such as a liposome or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Liposomes include water-in-oil-in-water CGF emulsions as well as conventional liposomes (Strejan et al., (1984) J. Neuroimmunol. 7:27).

[0265] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutical active substances is well known in the art.

[0266] Except insofar as any conventional media or agent is incompatible with the active compound, its use in the pharmaceutical compositions of the present disclosure is contemplated. Supplementary active compounds can also be incorporated into the compositions.

[0267] Therapeutic compositions must usually be sterile, substantially isotonic, and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes, or other ordered structures suitable for high-concentration dosages. The carrier can 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. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. It is often preferable to include isotonic agents, such as sugars, polyalcohols (e.g., mannitol, sorbitol), or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including in the composition an agent that delays absorption, such as monostearate salts and gelatin.

[0268] Sterile injectable solutions can be prepared by incorporating one or more antibodies or antibody derivatives disclosed herein, as needed, in the required amount with one or a combination of the above-listed ingredients in a suitable solvent, followed by sterilization, for example, by filtration through a sterile filtration membrane, by microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the required other ingredients from those listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying (lyophilization), which yields a powder of the active ingredient plus any additional desired ingredients from a previously sterile-filtered solution.

[0269] Therapeutic compositions can also be administered using medical devices known in the art. For example, the therapeutic compositions of the present disclosure can be administered by needleless hypodermic injection devices, 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. Implants and modules useful in the present disclosure include U.S. Patent No. 4,487,603, which discloses an implantable microinfusion pump for dispensing drugs at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering drugs through the skin; U.S. Patent No. 4,447,233, which discloses a drug infusion pump for delivering drugs at precise infusion rates; U.S. Patent No. 4,447,224, which discloses an implantable variable flow infusion device for continuous drug delivery; U.S. Patent No. 4,439,196, which discloses an osmotic drug delivery system having multiple chamber compartments; and U.S. Patent No. 4,475,196, which discloses an osmotic drug delivery system. Many other such implants, delivery systems, and modules are known.

[0270] With respect to therapeutic compositions, formulations of the present disclosure include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. These formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The amount of antibody or antibody derivative that can be combined with the carrier materials to produce a single dosage form will vary depending on the subject being treated and the particular mode of administration. The amount of antibody or antibody derivative that can be combined with the carrier materials to produce a single dosage form will generally be that amount of the composition that produces a therapeutic effect. Generally, out of 100%, this amount will range from about 0.01% to about 99%, about 0.1% to about 70%, or about 1% to about 30% of the active ingredient.

[0271] 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 with any preservatives, buffers, or propellants which may be required.

[0272] The phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include, but are not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

[0273] These pharmaceutical compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. Prevention of the presence of microorganisms may be ensured both by the above-mentioned sterilization procedures and by the inclusion of various antibacterial and antifungal agents (e.g., parabens, chlorobutanol, phenol sorbic acid, etc.). It may also be desirable to include isotonic agents, such as sugars and sodium chloride, in the compositions. In addition, prolonged absorption of injectable pharmaceutical formulations can be brought about by the inclusion of agents that delay absorption, such as aluminum monostearate and gelatin.

[0274] In certain embodiments, when the antibodies or antibody derivatives of the present disclosure are administered to humans and animals as pharmaceuticals, they can be administered alone or in combination with a pharmaceutically acceptable carrier, for example, as a pharmaceutical composition containing about 0.01% to about 99.5% (or about 0.1% to about 90%) of the antibody or antibody derivative.

[0275] 5. Manufactured products

[0276] The presently disclosed subject matter further provides articles of manufacture containing materials useful for the treatment, prevention, and / or diagnosis of the above-mentioned disorders.

[0277] In certain embodiments, the article of manufacture comprises a container and a label or package insert on or associated with the container. Non-limiting examples of suitable containers include bottles, vials, syringes, IV solution bags, and the like. The container can be formed from a variety of materials such as glass or plastic. The container can hold the composition by itself or in combination with other compositions effective for treating, preventing, and / or diagnosing a condition and can have a sterilization access port (e.g., the container can be an IV solution bag or a vial with a stopper pierceable by a hypodermic injection needle).

[0278] In certain embodiments, at least one active agent in the composition is an antibody or antibody derivative of the present disclosure. The label or package insert can indicate that the composition is used for treating the condition of choice.

[0279] In certain embodiments, the article of manufacture can include (a) a first container containing a composition, the composition comprising an antibody or antibody derivative of the present disclosure, and (b) a second container containing a composition, the second container containing a composition comprising an additional cytotoxic or another therapeutic agent. In certain embodiments, the article of manufacture can further include a package insert indicating that the composition can be used to treat a particular condition.

[0280] Alternatively, or additionally, the article of manufacture may comprise an additional container, e.g., a second or third container, containing a pharmaceutically acceptable buffer, including, but not limited to, bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. The article of manufacture may include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0281] [Table 3] [Table 4] [Table 5] [Table 6] [Table 7] [Table 8] [Table 9] [Table 10] [Table 11] [Table 12] [Table 13] [Table 14] [Table 15] [Table 16]

[0282] The following examples are merely illustrative of the subject matter disclosed herein and should not be construed as limiting in any way.

[0283] Example

[0284] Example 1. Immunization, generation and identification of anti-PDL1 VHH antibodies

[0285] Recombinant human PDL1 extracellular domain (ECD) protein antigen was purified in-house using a C-terminal Fc or polyhistidine tag. Llama immunization with PDL1 was performed using standard protocols known in the art. Serum antibody titers were measured by ELISA assay. After three rounds of immunization, high titers (1:100,000) were observed. Whole blood was then collected and PBMCs were isolated. Total RNA was then isolated from the PBMCs, and antibody V-region cDNAs were synthesized from the total RNA. VHH antibody genes were amplified by PCR using protocols known in the art, purified by DNA agarose gel, and constructed into the phagemid vector pADL-23c (Antibody Design Labs), which was then transformed into TG1 electrocompetent cells (from Lucigen). Transformed TG1 cells were cultured in Y2T medium. Phages bearing the displayed target VHH were produced by adding helper phage and co-culturing overnight. Phages in the culture supernatant were collected by centrifugation and panned for binders to human PDL1 (h-PDL1, prepared in-house) or cynomolgus monkey PDL1 (cyno-PDL1, Acro Biosystems) antigens using streptavidin-conjugated Dynabeads coated with biotinylated h-PDL1 or cyno-PDL1 ECD. After one round of panning, h-PDL1 or cyno-PDL1 binders were eluted and used to infect SS320 cells. SS320 cell colonies were selected and cultured in Y2T medium with IPTG added to secrete VHH antibodies. Supernatants containing VHH antibodies were screened by ELISA using h-PDL1 ECD-coated plates. Positive h-PDL1 binders were selected for sequencing. Twenty clones with different sequences were selected. The binding ability of the VHH antibodies to cyno-PDL1 was also examined by ELISA. The inhibitory effect of VHH antibody clones on PDL1 binding to PD1 was also determined using a PD1 / PDL1 whole-cell blocking assay. The top binder, 10A4, was selected for further engineering, and its CDRs and VH are shown in the sequence listing (SEQ ID NOs: 1-4).

[0286] Next, we used the 10A4 VHH to generate a bivalent antibody by adding human constant heavy chain 2 (CH2) and constant heavy chain 3 (CH3) domains. The constructed bivalent VHH antibody was expressed in ExpiCHO cells, and the supernatant protein was collected and purified with Protein A. For comparison, we also prepared two reference anti-PDL1 antibodies (both conventional IgG antibodies). Reference Ab 1 is an analog of MPDL3280A synthesized in-house based on the sequence disclosed in WO2016007235A1. Reference Ab 2 is PL3#7-43 Deglyco2 synthesized in-house based on the sequence disclosed in WO2018080812A1.

[0287] Biolayer interferometry was used to measure the association and dissociation constants of the binding kinetics of anti-PDL1 bivalent antibodies. Binding kinetics were measured at 30°C using a FortieBio Octet Red 96 and analyzed using FortieBio Data Analysis 9.0 software. Anti-PDL1 or control antibodies were captured using an anti-human IgG Fc (AHC) sensor. Wells containing only kinetic buffer were used as reference wells for subtraction during data processing. Data were fitted to a 1:1 Langmuir model of association and dissociation using a global Rmax-linked fit for each antibody-antigen binding event. Human PDL1-His is a recombinant PDL1 antigen (expressed in-house) fused to a polyhistidine tag at the C-terminus of human PDL1 antigen. Cyno PDL1-His is a recombinant PDL1 antigen (expressed in-house) fused to a polyhistidine tag at the C-terminus of cynomolgus monkey PDL1 antigen (Acro Biosystems).

[0288] As shown in Table 3 below, anti-PDL1 clone 10A4 effectively binds to human PDL1 and cynomolgus monkey PDL1 with KDs of 0.934 nM and 0.942 nM, respectively. Assays using mouse PDL1 did not demonstrate effective binding (data not shown).

[0289] [Table 17]

[0290] The ability of bivalent antibodies to block human PDL1 / PD1 interaction was tested in a whole-cell blocking assay. Chinese hamster ovary (CHO) cells stably expressing human PDL1 were established and confirmed by flow cytometry assay. CHO cells stably expressing human PDL1 were selected with 8 μg / mL puromycin during cell culture. Jurkat cells stably expressing human PD-1 and NFAT reporter genes were established. Specifically, Jurkat cells were co-transfected with human PD1 expression vector and NFAT expression vector by electroporation, and then selected with 300 μg / mL hygromycin during cell culture. CHO cells stably expressing h-PDL1 were plated in a 96-well plate at 100 μl / well (40,000 cells / well) in Ham's F-12 nutrient mixture medium containing 10% fetal bovine serum (FBS) and cultured for 18 hours. The following day, representative antibody clones were incubated at various concentrations with CHO cells stably expressing h-PDL1 for 1 hour. Human PDL1-expressing CHO cells were then co-incubated with Jurkat cells (50,000 cells / well) expressing human PD1 and NFAT reporter genes for 5 hours to determine the efficacy of antibodies in blocking the interaction between PD1-induced NFAT reporter activity and PDL1. Bright-Glo luciferase assay buffer containing substrate (Promega) was added, and luciferase activity was measured by chemiluminescence using a plate reader. The potency of antibodies in blocking PDL1 activity was calculated using nonlinear regression in GraphPad Prims. Representative results are shown in Figure 1. Compared to Reference Ab 1 and Reference Ab 2, 10A4 demonstrated comparable blocking ability with cells expressing human PDL1.

[0291] Example 2 - Humanization and hotspot mutations of anti-PDL1 antibodies

[0292] 10A4 was selected for framework humanization. Briefly, Igblast was performed using the sequence of this clone to search a database of human germline genes. The desired germline sequence was selected, and framework mutations were performed to change the framework sequence from llama to human. For the 10A4 clone, human germline IGHV3-23*04 was used, and two versions of humanized 10A4, 10A4-GLEF and 10A4-GLEL, were generated. 10A4-GLEF retained the llama signature residue (Phe47, Kabat numbering) in framework 2, while 10A4-GLEL replaced the llama signature residue with a human residue. Because the CDR2 region of the 10A4 sequence contains an asparagine site that could potentially cause deamidation, two hotspot mutant versions of 10A4 were created by mutating the asparagine to serine or threonine (10A4-GLEF-N2S or 10A4-GLEF-N2T). To avoid certain sequence liabilities and increase stability, sequence modifications were also made to Tyr100Phe and His101Asp (Kabat numbering). The constructs were cloned into expression vectors, and antibody proteins were produced by transient transfection of ExpiCHO cells and purified using Protein A. The binding affinities of the humanized and hotspot mutant antibodies to human PDL1 were determined by binding to recombinant h-PDL1 in an Octet binding assay as previously described. As shown in Table 4, the antigen-binding specificity of 10A4 was not altered by these modifications.

[0293] [Table 18]

[0294] The ability of the humanized bivalent antibodies to block human PDL1 / PD1 interaction was tested by whole-cell blocking assay as described above. The blocking activity of these antibodies against h-PDL1 was calculated using nonlinear regression in GraphPad Prism 8.0, as shown in Figure 2. Similar blocking activity was observed for all three versions of the 10A4 clone compared to the chimeric parent clone. 10A4-GLEF-N2S was also tested in the whole-cell blocking assay and showed similar blocking activity compared to Reference Ab 1 and Reference Ab 2, as shown in Figure 3.

[0295] The in vivo efficacy of the bivalent antibody 10A4-GLEF-N2S VHH-Fc was also investigated in a NOD-SCID mouse model bearing PBMCs and H292 xenografts. H292, a human lung cancer cell line, expresses relatively high levels of PDL1. In this tumor model, 5 million tumor cells were mixed with 1.7 million freshly isolated human PBMCs and then injected subcutaneously into immunodeficient (NOD-SCID) mice. The animals were then treated with different antibodies by intraperitoneal injection twice weekly for 4–5 weeks. Twenty-one days after the first treatment, a dose-dependent antitumor effect was observed. Tumor size was measured with a caliper, and tumor volume (mm) was calculated. 3 ) was estimated using the formula: TV = a × b2 / 2, where "a" and "b" are the long and short diameters of the tumor, respectively. TV was used to calculate tumor growth inhibition (TGI, an indicator of anti-tumor efficacy) using the formula: TGI(%) = [1-(Tt-T0) / (Ct-C0)] × 100%, where Tt = mean TV of treatment at time t, T0 = mean TV of treatment at time 0 (baseline), Ct = mean TV of control at time t, and C0 = mean TV of control at time 0 (baseline). As shown in Figure 4, the anti-PD-L1 VHH antibody 10A4 showed superior tumor growth inhibition at 16 mg / kg compared to the reference Ab 2 at 30 mg / kg.

[0296] Example 3 - Affinity maturation, selection and modification

[0297] Affinity maturation was performed on the 10A4-GLEF-N2S VHH-Fc clone. Primers were designed to generate single amino acid mutations in each CDR region. A library of mutations was generated using assembly PCR and cloned into a phagemid vector. The quality of the library was assessed by transforming TG1 cells and DNA sequencing of the clones. Phages were generated using helper phage, and phage panning was performed using streptavidin-conjugated Dynabeads coated with biotinylated h-PDL1 ECD. After one round of panning, elution of the panning products was used to infect SS320 cells. Colonies were selected and cultured in Y2T medium containing IPTG. VHH antibodies in the supernatant were tested by ELISA assay. Positive clones against human PDL1 were selected, cloned into an expression vector, and antibody protein was produced by transient transfection of ExpiCHO cells and purified using Protein A. The bivalent versions of the affinity-matured VHH constructs were then tested for binding to Octet using the hPDL1 ECD as described above. As shown in Table 5 below, all clones exhibited high affinity for human PD-L1, with KDs (dissociation constants) comparable to or superior to chimeric 10A4. The CDRs and VHHs of the affinity-matured clones are shown in the sequence listing.

[0298] [Table 19]

[0299] The whole-cell blocking activity of the affinity-matured bivalent VHH-Fc constructs was then tested in a PD1 / PDL1 whole-cell blocking assay as previously described. The results of the PD1 / PDL1 whole-cell blocking assay were graphed using GraphPad Prism, and representative data are shown in Figure 5. All clones showed similar blocking activity. 2H4 and 3D5 were selected for further testing.

[0300] The in vivo efficacy of the anti-PD-L1 VHH bivalent constructs 2H4-Fc and 3D5-Fc was tested in a syngeneic mouse model using B16F10 mouse melanoma cells transfected with human PDL1. B16F10 mouse melanoma cells were transfected with human PDL1 and implanted 4 days prior to antibody treatment. Tumor volumes of approximately 200 mm 3 Once tumor volume reached 1000 mg / kg, treatment was initiated with the drug administered subcutaneously (SC) or intraperitoneally (IP) at a dose of 1 mg / kg twice weekly for 4 weeks. On day 24 after tumor inoculation, the tumor volume of several mice in the control group reached the tumor size limit (1500 mm). 3 ) was reached. Therefore, day 20 after tumor inoculation was the data endpoint for the analysis. On day 20, tumor volumes were measured and analyzed for all groups. As shown in Figures 6A-7C and Tables 6 and 7, 2H4-Fc (subcutaneous administration, sc) and 3D5-Fc (intraperitoneal administration, ip) significantly reduced tumor growth compared with the vehicle control. Furthermore, both 2H4-Fc and 3D5-Fc demonstrated superior antitumor efficacy compared with reference Ab 2. Individual tumor volumes on day 20 after tumor inoculation are also shown in Figures 6B and 7B for subcutaneous and intraperitoneal injections, respectively. Body weight changes between groups were not significant throughout the study period (Figures 6C and 7C), indicating that the treatment was well tolerated.

[0301] [Table 20] a Mean ± SEM

[0302] [Table 21] a Mean ± SEM

[0303] Example 4 - Generation and in vivo characterization of anti-PDL1 / CD47 bispecific antibodies

[0304] PDL1 and CD47 are co-expressed in many tumors, including non-small cell lung cancer (NSCLC), head and neck squamous cell carcinoma (HNSCC), and ovarian cancer (OC). Therefore, simultaneously targeting both antigens using an anti-PDL1 / CD47 bispecific antibody (bsAb) can produce a synergistic effect that can more effectively block tumor growth and metastasis. Therefore, various anti-PDL1 / CD47 bispecific antibodies were generated using the anti-PDL1 antibody described above and the anti-CD47 antibody disclosed herein. For example, anti-PDL1 VHH clone 3D5 was fused to the N-terminus of each heavy chain of anti-CD47 antibody (#6 or deglyco#6). A schematic diagram of the bispecific antibody format is shown in Figure 8. The protein sequences are shown in SEQ ID NOs: 136-139. The bispecific antibodies were produced from transfected ExpiCHO cells and purified by protein A affinity column for further analysis. Data related to anti-PDL1 / CD47-2 are discussed below.

[0305] To measure the binding affinity of anti-PDL1 / CD47 antibodies to hPDL1 and hCD47 expressed on tumor or tumor-like cells, hPDL1-expressing PDL1-CHO-K1 cells (BPS Bioscience) and CD47-expressing Jurkat cells (a leukemia cell line) (ATCC) were used in a whole-cell binding assay. Binding was detected by FACS using a Cytoflex (Beckman). 1 × 10 cells were cultured in a 96-well plate. 4PDL1-CHO-K1 or Jurkat cells were seeded into each well with 100 μl of PBS (Hyclone). PDL1-CHO-K1 or Jurkat cells were incubated with serially diluted anti-PDL1 / CD47 (100 nM, 33 nM, 11 nM, 3.7 nM, 1.2 nM, 0.4 nM, 0.13 nM, 0.045 nM, 0.015 nM, 0.005 nM, 0.0016 nM) and a control for 15 min and then washed with PBS. The cells were then incubated with FITC-conjugated anti-human Fc antibody (Jackson Immuno-Research) diluted 1:500 for 15 min and then washed. The cells were suspended in 300 μl of PBS for FACS analysis. PF-07257876 is an anti-PDL1 / CD47 antibody disclosed in U.S. Patent Publication No. US 20210179716 A1. IBI322 is an anti-PDL1 / CD47 antibody disclosed in U.S. Patent Publication No. US 20220227870 A1. Both of these bispecific antibodies were developed for therapeutic purposes. Analogs of these bispecific antibodies were synthesized in-house and used as experimental controls. An anti-PDL1 monospecific antibody was used as a positive control, and an IgG isotype antibody was used as a negative control. As shown in Figure 9A, the anti-PDL1 / CD47 antibodies disclosed herein, like PF-07257876, IBI322, and the anti-PDL1 monospecific antibody control, showed effective binding to PDL1-CHO-K1 cells, whereas the negative control showed no binding. Similarly, as shown in Figure 9B, the anti-PDL1 / CD47 antibodies, like PF-07257876 and IBI322, showed effective binding to Jurkat cells, whereas the negative control showed no binding. The results demonstrated that the anti-PDL1 / CD47 antibodies have high binding affinity to tumor or tumor-like cells expressing human PDL1 and human CD47.

[0306] The anti-CD47 antibody used in the anti-PDL1 / CD47 antibody has been demonstrated to have reduced binding to normal tissues expressing CD47, such as red blood cells (RBCs). To test whether the anti-PDL1 / CD47 antibody has reduced binding to normal tissues, the total cell binding of the antibody to human primary red blood cells (RBCs) (Stanford Blood Center) was measured by FACS using Cytoflex (Beckman). 1 × 10 cells were cultured in a 96-well plate. 5 RBCs were seeded into each well with 100 μl of PBS (Hyclone). RBCs were incubated with serially diluted antibodies (200 nM, 20 nM, 2 nM, 0.2 nM, 0.02 nM, 0.002 nM, 0.0002 nM, 0.00002 nM, 0.000002 nM, 0.0000002 nM) and controls for 15 minutes, washed with PBS, and incubated with a 1:500 diluted FITC-conjugated anti-human Fc antibody (Jackson Immuno-Research) for 15 minutes, followed by washing. Each sample was suspended in 300 μl of PBS for FACS analysis. Hu5F9 (also known as magrolimab), a clinical-stage anti-CD47 antibody disclosed in Liu et al. (2015), Pre-Clinical Development of a Humanized Anti-CD47 Antibody with Anti-Cancer Therapeutic Potential, PLOS ONE 10(9):e0137345, was used as a positive control. As shown in Figure 10, the anti-PDL1 / CD47 antibody and the IgG isotype control showed no binding to RBCs. IBI322 and Hu5F9 showed strong binding to RBCs, while PF-07257876 showed relatively weak binding to RBCs compared to IBI322 and Hu5F9. The results indicated that the anti-CD47×PDL1 antibody may be therapeutically safer than the control bispecific antibody because it is less likely to cause antibody-mediated anemia due to its lack of binding to RBCs.

[0307] To test for reduced binding of anti-PDL1 / CD47 antibodies to normal tissues, the binding affinity of anti-PDL1 / CD47 antibodies to primary human platelets (Stanford Blood Center) was measured by FACS using a Cytoflex (Beckman). Platelets isolated from human PBMCs were plated at 1 × 10 per well with 100 μl of PBS (Hyclone) in each well. 5 Platelets were seeded into 96-well plates at 1000 x 1000 cells / well. Platelets were incubated with serially diluted antibodies (200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, 0.0128 nM, 0.00256 nM, 0.0005 nM, 0.0001 nM, 0.00002 nM, and 0.000004 nM) and controls for 15 minutes and then washed with PBS. Platelets were then incubated with FITC-conjugated anti-human Fc antibody (Jackson Immuno-Research) diluted 1:500 for 15 minutes, washed, and resuspended in 300 μl of PBS for FACS analysis. M1#21, an anti-CD47 antibody with reduced binding to normal tissues disclosed in International Application No. PCT / CN2021 / 121314 (published as WO2022063316A1), was used as an experimental control. As shown in Figure 11, the anti-PDL1 / CD47 antibodies and PF-07257876 showed low binding to platelets. IBI322 and Hu5F9 showed strong binding to platelets, while M1#21 showed relatively weak binding to platelets compared to IBI322 and Hu5F9. The results indicated that anti-PDL1 / CD47 antibodies may be safer for therapeutic use than IBI322, Hu5F9, and M1#21.

[0308] To further examine whether or not the anti-PDL1 / CD47 antibody reduces binding to normal tissues, the binding affinity of the anti-PDL1 / CD47 antibody to primary human CD3+ T cells, primary human CD56+ NK cells, primary human CD14+ monocytic cells, and primary human CD19+ B cells was measured by FACS using a Cytoflex (Beckman). Human PBMCs were cultured at 1 × 10 per well with 100 μl of PBS (Hyclone) in each well. 5PBMCs were seeded into 96-well plates at 1000 cells / well. PBMCs were incubated with serially diluted antibodies (200 nM, 40 nM, 8 nM, 1.6 nM, 0.32 nM, 0.064 nM, 0.0128 nM, 0.00256 nM, 0.0005 nM, 0.0001 nM, 0.00002 nM, 0.000004 nM) and controls for 15 minutes, and then washed with PBS. The cells were then incubated for 15 minutes with FITC-conjugated anti-human Fc antibody (Jackson Immuno-Research), Pacific Blue-conjugated mouse anti-human CD3 antibody (Biolegend), and APC-conjugated mouse anti-human CD56 antibody (Biolegend) at a 1:500 dilution, or with FITC-conjugated anti-human Fc antibody, Pacific Blue-conjugated mouse anti-human CD14 antibody (Biolegend), and APC-conjugated mouse anti-human CD19 antibody (Biolegend) at a 1:500 dilution, followed by washing. The cells were then suspended in 300 μl of PBS for FACS analysis. As shown in Figures 12A-12D, anti-PDL1 / CD47 antibodies and PF-07257876 showed lower binding to CD3+ T cells (12A), CD56+ NK cells (12B), CD14+ monocytic cells (12C), and CD19+ B cells (12D) compared with IBI322, Hu5F9, and M1#21. The results indicated that anti-PDL1 / CD47 antibodies may be safer for therapeutic use than IBI322, Hu5F9, and M1#21.

[0309] A mixed lymphocyte reaction (MLR) is a procedure in which T cells from one donor are activated in the presence of antigen-presenting cells (APCs) from another donor. Human leukocyte antigen (HLA) mismatches between the two donors induce potent alloreactivity. MLR responses can be further controlled by manipulating the activity of the PD1 / PDL1 axis in T cells. The MLR assay was used to test anti-CD47×PDL1 antibody-mediated cytokine release. Primary human monocytes were collected from human PBMCs using a monocyte isolation kit (Stemcell Technologies) and incubated in complete RPMI (ATCC) containing 50 ng / mL GM-CSF (Peprotech) and 50 ng / mL IL-4 (Peprotech). On day 3, the medium was replaced with freshly prepared complete RPMI containing 50 ng / mL GM-CSF and 50 ng / mL IL-4. The medium was replaced with complete RPMI containing 100 ng / mL LPS (FisherSci.) to induce maturation of human monocyte-derived dendritic cells (hDCs). Suspended hDCs were collected on day 7 and stored at -80°C. Prior to the MLR assay, human primary T cells were collected from human PBMCs of another donor using a CD3+ cell isolation kit (Invitrogen). 2 × 10 5 Primary human CD3+ cells were seeded into a 96-well plate at 100 ul per well. Primary human CD3+ cells were combined with 1 nM antibody and 1 x 10 4 hDCs were co-incubated with 1000 μl of PDL1 / CD47 antibody in a total of 200 μl per well. For each group, 25 μl of supernatant was collected at 24, 48, and 72 hours after co-incubation. AlphaLISA was used to detect cytokine release. Figure 13A shows a schematic diagram of the experimental procedure. As shown in Figure 13B, the anti-PDL1 / CD47 antibody induced stronger IL-2 release than its monospecific antibody components, i.e., anti-PDL1 (3D5) and anti-CD47 (#6). The results indicated that the anti-PDL1 / CD47 antibody had an enhanced ability to activate CD3+ T cells compared with the monospecific anti-PDL1 and anti-CD47 antibodies.

[0310] Example 5 - In vivo characterization of anti-PDL1 / CD47 bispecific antibodies

[0311] The in vivo anti-tumor efficacy of anti-PDL1 / CD47 antibodies was further evaluated in the A375 hPBMC model, comparing them with monospecific anti-PDL1 and anti-CD47 antibodies and their combination. Human A375 melanoma tumor cells were implanted subcutaneously into C57BL / 6 mice (Charles River). Tumors approximately 100 mm in size were observed. 3 At the time of tumor volume reaching 1000 mcg, mice were randomly assigned to receive intraperitoneal administration twice weekly of vehicle control (10 mL / kg), 4 mg / kg anti-PDL1 / CD47, 1.8 mg / kg anti-PDL1, 3.3 mg / kg anti-CD47, or a combination of 1.8 mg / kg anti-PDL1 and 3.3 mg / kg anti-CD47 (1.8 mg / kg anti-PDL1 and 3.3 mg / kg anti-CD47 represent the same number of molecules of anti-PDL1 and anti-CD47 antibody moieties in the anti-PDL1 / CD47 bispecific antibody, respectively). Tumor volume (TV) was calculated using the formula: V = 0.5 × (a × b2), where a and b are the longest and shortest diameters of the tumor, respectively. Tumor growth inhibition (TGI) was calculated based on the formula: TGI = [1 - (Tt - T0) / (Ct - C0)] × 100%, where T0 and Tt are the mean TVs at time 0 and time t in the antibody-treated group, and C0 and Ct are the mean TVs at time 0 and time t in the vehicle group. As shown in Figure 14A, on day 14 of treatment, anti-PDL1 / CD47 showed the most potent tumor growth inhibition (TGI = 38.9%, p < 0.001 vs. control) compared with anti-PDL1 (TGI = 0.2%), anti-CD47 (TGI = 17.2%), and their combination (TGI = 25.4%). As shown in Figure 14B, no significant weight loss was observed in any of the treatment groups, suggesting that the treatment was well tolerated. The results showed that the A375 melanoma tumor model was not sensitive to treatment with either anti-PDL1 or anti-CD47 antibodies alone, but the combination of both antibodies could be more effective than either antibody alone. Furthermore, the anti-PDL1 / CD47 bispecific antibody showed superior therapeutic benefits over anti-PDL1 and anti-CD47 monospecific antibodies and their combination.

[0312] The in vivo antitumor efficacy of anti-PDL1 / CD47 was further evaluated in the MDA-MB-231 model in comparison with PF-07257876. Human MDA-MB-231 triple-negative breast cancer cells were implanted subcutaneously in NSG mice (Jackson Lab). Tumors approximately 100 mm in size were observed. 3 At the time of tumor volume reaching 1000 mcg, mice were randomly assigned to receive intraperitoneal injections of vehicle control (10 mL / kg), 1 mg / kg PF-07257876, 10 mg / kg PF-07257876, 1.2 mg / kg anti-PDL1 / CD47, or 12 mg / kg anti-PDL1 / CD47 twice weekly for 3 weeks (1 mg / kg PF-07257876 and 1.2 mg / kg anti-PDL1 / CD47 have identical molecular weights). Tumor volume (TV) was calculated using the formula: V = 0.5 × (a × b²), where a and b are the longest and shortest diameters of the tumor, respectively. Tumor growth inhibition (TGI) was calculated based on the formula: TGI = [1 - (Tt - T0) / (Ct - C0)] × 100% (where T0 and Tt are the mean TVs at time 0 and time t for the antibody-treated group, and C0 and Ct are the mean TVs at time 0 and time t for the vehicle group). As shown in Figure 15A, on day 35 of treatment, both antibodies demonstrated dose-dependent tumor inhibition. Anti-PDL1 / CD47 at 12 mg / kg (TGI = 60.5%, p < 0.0001 vs. control) demonstrated greater tumor growth inhibition compared to PF-07257876 at 10 mg / kg (TGI = 40.9%, p < 0.05 vs. control), and anti-PDL1 / CD47 at 1.2 mg / kg (TGI = 20.1%) demonstrated greater tumor growth inhibition compared to PF-07257876 at 1 mg / kg (TGI = 3.0%). As shown in Figure 15B, no significant weight loss was observed in any of the treatment groups, suggesting that the treatment was well tolerated. The results demonstrated that the anti-PDL1 / CD47 antibody had superior in vivo antitumor efficacy compared to PF-07257876.

[0313] The in vivo safety of anti-PDL1 / CD47 was evaluated in comparison with the Hu5F9 analog in an hSIRPα / hCD47 double knock-in (DKI) mouse model. hSIRPα / hCD47 DKI mice (Biocytogen Boston) were randomly administered intraperitoneally with vehicle control (10 mL / kg), 10 mg / kg Hu5F9 analog, or 12 mg / kg anti-PDL1 / CD47 on days 0, 2, and 4. Blood was collected for RBC cell count and hemoglobulin measurement on days 1, 3, 6, and 13. As shown in Figure 16A (RBC count) and Figure 16B (hemoglobulin level), the Hu5F9 analog significantly depleted RBCs on days 1, 3, and 6 (p<0.05), whereas the anti-PDL1 / CD47 antibody did not. RBC depletion was reversible after treatment, as of day 13. The results indicated that anti-PDL1 / CD47 antibodies may be therapeutically safer in vivo compared to Hu5F9.

[0314] Example 6 - In vivo characterization of anti-PDL1 / CD47 bispecific antibodies

[0315] The tolerability of anti-PDL1 / CD47 was further evaluated in a cynomolgus monkey model compared with the IBI322 analog. Cynomolgus monkeys (Pharmaron) were randomly assigned to receive intraperitoneal administration of 12 mg / kg of anti-PDL1 / CD47 antibody or 8.5 mg / kg of the IBI322 analog on days 1, 8, and 15. Body weight (BW) was measured on days -2, 1, 8, and 15. Blood samples were collected on days 0, 1, 4, 7, 8, 11, 14, 15, 18, and 21 for cell counts and biochemical analysis.

[0316] As shown in Figure 17, anti-PDL1 / CD47 and IBI322 treatment did not result in weight loss on days 1, 8, and 15. As shown in Figure 18, red blood cell (RBC), white blood cell (WBC), hematocrit (HCT), and hemoglobin (HGB) results indicated no significant differences between anti-PDL1 / CD47 and IBI322 treatment. Reticulocyte (Abretic) analysis showed a mild upregulation with both anti-PDL1 / CD47 and IBI322 treatment. Platelet (PLT) analysis showed that IBI322 treatment increased PLTs on day 21, which was statistically significant (p<0.05). These results indicate that the anti-PDL1 / CD47 antibody was well tolerated in vivo.

[0317] In addition to the various embodiments described and claimed, the disclosed subject matter also relates to other embodiments having other combinations of the features disclosed and claimed herein. As such, specific features presented herein can be combined with each other in other ways within the scope of the disclosed subject matter, such that the disclosed subject matter includes any suitable combination of the features disclosed herein. The foregoing description of specific embodiments of the disclosed subject matter has been provided for purposes of illustration and description. It is not intended to be exhaustive or to limit the disclosed subject matter to these disclosed embodiments.

[0318] It will be apparent to those skilled in the art that various modifications and variations can be made to the compositions and methods of the disclosed subject matter without departing from the spirit or scope of the disclosed subject matter. Thus, it is intended that the disclosed subject matter include modifications and variations that come within the scope of the appended claims and their equivalents.

[0319] Various publications, patents, and patent applications are cited herein, the contents of which are incorporated by reference in their entireties.

Claims

1. A multispecific antibody that binds to PDL1 and CD47, i) a first antigen-binding moiety comprising a single domain anti-PDL1 antibody that binds to PDL1; ii) a second antigen-binding moiety comprising an anti-CD47 antibody that binds to CD47; and Including, Multispecific antibodies.

2. The multispecific antibody of claim 1 , wherein the single domain antibody comprises a VHH.

3. The multispecific antibody of claim 1 or 2, wherein the single domain antibody or the VHH comprises a heavy chain variable region (VH).

4. The single domain antibody is 1×10 －７ The multispecific antibody of any one of claims 1 to 3, which binds to PDL1 with a KD of M or less.

5. The single domain antibody is 5×10 －８ The multispecific antibody of any one of claims 1 to 4, which binds to PDL1 with a KD of M or less.

6. The single domain antibody is 1×10 －８ The multispecific antibody of any one of claims 1 to 5, which binds to PDL1 with a KD of M or less.

7. The single domain antibody is about 1 x 10 －１０ M ~ approx. 5 x 10 －８ The multispecific antibody of any one of claims 1 to 6, which binds to PDL1 with a KD of M.

8. The single domain antibody has the following properties with respect to binding to PDL1: a) a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 3; b) a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 8; c) a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 11, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 12, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 13; d) a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 17, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 18; e) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 23; f) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 26, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 27, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 28; g) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 33; h) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 38; i) a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 41, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 42, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 43; j) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 46, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 47, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 48; k) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 53; l) a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 56, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 57, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO: 58; m) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 61, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 62, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 63; or n) a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO: 68; cross-competes with a reference single domain antibody comprising a heavy chain variable region comprising The multispecific antibody according to any one of claims 1 to 7.

9. The single domain antibody comprises: a) a heavy chain variable region CDR1 comprising the amino acid sequence of any one of SEQ ID NOs: 1, 6, 11, 16, 21, 26, 31, 36, 41, 46, 51, 56, 61, or 66, or a variant thereof containing up to about three amino acid substitutions; b) a heavy chain variable region CDR2 comprising the amino acid sequence of any one of SEQ ID NOs: 2, 7, 12, 17, 22, 27, 32, 37, 42, 47, 52, 57, 62, or 67, or a variant thereof containing up to about three amino acid substitutions; and c) a heavy chain variable region CDR3 comprising the amino acid sequence of any one of SEQ ID NOs: 3, 8, 13, 18, 23, 28, 33, 38, 43, 48, 53, 58, 63, or 68, or a variant thereof containing up to about three amino acid substitutions; a heavy chain variable region comprising The multispecific antibody according to any one of claims 1 to 8.

10. 10. The multispecific antibody of any one of claims 1 to 9, wherein the single domain antibody comprises a heavy chain variable region comprising a CDR1 domain, a CDR2 domain and a CDR3 domain, wherein the CDR1 domain, the CDR2 domain and the CDR3 domain comprise the CDR1 domain, the CDR2 domain and the CDR3 domain contained in a reference heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64 or 69, respectively.

11. The multispecific antibody of any one of claims 1 to 10, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 1, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 2, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:

3.

12. The multispecific antibody of any one of claims 1 to 10, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 6, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 7, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:

8.

13. The multispecific antibody of any one of claims 1 to 10, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 11, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 12, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:

13.

14. The multispecific antibody of any one of claims 1 to 10, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 16, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 17, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:

18.

15. The multispecific antibody of any one of claims 1 to 10, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 21, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 22, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:

23.

16. The multispecific antibody of any one of claims 1 to 10, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence shown in SEQ ID NO: 26, a heavy chain variable region CDR2 comprising the amino acid sequence shown in SEQ ID NO: 27, and a heavy chain variable region CDR3 comprising the amino acid sequence shown in SEQ ID NO:

28.

17. The multispecific antibody of any one of claims 1 to 10, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 31, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 32, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

33.

18. The multispecific antibody of any one of claims 1 to 10, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 36, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 37, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

38.

19. The multispecific antibody of any one of claims 1 to 10, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 41, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 42, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

43.

20. The multispecific antibody of any one of claims 1 to 10, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 46, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 47, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

48.

21. The multispecific antibody of any one of claims 1 to 10, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 51, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 52, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

53.

22. The multispecific antibody of any one of claims 1 to 10, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 56, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 57, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

58.

23. The multispecific antibody of any one of claims 1 to 10, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 61, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 62, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

63.

24. The multispecific antibody of any one of claims 1 to 10, wherein the single domain antibody comprises a heavy chain variable region CDR1 comprising the amino acid sequence set forth in SEQ ID NO: 66, a heavy chain variable region CDR2 comprising the amino acid sequence set forth in SEQ ID NO: 67, and a heavy chain variable region CDR3 comprising the amino acid sequence set forth in SEQ ID NO:

68.

25. 25. The multispecific antibody of any one of claims 1 to 24, wherein said single domain antibody comprises a heavy chain variable region comprising an amino acid sequence having at least about 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 4, 9, 14, 19, 24, 29, 34, 39, 44, 49, 54, 59, 64 or 69.

26. The multispecific antibody of any one of claims 1 to 25, wherein the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:

4.

27. The multispecific antibody of any one of claims 1 to 25, wherein the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:

9.

28. The multispecific antibody of any one of claims 1 to 25, wherein the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:

14.

29. The multispecific antibody of any one of claims 1 to 25, wherein the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:

19.

30. The multispecific antibody of any one of claims 1 to 25, wherein the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:

24.

31. The multispecific antibody of any one of claims 1 to 25, wherein the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:

29.

32. The multispecific antibody of any one of claims 1 to 25, wherein the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:

34.

33. The multispecific antibody of any one of claims 1 to 25, wherein the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence shown in SEQ ID NO:

39.

34. The multispecific antibody of any one of claims 1 to 25, wherein the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

44.

35. The multispecific antibody of any one of claims 1 to 25, wherein the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

49.

36. The multispecific antibody of any one of claims 1 to 25, wherein the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

54.

37. The multispecific antibody of any one of claims 1 to 25, wherein the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

59.

38. The multispecific antibody of any one of claims 1 to 25, wherein the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

64.

39. The multispecific antibody of any one of claims 1 to 25, wherein the single domain antibody comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

69.

40. The multispecific antibody of any one of claims 1 to 39, wherein the single domain antibody comprises a humanized framework.

41. The multispecific antibody of any one of claims 1 to 40, wherein the second antigen-binding portion comprises a heavy chain variable region (VH) comprising: (1) CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 71; (2) CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 72; and (3) CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 73; and a light chain variable region (VL) comprising: (1) CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 74; (2) CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 75; and (3) CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:

76.

42. The multispecific antibody of any one of claims 1 to 40, wherein the second antigen-binding portion comprises a heavy chain variable region (VH) comprising: (1) CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 81; (2) CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 82; and (3) CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 83; and a light chain variable region (VL) comprising: (1) CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 84; (2) CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 85; and (3) CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:

86.

43. 42. The multispecific antibody of any one of claims 1 to 41, wherein the second antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 77 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

78.

44. 43. The multispecific antibody of any one of claims 1 to 42, wherein the second antigen-binding portion comprises a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 87 and a light chain variable region comprising the amino acid sequence set forth in SEQ ID NO:

88.

45. The multispecific antibody of any one of claims 1 to 44, wherein the anti-CD47 antibody comprises a human antibody.

46. 46. ​​The multispecific antibody of any one of claims 1 to 45, wherein the second antigen-binding portion comprises an anti-CD47 antibody comprising two antibody heavy chains and two antibody light chains.

47. The multispecific antibody of any one of claims 1 to 46, wherein the first antigen-binding portion comprises one or more anti-PDL1 antibodies.

48. The multispecific antibody of any one of claims 1 to 47, wherein the first antigen-binding portion comprises two anti-PDL1 antibodies.

49. The multispecific antibody of any one of claims 1 to 48, wherein the C-terminus of at least one of the two anti-CD47 light chains is linked to the anti-PDL1 antibody of the first antigen-binding moiety.

50. 50. The multispecific antibody of claim 49, wherein the C-terminus of each of the two anti-CD47 light chains is linked to an anti-PDL1 antibody of the first antigen-binding moiety.

51. 51. The multispecific antibody of any one of claims 1 to 50, wherein the N-terminus of at least one of the two anti-CD47 light chains is linked to the anti-PDL1 antibody of the first antigen-binding moiety.

52. 52. The multispecific antibody of claim 51 , wherein the N-terminus of each of the two anti-CD47 light chains is linked to the anti-PDL1 antibody of the first antigen-binding moiety.

53. 53. The multispecific antibody of any one of claims 1 to 52, wherein the C-terminus of at least one of the two anti-CD47 heavy chains is linked to the anti-PDL1 antibody of the first antigen-binding moiety.

54. 54. The multispecific antibody of claim 53, wherein the C-terminus of each of the two anti-CD47 heavy chains is linked to the anti-PDL1 antibody of the first antigen-binding moiety.

55. 55. The multispecific antibody of any one of claims 1 to 54, wherein the N-terminus of at least one of the two anti-CD47 heavy chains is linked to the anti-PDL1 antibody of the first antigen-binding moiety.

56. 56. The multispecific antibody of claim 55, wherein the N-terminus of each of the two anti-CD47 heavy chains is linked to an anti-PDL1 antibody of the first antigen-binding moiety.

57. 57. The multispecific antibody of any one of claims 1 to 56, wherein the first antigen-binding moiety is linked to the second antigen-binding moiety via a linker.

58. 58. The multispecific antibody of claim 57, wherein the linker is a peptide linker.

59. 59. The multispecific antibody of claim 58, wherein the peptide linker comprises from about 4 to about 30 amino acids.

60. 59. The multispecific antibody of claim 57 or 58, wherein the peptide linker comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 83 to 135.

61. 61. The multispecific antibody of any one of claims 1 to 60, wherein the anti-CD47 antibody of the second antigen-binding portion comprises an Fc region.

62. The multispecific antibody of any one of claims 1 to 61, wherein the Fc region comprises a human Fc region.

63. The multispecific antibody of any one of claims 1 to 62, wherein the Fc region comprises an Fc region selected from the group consisting of IgG, IgA, IgD, IgE and IgM Fc regions.

64. The multispecific antibody of any one of claims 1 to 63, wherein the Fc region comprises an Fc region selected from the group consisting of an IgG1, IgG2, IgG3, and IgG4 Fc region.

65. The multispecific antibody of any one of claims 1 to 64, wherein the Fc region comprises an IgG4 Fc region.

66. The multispecific antibody of any one of claims 1 to 64, wherein the Fc region comprises an IgG1 Fc region.

67. 67. The multispecific antibody of claim 66, wherein the IgGl Fc region comprises one or more mutations that enhance antibody-dependent cellular cytotoxicity (ADCC).

68. 68. The multispecific antibody of any one of claims 1 to 67, wherein the multispecific antibody comprises a full-length immunoglobulin, a single-chain Fv (scFv) fragment, a Fab fragment, a Fab' fragment, a F(ab')2, an Fv fragment, a disulfide-stabilized Fv fragment (dsFv), a (dsFv)2, a VHH, a VHH-Fc fusion, an Fv-Fc fusion, a scFv-Fc fusion, a scFv-Fv fusion, a diabody, a tribody, a tetrabody, or any combination thereof.

69. The multispecific antibody of any one of claims 1 to 68, wherein the multispecific antibody is a bispecific antibody.

70. i) a first antigen-binding moiety comprising a single domain anti-PDL1 antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 51, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 52, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 53; and ii) a second antigen-binding portion comprising an anti-CD47 antibody comprising a heavy chain variable region (VH) comprising: (1) CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO:71; (2) CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO:72; and (3) CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO:73; and a light chain variable region (VL) comprising: (1) CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO:74; (2) CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO:75; and (3) CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO:76; Including, 70. The multispecific antibody of any one of claims 1 to 69.

71. i) a first antigen-binding moiety comprising a single domain anti-PDL1 antibody comprising a heavy chain variable region CDR1 comprising amino acids having the sequence set forth in SEQ ID NO: 51, a heavy chain variable region CDR2 comprising amino acids having the sequence set forth in SEQ ID NO: 52, and a heavy chain variable region CDR3 comprising amino acids having the sequence set forth in SEQ ID NO: 53; and ii) a second antigen-binding portion comprising an anti-CD47 antibody comprising a heavy chain variable region (VH) comprising: (1) CDR-H1 comprising the amino acid sequence set forth in SEQ ID NO: 81; (2) CDR-H2 comprising the amino acid sequence set forth in SEQ ID NO: 82; and (3) CDR-H3 comprising the amino acid sequence set forth in SEQ ID NO: 83; and a light chain variable region (VL) comprising: (1) CDR-L1 comprising the amino acid sequence set forth in SEQ ID NO: 84; (2) CDR-L2 comprising the amino acid sequence set forth in SEQ ID NO: 85; and (3) CDR-L3 comprising the amino acid sequence set forth in SEQ ID NO: 86; Including, 70. The multispecific antibody of any one of claims 1 to 69.

72. i) a first antigen-binding moiety comprising a single domain anti-PDL1 antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 54; ii) a second antigen-binding portion comprising an anti-CD47 antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 77 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 78; and Including, A multispecific antibody according to any one of claims 1 to 71.

73. i) a first antigen-binding moiety comprising a single domain anti-PDL1 antibody comprising a heavy chain variable region comprising the amino acid sequence set forth in SEQ ID NO: 54; ii) a second antigen-binding portion comprising an anti-CD47 antibody comprising a heavy chain variable region (VH) comprising the amino acid sequence set forth in SEQ ID NO: 87 and a light chain variable region (VL) comprising the amino acid sequence set forth in SEQ ID NO: 88; and Including, A multispecific antibody according to any one of claims 1 to 71.

74. 74. The multispecific antibody of any one of claims 1 to 73, comprising a first chain comprising the amino acid sequence set forth in SEQ ID NO: 136 and a second chain comprising the amino acid sequence set forth in SEQ ID NO:

137.

75. 74. The multispecific antibody of any one of claims 1 to 73, comprising a first chain comprising the amino acid sequence set forth in SEQ ID NO: 138 and a second chain comprising the amino acid sequence set forth in SEQ ID NO:

139.

76. 74. The multispecific antibody of any one of claims 1 to 73, comprising a first chain comprising the amino acid sequence set forth in SEQ ID NO: 140 and a second chain comprising the amino acid sequence set forth in SEQ ID NO:

141.

77. 77. An immunoconjugate comprising a multispecific antibody according to any one of claims 1 to 76, linked to a therapeutic agent.

78. 78. The immunoconjugate of claim 77, wherein the therapeutic agent is a cytotoxin.

79. 79. The immunoconjugate of claim 78, wherein the therapeutic agent is a radioisotope.

80. An antigen-recognizing receptor comprising an extracellular antigen-binding domain comprising the multispecific antibody of any one of claims 1 to 79.

81. 81. The antigen recognition receptor of claim 80, which is a chimeric antigen receptor (CAR) or a recombinant T cell receptor.

82. The antigen recognition receptor of claim 80 or 81, which is a CAR.

83. The antigen-recognizing receptor according to any one of claims 80 to 82, wherein the multispecific antibody contained in the extracellular antigen-binding domain comprises a VHH, scFv, Fab, Fab', di-scFv, or any combination thereof.

84. The antigen-recognizing receptor according to any one of claims 80 to 83, wherein the multispecific antibody comprised in the extracellular antigen-binding domain comprises an anti-PDL1 VHH and an anti-CD47 scFv.

85. The antigen-recognizing receptor of claim 84, wherein the anti-PDL1 VHH and the anti-CD47 scFv are linked by a peptide linker.

86. An immune response cell comprising the antigen-recognizing receptor according to any one of claims 80 to 85.

87. 87. The immune response cell of claim 86, wherein the immune response cell is selected from the group consisting of a T cell, a natural killer (NK) cell, a cytotoxic T lymphocyte (CTL), a regulatory T cell, a natural killer T (NKT) cell, and a myeloid cell.

88. 88. The immune response cell of claim 87, wherein the immune response cell is a T cell.

89. 100. A pharmaceutical composition comprising: a) a multispecific antibody according to any one of claims 1 to 76; an immunoconjugate according to any one of claims 77 to 79; an immune response cell according to any one of claims 86 to 88; and b) a pharmaceutically acceptable carrier.

90. One or more nucleic acids encoding the multispecific antibody of any one of claims 1 to 76.

91. 91. One or more vectors comprising the nucleic acid of claim 90.

92. 92. A host cell comprising the nucleic acid of claim 90 or the vector of claim 91.

93. 93. A method for preparing a multispecific antibody according to any one of claims 1 to 76, comprising expressing said multispecific antibody in a host cell according to claim 92 and isolating said multispecific antibody from said host cell.

94. 90. A method of reducing tumor burden in a subject, comprising administering to the subject an effective amount of a multispecific antibody according to any one of claims 1 to 76, an immunoconjugate according to any one of claims 77 to 79, or a pharmaceutical composition according to claim 89.

95. 95. The method of claim 94, wherein the method reduces the number of tumor cells.

96. 96. The method of claim 94 or 95, wherein the method reduces tumor size.

97. 97. The method of any one of claims 94 to 96, wherein the method eradicates the tumor in the subject.

98. 98. The method of any one of claims 94 to 97, wherein the tumor exhibits high microsatellite instability (MSI).

99. 99. The method of any one of claims 94 to 98, 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 cancer, endometrial cancer, stomach cancer, bile duct cancer, head and neck cancer, blood cancer, and combinations thereof.

100. 90. A method for treating and / or preventing cancer, comprising administering to a subject an effective amount of a multispecific antibody according to any one of claims 1 to 76, an immunoconjugate according to any one of claims 77 to 79, or a pharmaceutical composition according to claim 89.

101. 90. A method for extending the survival of a subject with cancer, comprising administering to said subject an effective amount of a multispecific antibody according to any one of claims 1 to 76, an immunoconjugate according to any one of claims 77 to 79, or a pharmaceutical composition according to claim 89.

102. 102. The method of claim 100 or 101, wherein the cancer exhibits high microsatellite instability (MSI).

103. 103. The method of any one of claims 100 to 102, 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, stomach cancer, bile duct cancer, head and neck cancer, blood cancer, and combinations thereof.

104. A multispecific antibody according to any one of claims 1 to 76 for use as a medicament.

105. 77. The multispecific antibody of any one of claims 1 to 76 for use in the treatment of cancer.

106. 90. The pharmaceutical composition of claim 89 for use as a medicament.

107. 90. The pharmaceutical composition of claim 89 for use in the treatment of cancer.

108. 108. The multispecific antibody of claim 105 or the pharmaceutical composition of claim 107, wherein the cancer exhibits high microsatellite instability (MSI).

109. 108. The multispecific antibody of claim 105 or the pharmaceutical composition of claim 107, 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 cancer, bile duct cancer, head and neck cancer, blood cancer, and combinations thereof.

110. 90. A kit comprising an antibody according to any one of claims 1 to 76, an immunoconjugate according to any one of claims 77 to 79, a pharmaceutical composition according to claim 89, one or more nucleic acids according to claim 90, one or more vectors according to claim 91, or an immune response cell according to claims 86 to 88.

111. 111. The kit of claim 110, further comprising written instructions for treating and / or preventing a neoplasm.

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