JP2019506863A - Double-binding protein for PD-L1 and KDR - Google Patents

Abstract

Bispecific binding proteins such as bispecific antibodies that bind to both human PD-L1 and human KDR are provided. This bispecific binding protein is useful for treating diseases and conditions characterized by immunosuppression and / or excessive angiogenesis.

Description

This application claims priority to US Provisional Application No. 62 / 290,350, filed February 2, 2016. The contents of this application are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION Provided herein are bispecific binding proteins comprising a first binding region that binds human PD-L1 and a second binding region that binds human KDR. Also, methods of making bispecific binding proteins and using bispecific binding proteins to treat diseases or conditions where it is desirable to reduce or inhibit immunosuppression or reduce or inhibit angiogenesis A method is also provided.

  Program death 1 (PD-1) is a member of the CD28 family of receptors including CD28, CTLA-4, PD-1, ICOS and BTLA (Freeman et al. (2000) J Exp Med 192: 1027-34. Latchman et al. (2001) Nat Immunol 2: 261-8). PD-1 is an inducible immunosuppressive receptor that is primarily upregulated on activated T cells and B cells during the progression of immunopathological conditions. Interaction of PD-1 with its ligand PD-L1 is through inhibition of TCR and BCR-mediated proliferation, and endogenous PD-1-mediated negative signaling of the immunoreceptor tyrosine-based inhibitory motif (ITIM) Induction of cytokine production and apoptosis of antigen-specific T cells (Agata et al. (1996) Int. Immunol. 8: 765, Unkeless and Jin. (1997) Curr. Opin. Immunol. 9: 338-343, Okzaki et al., (2001) PNAS 98: 13866-71, Dong et al. (2002) Nat. Med. 8: 793-800). PD-L1 is a cell surface glycoprotein and is a major ligand for PD-1. PD-L1 is also inducible on lymphoid and non-lymphoid peripheral tissues after cell activation. PD-L1 is up-regulated in various diseased cell types including cancer cells and stromal cells in addition to immune cells and plays an active role in immune suppression during the course of disease progression (Iwai et al. (2002) PNAS 99: 12293-7, Ohigashi et al. (2005) Clin Cancer Res 11: 2947-53). Up-regulation of PD-L1 has been associated with poor clinical outcome in various cancers and viral infections (Hofmeyer et al. (2011) J. BioMed. Biotech. 2011-1-9, McDermott and Atkins. (2013) Cancer Med. 2: 662-73). Blocking PD-1 or PD-L1 by antibodies promoted increased CD8 T cell infiltration, CTL activity and the presence of Th1 cytokine IFN-γ in the preclinical and clinical settings (Zhou et al. (2010) J Immunol.185: 5082-92, Nomi et al. (2007) Clin Cancer Res.13: 2152-7, Flies et al. (2011) Yale J. Bio.Med.48: 409-21, Zitvogel and Kroemer. (2012) Onco Immunol. 1: 1223-25). PD-L1 antibodies as immunomodulators have been shown to be effective when used as monotherapy or in combination with antibodies against other immunosuppressive molecules.

  VEGFR is a receptor tyrosine kinase and belongs to the same receptor family as PDGF and fibroblast growth factor (FGF). KDR (also known as VEGFR2) is a receptor that binds to VEGF isoforms A, C, D, and E. This plays a role in the endothelial cell differentiation and mitogenic, vascularization and permeation promoting effects of VEGF. KDR is a 200 kDa glycoprotein consisting of seven Ig-like loops in the extracellular domain, a transmembrane domain, and two intracellular tyrosine kinase domains separated by a kinase insert. The second and third Ig-like loops are high affinity ligand binding domains of VEGF, while the first and fourth Ig-like loops regulate ligand binding and receptor dimerization, respectively. VEGF binds to KDR with a Kd of 75-250 pM compared to the 25 pM Kd of VEGFR1. KDR is mainly expressed on the cell surface of vascular endothelial cells. KDR is also found on the cell surface of hematopoietic cells, vascular smooth muscle cells (VSMC), and some malignant cells.

  Angiogenesis is a highly complex process that develops new blood vessels, including proliferation and migration of capillary endothelial cells from existing blood vessels and tissue infiltration, cell assembly into tubular structures, newly formed tubular Includes the connection of the assembly to the closed circuit vasculature, as well as the maturation of newly formed capillaries.

  Angiogenesis is important in normal physiological processes including embryonic development, follicular growth and wound healing. Excessive neovascularization also results in neovascularization in neoplastic diseases and in non-neoplastic diseases such as age-related macular degeneration (AMD), diabetic retinopathy and neovascular glaucoma. Anti-angiogenic therapy targeting vascular endothelial growth factor (VEGF) with ranibizumab (LUCENTIS®) has been shown to be effective in slowing the progression of AMD.

Angiogenesis plays an important role in primary tumor growth and metastasis because growth and metastasis depend on the formation of new blood vessels. Without angiogenesis due to angiogenesis, the tumor becomes necrotic, apoptotic, or cannot grow to an appreciable size. Tumor angiogenesis includes several processes including endothelial cell activation, proliferation, migration, and tissue invasion from existing blood vessels. These processes are triggered by the production of angiogenic growth factors such as VEGF by tumor cells and their surrounding stroma.
KDR has become an important target for anticancer therapy. Targeting KDR increases the probability of suppressing VEGF signaling, and thus tumor growth, since many tumors increase the amount of VEGF secreted, but the number of KDR molecules remains relatively constant. Inhibits.

Freeman et al. (2000) J Exp Med 192: 1027-34. Latchman et al. (2001) Nat Immunol 2: 261-8 Agata et al. (1996) Int. Immunol. 8: 765 Unkeless and Jin. (1997) Curr. Opin. Immunol. 9: 338-343 Okzaki et al. (2001) PNAS 98: 13866-71. Dong et al. (2002) Nat. Med. 8: 793-800 Iwai et al. (2002) PNAS 99: 12293-7 Ohashi et al. (2005) Clin Cancer Res 11: 2947-53. Hofmeyer et al. (2011) J. Org. BioMed. Biotech. 2011-1-9 McDermott and Atkins. (2013) Cancer Med. 2: 662-73 Zhou et al. (2010) J. et al. Immunol. 185: 5082-92 Nomi et al. (2007) Clin Cancer Res. 13: 2152-7 Flies et al. (2011) Yale J. et al. Bio. Med. 48: 409-21 Zitvogel and Kroemer. (2012) Onco Immunol. 1: 1223-25

  Bispecific binding proteins that bind to human PD-L1 and human KDR are provided. In certain embodiments, the bispecific binding protein binds to PD-L1 and blocks the interaction between PD-L1 and PD-1. By blocking the interaction between PD-L1 and PD-1, such bispecific binding proteins are useful for reducing or inhibiting immune suppression. By blocking the interaction between VEGF and KDR, such bispecific binding proteins are useful for reducing or inhibiting angiogenesis. Bispecific binding proteins are particularly useful in combining the ability to inhibit both immunosuppression and angiogenesis in a single agent.

  In one embodiment, a bispecific antibody that binds human PD-L1 and human KDR is provided. Bispecific antibodies comprise a first antigen binding site that binds human PD-L1 and a second antigen binding site that binds human KDR. Also, a nucleic acid molecule encoding a bispecific antibody, as well as an expression vector comprising the nucleic acid, capable of expressing the nucleic acid in a prokaryotic or eukaryotic host cell and thus resulting in production of the bispecific antibody Vectors are also provided. Also provided are host cells comprising expression vectors for recombinant production of bispecific antibodies.

  Provided herein are bispecific antibodies comprising a scFv that binds PD-L1 linked to an antibody that binds KDR. In one embodiment, PD-L1 scFv is linked to the carboxy terminus of the heavy chain constant domain of IgG that binds to KDR. In another embodiment, the PD-L1 scFv is linked to the carboxy terminus of the light chain constant domain of IgG that binds to KDR. In another embodiment, PD-L1 scFv is linked to the amino terminus of an IgG heavy chain variable domain that binds to KDR. In another embodiment, the PD-L1 scFv is linked to the amino terminus of the light chain variable domain of IgG that binds to KDR.

  Provided herein are bispecific antibodies comprising a scFv that binds KDR linked to an antibody that binds PD-L1. In one embodiment, the KDR scFv is linked to the carboxy terminus of the heavy chain constant domain of IgG that binds to PD-L1. In another embodiment, the KDR scFv is linked to the carboxy terminus of the light chain constant domain of IgG that binds to PD-L1. In another embodiment, the KDR scFv is linked to the amino terminus of the IgG heavy chain variable domain that binds to PD-L1. In another embodiment, the PD-L1 scFv is linked to the amino terminus of an IgG light chain variable domain that binds to PD-L1.

In one embodiment, the amino acid sequence of scFv that binds to PD-L1 is:
(SEQ ID NO: 1). CDRs are underlined.

In one embodiment, the amino acid sequence of the scFv that binds to KDR is:
(SEQ ID NO: 2). CDRs are underlined.

In one embodiment, the amino acid sequence of the scFv that binds to KDR is:
(SEQ ID NO: 3). CDRs are underlined.

In one embodiment, the amino acid sequence of the heavy chain variable domain of IgG that binds to PD-L1 is:
(SEQ ID NO: 4). CDRs are underlined.

In one embodiment, the amino acid sequence of the light chain variable domain of IgG that binds to PD-L1 is:
(SEQ ID NO: 5). CDRs are underlined.

In one embodiment, the amino acid sequence of the heavy chain variable domain of IgG that binds to KDR is:
(SEQ ID NO: 6). CDRs are underlined.

In one embodiment, the amino acid sequence of the light chain variable domain of IgG that binds to KDR is:
(SEQ ID NO: 7). CDRs are underlined.

In one embodiment, the amino acid sequence of the heavy chain variable domain of IgG that binds to KDR is:
(SEQ ID NO: 8). CDRs are underlined.

In one embodiment, the amino acid sequence of the light chain variable domain of IgG that binds to KDR is:
(SEQ ID NO: 9). CDRs are underlined.

  In one embodiment, the bispecific antibody comprises a heavy chain variable domain that binds human PD-L1 and has three complementarity determining regions (CDRs). Therefore, the amino acid sequence of CDR1 is GFTFSAYRMF (SEQ ID NO: 10), the amino acid sequence of CDR2 is SIYPSGGITFYADSVKG (SEQ ID NO: 11), and the amino acid sequence of CDR3 is IKLGTVTTVDY (SEQ ID NO: 12).

  In one embodiment, the bispecific antibody comprises a light chain variable domain that binds human PD-L1 and has three CDRs. Therefore, the amino acid sequence of CDR1 is TGTSSDVGAYNYVS (SEQ ID NO: 13), the amino acid sequence of CDR2 is DVSNRPS (SEQ ID NO: 14), and the amino acid sequence of CDR3 is SSYTSSSTRV (SEQ ID NO: 15).

  In one embodiment, the bispecific antibody comprises a heavy chain variable domain that binds human KDR and has three CDRs. Here, the amino acid sequence of CDR1 is GFTFSWYVMG (SEQ ID NO: 16), the amino acid sequence of CDR2 is SIYPSGGATNYADSVKG (SEQ ID NO: 17), and the amino acid sequence of CDR3 is GNYFDY (SEQ ID NO: 18).

  In one embodiment, the bispecific antibody comprises a light chain variable domain that binds human KDR and has three CDRs. Here, the amino acid sequence of CDR1 is SGEKLGDEYAS (SEQ ID NO: 19), the amino acid sequence of CDR2 is QDNKRPS (SEQ ID NO: 20), and the amino acid sequence of CDR3 is QAWDSTLL (SEQ ID NO: 21).

  In one embodiment, the bispecific antibody comprises a heavy chain variable domain that binds to human KDR and has three CDRs. Here, the amino acid sequence of CDR1 is GFTFSWYVMG (SEQ ID NO: 22), the amino acid sequence of CDR2 is SIYPQGGATSYADSVK (SEQ ID NO: 23), and the amino acid sequence of CDR3 is GNYFDY (SEQ ID NO: 24).

  In one embodiment, the bispecific antibody comprises a light chain variable domain that binds human KDR and has three CDRs. Here, the amino acid sequence of CDR1 is RASQSVSSNYFG (SEQ ID NO: 25), the amino acid sequence of CDR2 is GASSRAT (SEQ ID NO: 26), and the amino acid sequence of CDR3 is QQFDSLPLT (SEQ ID NO: 27).

In one embodiment, the amino acid sequence of the heavy chain of IgG that binds to PD-L1 is:
(SEQ ID NO: 28). CDRs are underlined. In some examples, LL (bold) may be mutated to other residues such as AA.

In one embodiment, the amino acid sequence of the light chain of IgG that binds to PD-L1 is:
(SEQ ID NO: 29). CDRs are underlined.

In one embodiment, the amino acid sequence of the heavy chain of IgG that binds to KDR is:
(SEQ ID NO: 30). CDRs are underlined. In some examples, LL (bold) may be mutated to other residues such as AA.

In one embodiment, the amino acid sequence of the light chain of IgG that binds to KDR is:
(SEQ ID NO: 31). CDRs are underlined.

In one embodiment, the amino acid sequence of the heavy chain of IgG that binds to KDR is:
(SEQ ID NO: 32). CDRs are underlined. In some examples, LL (bold) may be mutated to other residues such as AA.

In one embodiment, the amino acid sequence of the light chain of IgG that binds to KDR is:
(SEQ ID NO: 33). CDRs are underlined.

In one embodiment, the amino acid sequence of scFv that binds to PD-L1 is:
(SEQ ID NO: 57). CDRs are underlined. Also underlined with two cysteines “C”, which are mutated from G.

  In one embodiment, the bispecific antibody comprises a heavy chain variable domain that binds human PD-L1 and has three complementarity determining regions (CDRs). Here, the amino acid sequences of CDR1, CDR2 and CDR3 are as follows: GFTFSWYLMK, YIGSSGGFTAYADSVKG, and EDDFGAMDV (SEQ ID NOs: 58 to 60).

  In one embodiment, the bispecific antibody comprises a light chain variable domain that binds human PD-L1 and has three complementarity determining regions (CDRs). Thus, the amino acid sequences of CDR1, CDR2 and CDR3 are as follows: RASQTVSKYFNW, ATSTLQS, and QQSYTTPWT (SEQ ID NOs: 61 to 63).

In one embodiment, the amino acid sequence of the heavy chain variable domain of IgG that binds to PD-L1 is:
(SEQ ID NO: 64). CDRs are underlined.

In one embodiment, the amino acid sequence of the heavy chain of IgG that binds to PD-L1 is:
(SEQ ID NO: 66). CDRs are underlined. It is underlined with two alanines [AA], which is mutated from leucine LL.

In one embodiment, the amino acid sequence of the light chain variable domain of IgG that binds to PD-L1 is:
(SEQ ID NO: 65). CDRs are underlined.

In one embodiment, the amino acid sequence of the light chain variable domain of IgG that binds to PD-L1 is:
(SEQ ID NO: 67). CDRs are underlined.

  The present invention also provides conjugates of bispecific binding proteins to, for example but not limited to, imaging agents, therapeutic agents or cytotoxic agents.

  The present invention further provides a composition comprising a bispecific binding protein and at least one pharmaceutically acceptable carrier.

  In one embodiment, a fusion protein capable of binding to human PDL1 and human KDR is provided. The fusion protein may comprise a moiety that binds to human PD-L1 and a moiety that binds to human KDR. In one embodiment, the portion of the fusion protein that binds human PD-L1 is an antibody or a PD-L1 binding fragment thereof. In one embodiment, the portion of the fusion protein that binds human KDR is an antibody or KDR-binding fragment thereof. In one embodiment, the portion of the fusion protein that binds to human PD-L1 is an antibody or a PD-L1 binding fragment thereof, and the portion of the fusion protein that binds to human KDR is an antibody or a KDR binding fragment thereof.

  Provided is a method of inhibiting the interaction of human PD1 and human PD1L in a subject comprising administering an effective amount of a bispecific antibody or fragment thereof disclosed herein. A method of inhibiting immunosuppression mediated by human PD-L1, comprising administering an effective amount of a bispecific antibody or fragment thereof disclosed herein, or a fusion protein disclosed herein Is further provided.

  A method of stimulating an immune response against a cell or tissue expressing human PD-L1, comprising an effective amount of a bispecific antibody or fragment thereof disclosed herein, or a fusion disclosed herein. Further provided is a method comprising administering a protein to a subject. In certain embodiments, the cells or tissues that express human PD-L1 are neoplastic cells or infected cells.

  A method of neutralizing human KDR or mouse KDR activation comprising contacting a cell with an effective amount of a bispecific antibody or fragment thereof of the present invention is provided.

  Also provided is a method of inhibiting angiogenesis comprising administering to a subject an effective amount of a bispecific antibody or fragment thereof of the present invention.

  Also provided is a method of reducing tumor growth comprising administering to a subject an effective amount of a bispecific antibody or fragment thereof of the present invention.

  A method of treating a neoplastic disease in a subject comprising administering to the subject an effective amount of a bispecific antibody or fragment thereof disclosed herein, wherein the neoplastic disease is lung cancer, colorectal Disclosed herein is a method selected from the group consisting of cancer, renal cell carcinoma, glioblastoma, ovarian cancer, bladder cancer, gastric cancer, multiple myeloma, non-small cell lung cancer and pancreatic cancer.

Provided herein are the following numbered, non-limiting embodiments of the present invention.
1. A bispecific binding protein comprising a first region that binds to human PDL1 and a second region that binds to human KDR.
2. The bispecific binding protein of embodiment 1, which is a bispecific antibody.
3. The bispecific binding protein of embodiment 1, which is a fusion protein.
4). Bispecific antibodies comprising IgG, IgA, IgE, or IgD and scFv.
5. The bispecific antibody of embodiment 4, comprising an IgG that binds PD-L1 and an scFv that binds KDR.
6). The bispecific antibody of embodiment 4, comprising an IgG that binds to KDR and an scFv that binds PD-L1.
7). It comprises a heavy chain variable domain that binds to human PD-L1 and has three complementarity determining regions (CDRs), wherein the CDR1, CDR2, and CDR3 amino acid sequences are SEQ ID NOs: 10-12, or SEQ ID NOs: 58-60, respectively The bispecific antibody of embodiment 5 or 6, wherein
8). Embodiment 5 comprising a light chain variable domain that binds to human PD-L1 and has three CDRs, wherein the amino acid sequences of CDR1, CDR2, and CDR3 are SEQ ID NO: 13-15, or SEQ ID NO: 61-63, respectively. Or 6 bispecific antibodies.
9. The bispecific antibody of embodiment 5 or 6, comprising a heavy chain variable domain that binds to human KDR and has three CDRs, wherein the amino acid sequences of CDR1, CDR2, and CDR3 are SEQ ID NOs: 16-18, respectively .
10. The bispecific antibody of embodiment 5 or 6, comprising a light chain variable domain that binds human KDR and has three CDRs, wherein the amino acid sequences CDR1, CDR2, and CDR3 are SEQ ID NOs: 19-21, respectively.
11. The bispecific antibody of embodiment 5 or 6, comprising a heavy chain variable domain that binds to human KDR and has three CDRs, wherein the amino acid sequences CDR1, CDR2, and CDR3 are SEQ ID NOs: 22-24, respectively.
12 The bispecific antibody of embodiment 5 or 6, comprising a light chain variable domain that binds human KDR and has three CDRs, wherein the amino acid sequences CDR1, CDR2, and CDR3 are SEQ ID NOs: 25-27, respectively.
13. Comprising heavy and light chains, the heavy and light chains comprising SEQ ID NOs: 34 and 31, SEQ ID NOs: 30 and 35, SEQ ID NOs: 36 and 31, SEQ ID NOs: 30 and 37, SEQ ID NOs: 38 and 33, SEQ ID NOs: 32 and 39 SEQ ID NOS: 40 and 33, SEQ ID NOS: 32 and 41, SEQ ID NOS: 42 and 29, SEQ ID NOS: 28 and 43, SEQ ID NOS: 44 and 29, SEQ ID NOS: 28 and 45, SEQ ID NOS: 46 and 29, SEQ ID NOS: 28 and 47, SEQ ID NO: 48 and 29, SEQ ID NO: 28 and 49, SEQ ID NO: 51 and 50, SEQ ID NO: 52 and 50, SEQ ID NO: 53 and 50, SEQ ID NO: 54 and 29, SEQ ID NO: 55 and 29, and SEQ ID NO: 56 and 29 The bispecific antibody of embodiment 5 or 6, comprising the sequence of each heavy / light chain pair selected from the group.
14 A method for treating a patient in need of reduced immunosuppression or angiogenesis, wherein the bispecific of any one of embodiments 1-3 is used for a patient in need of such reduction of immunosuppression or angiogenesis. Administering a sex binding protein or a bispecific binding protein of any one of embodiments 4-13.
15. A method for treating cancer, wherein a patient in need thereof is administered any one of the bispecific binding proteins of embodiments 1-3 or any one of the bispecific antibodies of embodiments 4-13 Including the method.
16. Embodiment 15 wherein the cancer is selected from the group consisting of lung cancer, colorectal cancer, renal cell cancer, glioblastoma, ovarian cancer, bladder cancer, gastric cancer, multiple myeloma, non-small cell lung cancer, and pancreatic cancer. The method described in 1.
17. An isolated nucleic acid molecule encoding any one of the bispecific binding proteins of embodiments 1-3, any one of the bispecific antibodies of embodiments 4-13, or a polypeptide chain thereof.
18. A vector comprising the nucleic acid molecule of embodiment 17.
19. A cultured host cell comprising the vector of embodiment 18.
20. 20. A method of producing a polypeptide comprising culturing the host cell of embodiment 19 under conditions that allow expression of the nucleic acid molecule.
21. Embodiment 1. A bispecific binding protein of any one of embodiments 1-3, or a conjugate of the bispecific antibody of any one of embodiments 4-13, wherein the bispecific binding protein or bispecific A conjugate wherein the sex antibody is conjugated to an agent selected from the group consisting of a contrast agent, a therapeutic agent, and a cytotoxic agent.
22. A pharmaceutical composition comprising:
A bispecific binding protein of any one of embodiments 1-3, a bispecific antibody of any one of embodiments 4-13, or a conjugate of embodiment 21, and a pharmaceutically acceptable carrier. A pharmaceutical composition comprising.

  The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will be apparent from the description and from the claims.

FIGS. 1A, 1B, 1C and 1D show four possible ways in which bispecific antibodies can be constructed that include a PD-L1 binding region and a KDR binding region. In each case, the bispecific antibody comprises an IgG comprising one of the binding regions covalently bound to the scFv comprising the other binding region. IgG is shown using conventional two-arm antibody delineation. scFv is an elongated ellipse. FIG. 1A shows an scFv linked to the carboxy terminus of an IgG heavy chain constant domain. FIG. 1B shows the scFv linked to the carboxy terminus of the IgG light chain constant domain. FIG. 1C shows the scFv linked to the amino terminus of the IgG light chain variable domain. FIG. 1D shows the scFv linked to the amino terminus of the IgG heavy chain variable domain. Bispecific antibody heavy and light chain amino acid sequences in which a PD-L1-specific scFv, designated D7A8, is linked to the carboxy terminus of the heavy chain constant domain of a KDR-specific IgG antibody, designated B1A1 (SEQ ID NOs: 34 and 31) are shown. A heavy and light chain amino acid sequence of a bispecific antibody in which a PD-L1-specific scFv called D7A8 is linked to the carboxy terminus of a KDR-specific IgG antibody light chain constant domain called B1A1 (SEQ ID NO: 30 and 35). The amino acid sequences of the heavy and light chains of the bispecific antibody in which a PD-L1-specific scFv called D7A8 is linked to the amino terminus of the heavy chain variable domain of the KDR-specific IgG antibody called B1A1 Numbers 36 and 31) are shown. The amino acid sequences of the heavy and light chains of the bispecific antibody in which the PD-L1-specific scFv called D7A8 is linked to the amino terminus of the light chain variable domain of the KDR-specific IgG antibody called B1A1 Numbers 30 and 37) are shown. The amino acid sequences of the heavy and light chains of the bispecific antibody in which the PD-L1-specific scFv called D7A8 is linked to the carboxy terminus of the heavy chain constant domain of the KDR-specific IgG antibody called B1C4A7 (SEQ ID NO: 38 And 33). The amino acid sequences of the heavy and light chains of the bispecific antibody in which the PD-L1-specific scFv called D7A8 is linked to the carboxy terminus of the light chain constant domain of the KDR-specific IgG antibody called B1C4A7 Numbers 32 and 39) are shown. The amino acid sequences of the heavy and light chains of the bispecific antibody in which the PD-L1 specific scFv called D7A8 is linked to the amino terminus of the heavy chain variable domain of the KDR specific IgG antibody called B1C4A7 (SEQ ID NO: 40 And 33). The amino acid sequences of the heavy and light chains of the bispecific antibody in which the PD-L1-specific scFv called D7A8 is linked to the amino terminus of the light chain variable domain of the KDR-specific IgG antibody called B1C4A7 Numbers 32 and 41) are shown. The heavy and light chain amino acid sequences of the bispecific antibody in which the KDR specific scFv called B1A1 is linked to the carboxy terminus of the heavy chain constant domain of the PD-L1 specific IgG antibody called D7A8 (SEQ ID NO: 42 And 29). The amino acid sequences of the heavy and light chains of the bispecific antibody in which the KDR specific scFv called B1A1 is linked to the carboxy terminus of the light chain constant domain of the KDR specific IgG antibody called D7A8 (SEQ ID NO: 28 and 43 ). The amino acid sequences of the heavy and light chains of the bispecific antibody in which the KDR-specific scFv called B1A1 is linked to the amino terminus of the heavy chain variable domain of the KDR-specific IgG antibody called D7A8 (SEQ ID NOs: 44 and 29 ). The amino acid sequences of the heavy and light chains of the bispecific antibody in which the KDR-specific scFv called B1A1 is linked to the amino terminus of the light chain variable domain of the KDR-specific IgG antibody called D7A8 (SEQ ID NOs: 28 and 45 ). The amino acid sequences of the heavy and light chains of the bispecific antibody in which the KDR-specific scFv called B1C4A7 is linked to the carboxy terminus of the heavy chain constant domain of the PD-L1 specific IgG antibody called D7A8 Numbers 46 and 29) are shown. The bispecific antibody heavy and light chain amino acid sequences (SEQ ID NOs: 28 and 47), wherein a KDR-specific scFv called B1C4A7 is linked to the carboxy terminus of the light chain constant domain of a KDR-specific IgG antibody called D7A8. ). The amino acid sequences of the heavy and light chains of the bispecific antibody in which the KDR-specific scFv called B1C4A7 is linked to the amino terminus of the heavy chain variable domain of the KDR-specific IgG antibody called D7A8 (SEQ ID NOs: 48 and 29 ) The amino acid sequences of the heavy and light chains of the bispecific antibody in which the KDR specific scFv called B1C4A7 is linked to the amino terminus of the light chain variable domain of the KDR specific IgG antibody called D7A8 (SEQ ID NOs: 28 and 49 ). 2 shows the structure of a bispecific antibody (BsAb) against VEGFR2 and PDL1. Anti-VEGFR2 antibodies B1A1 (binding to human, rat and monkey VEGFR2) and B1C4A7 (binding to human, mouse, rat and monkey VEGFR2) were used as conventional IgG1. Anti-PDL1 antibody D7A8 (binding to mouse, rat and monkey PDL1) was reformatted into a single chain variable fragment and added to the c-terminus of either B1A1 or B1C4A7. “LALA version” means an antibody in which the CH2 region is mutated by substituting two leucine residues (eg, LL bold letters in SEQ ID NOs: 28, 30, and 32 above) with two alanines (AA). To do. 2 shows SDS-PAGE analysis of bispecific antibodies after protein A purification. Figure 2 shows a size exclusion chromatogram (UV trace at 280 nm) of protein A purified bispecific antibody in a UPLC system. Figure 2 shows a size exclusion chromatogram (UV trace at 280 nm) of protein A purified bispecific antibody in a UPLC system. Results of DSC scanning of bispecific antibody in PBS buffer are shown. Results of DSC scanning of bispecific antibody in PBS buffer are shown. The result of the Western blotting of the bispecific antibody which was processed at 37 ° C. for 5 days in mouse serum is shown. Shown are the results of a dose response ELISA of bispecific antibody A7-A8 to determine the EC50 for human and mouse VEGFR2, and human and mouse PDL1. The results of a dose response blocking ELISA to determine the IC50 of the bispecific antibody A7-A8 for VEGF-VEGFR2 and PDL1-PD1 are shown. FIG. 2 shows binding of cell expression to human or mouse VEGFR2 and human or mouse PDL1. FIG. 5 shows the interaction of bispecific antibody A7-A8 with receptors hVEGFR2 and hPDL1 investigated by surface plasmon resonance (Biacore). Figure 2 shows a scheme showing complexes on immobilized hVEGFR2 surface and complexes in solution of cross-linked ELISA. The ability of bispecific antibody A7-A8 to bind simultaneously to VEGFR2 and PDL1 is examined by cross-linking ELISA. Inhibition of VEGF-stimulated phosphorylation of VEGFR2 and downstream molecules in KDR-PAE (hVEGFR2) and EOMA (mVEGFR2) by bispecific antibody A7-A8. 2 shows the secretion of cytokines IL2 and INFγ in the presence of bispecific antibody A7-A8. Shown are dose response ELISA results of bispecific antibody A1-A8 to determine EC50 for human and mouse VEGFR2, and human and mouse PDL1. Shown are dose response blocking ELISA results to determine the IC50 of bispecific antibody A1-A8 for VEGF-VEGFR2 and PDL1-PD1. FIG. 5 shows binding to cells expressing human or mouse VEGFR2 and human or mouse PDL1. FIG. 5 shows the interaction of bispecific antibody A1-A8 with receptors hVEGFR2 and hPDL1 investigated by surface plasmon resonance (Biacore). The bispecific antibody A1-A8 is shown to be able to bind to VEGFR2 and PDL1 simultaneously by examination by cross-binding ELISA. In the presence of 1.5 μg / ml (7.5 nM) of BsAb (A1-A8) -LALA and BsAb (A7-A8) -LALA, both VEGFR2 and MAPK phosphoprotein bands are better than those without antibody treatment. It is a figure which shows that it was also quite weak. 2 shows the secretion of cytokines IL2 and INFγ in the presence of bispecific antibodies A1-A8 and A7-A8. The CT26 test result regarding BsAb (A7-A8) is shown. 39A and 39B show the MC38 test results for BsAb (A7-A8). The SDS-PAGE results for BsAb (B1A1-A11) and BsAb (B1C4A7-A11) variants in which degraded bands were observed are shown. The SDS-PAGE results are shown for BsAb (A11-B1A1) and BsAb (D7A8-B1A1) variants in which no resolved bands were observed after changing the orientation. Shows binding to PDL1 and VEGFR2. Shows blocking interaction of ligand and receptor. A common light chain sequence (SEQ ID NO: 50) and three heavy chain sequences (SEQ ID NO: 51-53) of BsAb (A11-B1A1), BsAb (A11-B1A1) _30 and BsAb (A11-B1A1) _30cc are shown. A common light chain sequence (SEQ ID NO: 50) and three heavy chain sequences (SEQ ID NO: 51-53) of BsAb (A11-B1A1), BsAb (A11-B1A1) _30 and BsAb (A11-B1A1) _30cc are shown. The common light chain sequence (SEQ ID NO: 29) and three heavy chain sequences (SEQ ID NO: 54-56) of BsAb (D7A8-B1A1), BsAb (D7A8-B1A1) _30 and BsAb (D7A8-B1A1) _30cc are shown. The common light chain sequence (SEQ ID NO: 29) and three heavy chain sequences (SEQ ID NO: 54-56) of BsAb (D7A8-B1A1), BsAb (D7A8-B1A1) _30 and BsAb (D7A8-B1A1) _30cc are shown.

  The interaction between PD-1 and PD-L1 on immune cells inhibits proliferation and cytokine production by immune cells. PD-L1 is also inducible and upregulated in various tissues including cancer. Both PD-1 and PD-L1 play a role in immunosuppression. As used herein, novel bispecifics such as bispecific antibodies or antigen binding fragments of such bispecific antibodies that bind to human PD-L1 and block its interaction with human PD-1. A sex binding protein is provided.

  Bispecific antibodies also bind to human KDR and block its interaction with human VEGF. In some embodiments, the bispecific antibody blocks ligand binding to KDR (eg, binding of one or more of VEGF-A, VEGF-C, VEGF-D, or VEGF-E). In some embodiments, the bispecific antibody neutralizes KDR activation. Bispecific antibodies can be used, for example, to treat neoplastic diseases including solid and non-solid tumors, and hyperproliferative disorders. Accordingly, provided are methods for neutralizing KDR activation, methods for inhibiting tumor growth, including inhibition of tumor-related angiogenesis, and methods for treating angiogenesis-related disorders.

  Also provided is a kit comprising a bispecific antibody or antibody fragment that binds to PDL1 and KDR.

  Bispecific antibodies are not limited by any particular mechanism of KDR inhibition. The mechanism followed by one bispecific antibody is not necessarily the same as the mechanism followed by another bispecific antibody. Some possible mechanisms include prevention of binding of the VEGF ligand to the extracellular binding domain of KDR and prevention of receptor dimerization or oligomerization. However, other mechanisms cannot be excluded.

  Bispecific antibodies inhibit KDR activation. One measure of KDR inhibition is a decrease in receptor tyrosine kinase activity. Tyrosine kinase inhibition can be determined using well-known methods such as measuring the level of receptor autophosphorylation. Inhibition of KDR can also be observed by inhibition or modulation of phosphorylation events of natural or synthetic KDR substrates and other components of the KDR signaling pathway. Phosphorylation can be detected, for example, using an antibody specific for phosphotyrosine in an ELISA assay or Western blot. Several assays for tyrosine kinase activity are described in Panek et al. , J .; Pharmacol. Exp. Thera. 283: 1433-44 (1997) and Batley et al. , Life Sci. 62: 143-50 (1998).

  In vivo assays can also be utilized. For example, receptor tyrosine kinase inhibition can be observed by mitogenic assays using cell lines stimulated with receptor ligand in the presence or absence of inhibitors. For example, KUV inhibition may be assayed using VEGF-stimulated HUVEC cells (ATCC). Another method involves testing for inhibition of proliferation of VEGF-expressing tumor cells, eg, using human tumor cells injected into mice. See, for example, US Pat. No. 6,365,157 (Rockwell et al.).

  The bispecific binding proteins (eg, bispecific antibodies) disclosed herein can be made by methods known in the art. Such methods can include the use of nucleic acids encoding binding proteins (eg, bispecific antibodies) or portions thereof. Vectors that can be used to make the binding proteins disclosed herein (eg, bispecific antibodies) include pBh1 (Dyax) or pcDNA ™ 3.4 TOPO® vector (Thermo). Transient expression vectors suitable for expressing proteins in HEK293 cells, such as Fisher Scientific). PCHO. In CHO-S (Thermo Fisher Scientific). A stable expression vector suitable for protein expression in mammalian cells, such as GS vector in 1 or CHO-K (Lonza). One skilled in the art may refer to the following publications for guidance on making the binding proteins (eg, bispecific antibodies) disclosed herein or portions thereof: Lu D and Zhu Z. (2014); Construction and production of an IgG-like tetravalent bivalent antigen, IgG-single-chain Fv fusion. Human Monoclonal antigens, methods and protocols; Humanna press; ISSN 1064-3745, Marvin J. et al. S. , Zhu Z. (2006) Bisificial anti-bodies for dual-modality cancer therapy: killing two signaling cascades. Curr. Opin. Drug Discov. Dev. 9, 184-193, Lu, D .; Zhang, H .; , Koo, H .; , Et al. (2005) A fully human recombinant IgG-like bispecific anti-body to both the epithelial growth factor and the instinct of the human population. J. et al. Biol. Chem. 280, 19665-19672, Demarest S. et al. J (2011) Emerging antibody combinations in oncology. mAbs 3,338-351, Spies C.I. , Zhai Q. , Carter P. (2015) Alternative molecular formats and thermal applications for bispecific antigens. Molecular Immunology 67, 95-106, Crosdale R.M. , Et al. (2012) Development of tetravalent IgG1 dual targeting IGF-1R-EGFR antibodies with potent tumor inhibition. Archives of Biochem. and Biophys. 526, 206-218.

  The amino acid sequences of the heavy and light chain CDRs described herein are specified according to the Kabat and Chothia specification scheme. The first two heavy chain CDRs are identified according to the general format of Kabat and Chothia, which provides distinct but overlapping positions for the CDRs. Comparison of multiple heavy and light chains shows significant similarity between many of the CDR sequences. Thus, many CDRs would be expected to be mixed and matched between sequences.

  The bispecific binding protein or bispecific antibody described herein may have one or more amino acid substitutions, deletions, insertions and / or additions. In certain embodiments, the bispecific antibody or protein comprises one of the heavy chain variable domains disclosed above and one of the light chain variable domains described above. In certain embodiments, the bispecific antibody or binding fragment thereof is at least 85%, at least 90%, at least 95%, at least 96%, relative to one of the variable domain sequences disclosed above, It includes one or more of the variable domains disclosed above having an amino acid sequence that is at least 97%, at least 98%, or at least 99% identical. To reduce effector function, a leucine at a position corresponding to 234 or 235 of IgG1 (in the CH2 domain) may be mutated to alanine (eg, the LALA version or LALA mutation described above). In addition, mutations may be made to introduce one or more disulfide bonds between two domains, such as two variable domains, to improve thermal stability. For example, residues corresponding to residues 44 and 248 of SEQ ID NO: 57 may be mutated to cysteine to introduce disulfide bonds.

As used herein, the term “antibody” includes whole antibodies and any antigen-binding fragment (ie, “antigen-binding portion”) or single chains thereof, unless otherwise indicated or apparent in context. “Antibody” refers, in one embodiment, to a glycoprotein or antigen-binding fragment thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as V _H ) and a heavy chain constant region. In certain naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region consists of three domains: CH1, CH2 and CH3. In certain naturally occurring antibodies, each light chain is comprised of a light chain variable region (abbreviated herein as _VL ) and a light chain constant region. The light chain constant region is comprised of one domain, C _L. The V _H and V _L regions are more conserved and can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed with regions called framework regions (FR). Each V _H and V _L is composed of three CDRs and four framework regions (FR) arranged from the amino terminus to the carboxy terminus in the order FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. . The variable region of the heavy and light chains contains a binding domain that interacts with an antigen. The constant region of the antibody may mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component of the classical complement system (C1q).

  As used herein, “antibody” is used in the broadest sense and specifically includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, multispecifics as long as they exhibit the desired biological activity. Antibody (eg, bispecific antibody) and antibody fragments. Examples include human antibodies, humanized antibodies, and chimeric antibodies. As used herein, an “antibody fragment” is an intact antibody that generally comprises the antigen-binding and / or variable region of an intact antibody and / or the Fc region of an antibody that retains the ability to bind FcR. Part may be included. Examples of antibody fragments include linear antibodies, single chain antibody molecules, and multispecific antibodies formed from antibody fragments. Preferably, the antibody fragment retains the entire constant region of an IgG heavy chain and includes an IgG light chain.

As used herein, the term “antigen-binding portion” or “antigen-binding fragment” of an antibody refers to one or more of the antibodies that retain the ability to specifically bind to an antigen (eg, human PD-L1 or KDR). Refers to a fragment of Examples of binding fragments encompassed within the term “antigen-binding portion / fragment” of an antibody include (i) a Fab fragment that is a monovalent fragment consisting of V _L , V _H , C _L and CH1 domains, (ii) An F (ab ′) 2 fragment, which is a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region, (iii) an Fd fragment consisting of the V _H and CH1 domains, (iv) a single arm of the antibody Fv fragments consisting of _VL and _VH domains, and (v) dAb fragments consisting of _VH domains (Ward et al. (1989) Nature 341: 544-546). An isolated complementarity-determining region (CDR), or a combination of two or more isolated CDRs joined by a synthetic linker, can contain the antigen-binding domain of an antibody if it can bind to an antigen.

  “Human” antibodies (HuMAbs) refer to antibodies having variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. Furthermore, if the antibody contains a constant region, this constant region is also derived from human germline immunoglobulin sequences. The human antibodies of the present invention are amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, suddenly introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo. Mutation). However, as used herein, the term “human antibody” is intended to include antibodies in which a CDR sequence derived from the germline of another mammalian species, such as a mouse, is grafted to a human framework sequence. It is not a thing. The terms “human” antibody and “fully human” antibody are used synonymously.

  A “humanized” antibody refers to a non-human antibody, eg, an antibody in which some, most or all of the amino acids outside the CDR domains of a murine antibody are replaced with corresponding amino acids derived from human immunoglobulins. . In one embodiment of a humanized antibody, most or all of the amino acids outside the CDR domains are replaced with amino acids from a human immunoglobulin, but to some extent most or all of the amino acids within one or more CDR regions. Is immutable. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible unless they negate the ability of the antibody to bind to a particular antigen. A “humanized” antibody retains similar antigenic specificity as the original antibody.

  “Chimeric antibody” refers to an antibody in which the variable region is derived from one species and the constant region is derived from another species, for example, an antibody in which the variable region is derived from a mouse antibody and the constant region is derived from a human antibody. By “hybrid” antibody is meant an antibody having different types of heavy and light chains, such as a mouse (parent) heavy chain and a humanized light chain (or vice versa).

  “Bispecificity” refers to a protein (eg, an antibody) that binds to both PD-L1 and KDR. A “bispecific antibody” is an artificial hybrid antibody having two different heavy / light chain pairs, resulting in two antigen binding sites having specificity for different antigens. Bispecific antibodies can be produced by a variety of methods including fusion of proteins or linking of Fab 'fragments. See, eg, Songsivirai & Lachmann (1990) Clin. Exp. Immunol. 79: 315-321, Kostelny et al. (1992) J. MoI. Immunol. 148, 1547-1553.

  The bispecific molecules described herein can be prepared by conjugating constitutive binding specificities using other methods known in the art. For example, each binding specificity of a bispecific molecule may be generated separately and then conjugated to each other. Various coupling or cross-linking agents may be used for covalent bonding. Examples include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB), o-phenylene dimaleimide (oPDM), N -Succinimidyl-3- (2-pyridyldithio) propionate (SPDP) and sulfosuccinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) (eg Karpovsky et al. (1984). J. Exp. Med. 160: 1686; Liu MA et al. (1985) Proc. Natl. Acad. Sci. (USA) 82: 8648). Other methods include Paulus (1985) Behring Ins. Mitt. No. 78, 118-132, Brennan et al. (1985) Science 229: 81-83), and Glennie et al. (1987) J. MoI. Immunol. 139: 2367-2375). Preferred complexing agents are SATA and sulfo-SMCC, both of which are Pierce Chemical Co. (Rockford, IL). The antibody may also be conjugated via a sulfhydryl bond in the C-terminal hinge region of the two heavy chains. In a particularly preferred embodiment, the hinge region is modified to contain an odd number, preferably one sulfhydryl residue, prior to conjugation.

  Alternatively, both binding moieties may be encoded in the same vector and expressed and assembled in the same host cell. The bispecific molecule described herein can be a single chain molecule comprising two single chain antibodies, or a complex having at least two antibody chains, or a combination thereof. Methods for preparing bispecific molecules are described, for example, in US Pat. No. 5,260,203, US Pat. No. 5,455,030, US Pat. No. 4,881,175, US Pat. 405, US Pat. No. 5,091,513, US Pat. No. 5,476,786, US Pat. No. 5,013,653, US Pat. No. 5,258,498, and US Pat. No. 5,482. 858. Binding of bispecific molecules to specific targets is known in the art or as described herein, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), FACS analysis, bioassay (Eg, growth inhibition), or can be confirmed using art-recognized methods such as Western blot assays.

  “Inhibiting a receptor” refers to reducing and / or inactivating a receptor's ability to transmit a signal, for example, by reducing and / or inactivating the receptor's endogenous kinase activity. means.

  “Identity” refers to the identity shared by two amino acid or nucleic acid sequences, taking into account the number of gaps and the length of each gap, that need to be introduced for optimal alignment of the two sequences. Refers to the number or percentage of positions.

  The terms “peptide”, “polypeptide” and “protein” are used interchangeably to describe the arrangement of amino acid residues in a polymer. A peptide, polypeptide, or protein can be composed of the standard 20 naturally occurring amino acids, plus rare amino acids and synthetic amino acid analogs. They may be any amino acid chain, regardless of length or post-translational modification (eg, glycosylation or phosphorylation).

  In some cases, (a) the structure of the peptide backbone in the region of substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) their effect on maintaining the bulk of the side chain is significantly different. Amino acid substitutions can be made by selecting substituents without any. For example, naturally occurring residues can be grouped based on side chain properties: (1) hydrophobic amino acids (norleucine, methionine, alanine, valine, leucine, and isoleucine), (2) neutral hydrophilicity Amino acids (cysteine, serine and threonine), (3) Acidic amino acids (aspartic acid and glutamic acid), (4) Basic amino acids (asparagine, glutamine, histidine, lysine and arginine), (5) Influence on chain orientation Amino acids (glycine and proline), and (6) aromatic amino acids (tryptophan, tyrosine, phenylalanine). Substitutions made within these groups can be considered conservative substitutions. Examples of substitutions include, but are not limited to, valine for alanine, lysine for arginine, glutamine for asparagine, glutamic acid for aspartic acid, serine for cysteine, asparagine for glutamine, aspartic acid for glutamic acid, proline for glycine, arginine for histidine, Leucine for isoleucine, isoleucine for leucine, arginine for lysine, leucine for methionine, leucine for phenylalanine, glycine for proline, threonine for serine, serine for threonine, tyrosine for tryptophan, phenylalanine for tyrosine, and / or leucine for valine. It is done.

  Methods and computer programs for determining sequence similarity are publicly available, including but not limited to the GCG program package (Devereux et al., Nucleic Acids Research 12: 387, 1984). , BLASTP, BLASTN, FASTA (Altschul et al. J. Mol. Biol. 215: 403 (1990)), and the ALIGN program (version 2.0). The well-known Smith Waterman algorithm may also be used to determine similarity. The BLAST program is available from NCBI and other information sources (BLAST Manual, Altschul et al. NCBI NLM NIH, Bethesda, Md. 20894; BLAST 2.0, http://www.ncbi.nlm.nih.gov/blast/ Is publicly available. In sequence comparison, these methods allow for various substitutions, deletions and other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; Tyrosine.

  Bispecific antibodies provided herein also include antibodies with improved binding properties by direct mutagenesis, affinity maturation methods, phage display, or chain shuffling. Affinity and specificity can be altered or improved by mutating the CDRs and screening for antigen binding sites with the desired properties. CDRs are mutated in various ways. One method is to randomize individual residues or combinations of residues so that all 20 amino acids are found at a particular position in a population of otherwise identical antigen binding sites. is there. Alternatively, mutations are induced over a range of CDR residues by error-prone PCR methods (see, eg, Hawkins et al., J. Mol. Biol., 226: 889-896 (1992)). For example, phage display vectors containing heavy and light chain variable region genes can be propagated in E. coli mutator strains (eg, Low et al., J. Mol. Biol., 250). : 359-368 (1996)). These mutagenesis methods are illustrative of many methods known to those skilled in the art.

  In order to minimize immunogenicity, bispecific antibodies comprising human constant domain sequences are preferred. Bispecific antibodies may be members of any immunoglobulin class, such as IgG, IgM, IgA, IgD, or IgE, and subclasses thereof, may include members thereof, or combinations thereof. Also good. The antibody class may be selected to optimize effector function, such as complement dependent cytotoxicity (CDC) and antibody dependent cellular cytotoxicity (ADCC).

One particular embodiment of a bispecific binding protein involves the use of PD-L1- and KDR binding antibody fragments. Fv is the smallest fragment that contains complete heavy and light chain variable domains, including all six hypervariable loops (CDRs). Lacking constant domains, variable domains are non-covalently associated. The heavy and light chains are single polypeptide chains (“single-chain Fv” or “scFv”) using linkers that allow the V _H and V _L domains to associate to form an antigen binding site. ) May be connected. In one embodiment, the linker is (Gly-Gly-Gly-Gly-Ser) ₃ . scFv fragments are much smaller than whole antibodies because they lack the constant domains of whole antibodies. The scFv fragment also does not have normal heavy chain constant domain interaction with other biomolecules that are undesirable in certain embodiments.

Fragments of antibodies comprising V _H , V _L , and optionally C _L , C _H 1 or other constant domains can also be used in bispecific binding proteins. The monovalent fragment of an antibody produced by papain digestion is called Fab and lacks the heavy chain hinge region. The fragment generated by pepsin digestion called F (ab ′) ₂ retains the heavy chain hinge and is bivalent. Such fragments may also be produced recombinantly. Many other useful antigen-binding antibody fragments are known in the art including, but not limited to, diabodies, triabodies, single domain antibodies, and other monovalent and multivalent A type is mentioned.

In one embodiment, the PD-L1 binding region of a bispecific binding protein, such as a bispecific antibody, is at least about 10 ² M ⁻¹ s ⁻¹ , at least about 10 ^{3 as} measured by surface plasmon resonance. An on rate constant of M ⁻¹ s ⁻¹ , at least about 10 ⁴ M ⁻¹ s ⁻¹ , at least about 10 ⁵ M ⁻¹ s ⁻¹ , or at least about 10 ⁶ M ⁻¹ s ⁻¹ ( Kon). In one embodiment, the PD-L1 binding region is 10 ² M ⁻¹ s ⁻¹ to 10 ³ M ⁻¹ s ⁻¹ , 10 ³ M ⁻¹ s ⁻¹ to 10 ⁴ M as measured by surface plasmon resonance. ⁻¹ s ⁻¹ , 10 ⁴ M ⁻¹ s ⁻¹ to 10 ⁵ M ⁻¹ s ⁻¹ , or 10 ⁵ M ⁻¹ s ⁻¹ to 10 ⁶ M ⁻¹ s ^−1. Have.

In one embodiment, the KDR binding region of a bispecific binding protein, such as a bispecific antibody, is at least about 10 ² M ⁻¹ s ⁻¹ , at least about 10 ³ M ^{− as} measured by surface plasmon resonance. ^{It has} a binding rate constant (Kon) of ¹ s ⁻¹ , at least about 10 ⁴ M ⁻¹ s ⁻¹ , at least about 10 ⁵ M ⁻¹ s ⁻¹ , or at least about 10 ⁶ M ⁻¹ s ⁻¹ . In certain embodiments, the KDR binding region is 10 ² M ⁻¹ s ⁻¹ to 10 ³ M ⁻¹ s ⁻¹ , 10 ³ M ⁻¹ s ⁻¹ to 10 ⁴ M ^{−1 as} measured by surface plasmon resonance. It has a binding rate constant (Kon) of s ⁻¹ , 10 ⁴ M ⁻¹ s ⁻¹ to 10 ⁵ M ⁻¹ s ⁻¹ , or 10 ⁵ M ⁻¹ s ⁻¹ to 10 ⁶ M ⁻¹ s ⁻¹ .

In one embodiment, the PD-L1 binding region of a bispecific binding protein, such as a bispecific antibody, is at most about 10 ⁻³ s ⁻¹ , at most about 10 ^{− as} measured by surface plasmon resonance. ^{It has an} off rate constant (Koff) of ⁴ s ⁻¹ , at most about 10 ⁻⁵ s ⁻¹ , or at most about 10 ⁻⁶ s ⁻¹ . In one embodiment, the PD-L1 binding region is 10 ⁻³ s ⁻¹ to 10 ⁻⁴ s ⁻¹ , 10 ⁻⁴ s ⁻¹ to 10 ⁻⁵ s ⁻¹ , or 10 as measured by surface plasmon resonance. ^{It has} a dissociation rate constant (Koff) of ⁻⁵ s ⁻¹ to 10 ⁻⁶ s ⁻¹ .

In one embodiment, the KDR binding region of a bispecific binding protein, such as a bispecific antibody, is at most about 10 ⁻³ s ⁻¹ , at most about 10 ⁻⁴ s as measured by surface plasmon resonance. ⁻¹ , at most about 10 ⁻⁵ s ⁻¹ , or at most about 10 ⁻⁶ s ^{−1 with} a dissociation rate constant (Koff). In one embodiment, the KDR binding region is 10 ⁻³ s ⁻¹ to 10 ⁻⁴ s ⁻¹ , 10 ⁻⁴ s ⁻¹ to 10 ⁻⁵ s ⁻¹ , or 10 ^{−5 as} measured by surface plasmon resonance. It has a dissociation rate constant (Koff) of s ⁻¹ to 10 ⁻⁶ s ⁻¹ .

In one embodiment, the PD-L1 binding region of a bispecific binding protein, such as a bispecific antibody, is at most about 10 ⁻⁷ M, at most about 10 ⁻⁸ M, at most about 10 ⁻⁹ M. A dissociation constant (K _D ) of at most about 10 ⁻¹⁰ M, at most about 10 ⁻¹¹ M, at most about 10 ⁻¹² M, or at most 10 ⁻¹³ M. In one embodiment, PD-L1 binding region of the bispecific binding proteins such as bispecific ^{antibodies, 10 -7 M~10 -8 M, 10} -8 M~10 -9 M, 10 -9 M to have a ^{-10 M, 10 -10 M~10 -11 M} , 10 -11 M~10 -12 M or ¹⁰ -12 M to ^-13 dissociation constant for its target of M, _{(K D).}

In one embodiment, the KDR binding region of a bispecific binding protein, such as a bispecific antibody, has at most about 10 ⁻⁷ M, at most about 10 ⁻⁸ M, at most about 10 ⁻⁹ M, at most It has a dissociation constant (K _D ) of at least about 10 ⁻¹⁰ M, at most about 10 ⁻¹¹ M, at most about 10 ⁻¹² M, or at most 10 ⁻¹³ M. In one embodiment, KDR binding region of the bispecific binding proteins such as bispecific ^{antibodies, 10 -7 M~10 -8 M, 10} -8 M~10 -9 M, 10 -9 M~ 10 has a ^{-10 M, 10 -10 M~10 -11 M} , 10 -11 M~10 -12 M or ¹⁰ -12 M to ^-13 dissociation constant for its target of M, _{(K D).}

The bispecific binding protein described herein may be a conjugate further comprising a contrast agent, a therapeutic agent, or a cytotoxic agent. In one embodiment, the contrast agent is a radioactive label, an enzyme, a fluorescent label, a luminescent label, a bioluminescent label, a magnetic label, or biotin. In another embodiment, the radiolabel is ³ H, ¹⁴ C, ³⁵ S, ⁹⁰ Y, ⁹⁹ Tc, ¹¹¹ In, ¹²⁵ I, ¹³¹ I, ¹⁷⁷ Lu, or ¹⁵³ Sm. In yet another embodiment, the therapeutic or cytotoxic agent is an antimetabolite, alkylating agent, antibiotic, growth factor, cytokine (eg, immunostimulatory cytokine), anti-angiogenic agent, anti-mitotic agent, An anthracycline, toxin, or apoptotic agent. As discussed below, immunostimulatory cytokines are particularly important.

  Molecules that bind to human PD-L1 to inhibit immunosuppression and also bind to human KDR to inhibit angiogenesis are provided. In one embodiment, such a molecule combines the PD-L1 binding domain of a first antibody that binds human PD-L1 with the KDR binding domain of a second antibody that binds human KDR.

  The PD-L1 binding portion of the molecule may be the antigen binding domain of an antibody, such as a heavy and light chain variable domain pair. Suitable antibody heavy and light chain variable domains and antibodies comprising them are provided herein. The PD-L1 binding portion of the bispecific binding protein may be any agent that binds to PD-L1 and blocks immunosuppression. These include anti-PD-L1 antibodies and fragments that are not limited to the antibodies disclosed herein, and peptides and proteins derived from human PD1, the natural ligand for human PD-L1. As disclosed herein, the PD-L1 binding region is linked to a region that binds human KDR.

  Accordingly, in certain embodiments, a hybrid molecule is provided that comprises a region that binds to human PD-L1 and blocks binding to human PD1, and a region that binds to human KDR and blocks binding to human KDR. PD-L1 and the KDR binding region may be connected as a single polypeptide by a linker. Accordingly, a human PD-L1 binding region linked to a human KDR binding region is provided.

  Another aspect of the present invention provides a nucleic acid molecule encoding the above antibody or a chain thereof. The nucleic acid may be present in whole cells, cell lysate, or partially purified or substantially pure form. Other cellular components or other contaminants, such as other cellular nucleic acids or by standard techniques such as alkali / SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis and other techniques well known in the art A nucleic acid is “isolated” or “substantially purified” when purified from a protein. F. Ausubel, et al. , Ed. (1987) Current Protocols in Molecular Biology, Green Publishing and Wiley Interscience, New York. The nucleic acids of the invention can be, for example, DNA or RNA and may or may not contain intron sequences. In a preferred embodiment, the nucleic acid is a cDNA molecule.

  The nucleic acids of the invention can be obtained using standard molecular biology techniques. For antibodies expressed by a hybridoma (eg, a hybridoma prepared from a transgenic mouse carrying a human immunoglobulin gene), the cDNA encoding the light and heavy chains of the antibody produced by the hybridoma can be expressed by standard PCR amplification. Alternatively, it can be obtained by cDNA cloning techniques. For antibodies obtained from an immunoglobulin gene library (eg, using phage display technology), nucleic acid encoding the antibody can be recovered from the library. The above-described antibody or its chain can be expressed using a nucleic acid and a vector containing the nucleic acid.

  The term “vector” as used herein is intended to refer 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 additional DNA segments can be ligated. Another type of vector is a viral vector that can link additional DNA segments to the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the host cell genome upon introduction into the host cell, and thereby replicated along with the host genome. In addition, certain vectors can direct the expression of genes to which they are operably linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”). In general, expression vectors useful in recombinant DNA technology are often in the form of plasmids. In the present specification, “plasmid” and “vector” may be used interchangeably as the plasmid is the most commonly used form of vector. However, other forms of expression vectors are also included, such as viral vectors that perform equivalent functions (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses).

  The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell containing nucleic acid that is not naturally present in the cell, and It may be a cell into which a vector has been introduced. It should be understood that such terms are intended to refer not only to a particular subject cell, but also to the progeny of such a cell. Such progeny are not actually identical to the parent cell, but are also used herein because certain modifications may occur in subsequent generations due to mutations or environmental effects Included within the scope of the term “host cell”.

  It will be appreciated that bispecific binding proteins, when used in mammals for prophylactic or therapeutic purposes, are generally administered in the form of a composition further comprising at least one pharmaceutically acceptable carrier. . Suitable pharmaceutically acceptable carriers include, for example, one or more of water, saline, phosphate buffered saline, dextrose, glycerol, sucrose, polysorbate, ethanol, and the like, and combinations thereof. Can be mentioned. Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary substances such as wetting or emulsifying agents, preservatives, or buffers that enhance the shelf life or effectiveness of the bispecific binding protein.

  In the methods described herein, a therapeutically effective amount of a bispecific binding protein, such as a bispecific antibody, is administered to a mammal in need thereof. The term “administering” as used herein means delivering a bispecific binding protein, such as a bispecific antibody, to a mammal by any method that can achieve the desired result. . Administration can be, for example, intravenous or intramuscular. Bispecific binding proteins such as bispecific antibodies are particularly useful for human administration, but may be administered to other mammals as well. The term “mammal” as used herein is intended to include, but is not limited to, humans, laboratory animals, domestic pets, and livestock. “Therapeutically effective amount” means the amount of a bispecific binding protein, such as a bispecific antibody, that is effective to produce a desired therapeutic effect when administered to a mammal.

  Bispecific binding proteins such as bispecific antibodies are useful for inhibiting tumors and other neoplastic diseases and for treating other pathological conditions associated with immunosuppression. Tumors that can be treated include primary tumors, metastatic tumors, and refractory tumors. Refractory tumors include tumors that cannot respond or are resistant to treatment with chemotherapeutic agents alone, antibodies alone, radiation alone, or combinations thereof. Refractory tumors also include tumors that appear to be inhibited by treatment with such agents, but relapse up to 5 years, sometimes up to 10 years or more after treatment is discontinued. Bispecific binding proteins such as bispecific antibodies are effective in the treatment of angiogenic tumors and non-angiogenic or substantially non-angiogenic tumors.

  Examples of solid tumors that can be treated include breast cancer, lung cancer, colorectal cancer, pancreatic cancer, glioma and lymphoma. Some examples of such tumors are epidermal tumors, squamous tumors, eg head and neck tumors, colorectal tumors, prostate tumors, breast tumors, lung tumors, eg small cell and non-small cell lung tumors Pancreatic tumors, thyroid tumors, ovarian tumors, and liver tumors. Other examples include Kaposi's sarcoma, CNS neoplasm, neuroblastoma, hemangioblastoma, meningiomas and brain metastases, melanoma, gastrointestinal and renal cancer and sarcoma, rhabdomyosarcoma, glioblastoma, Preferable examples include pleomorphic glioblastoma and leiomyosarcoma. Examples of angiogenic skin cancers where bispecific binding proteins such as bispecific antibodies are effective include squamous cell carcinoma, basal cell carcinoma and skin cancer (proliferation of malignant keratinocytes such as human malignant keratinocytes) Can be treated by inhibiting).

  Examples of non-solid tumors include leukemia, multiple myeloma and lymphoma that are non-responsive to cytokines. Some examples of leukemia include acute myeloid leukemia (AML), chronic myeloid leukemia (CML), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), erythroid leukemia or monocytic leukemia. Is mentioned. Some examples of lymphomas include Hodgkin's disease and non-Hodgkin's lymphoma.

Bispecific binding proteins such as bispecific antibodies are also used to treat viral infections. PD-1 expression in T cells correlates with viral load in HIV and HCV infected patients, and PD-1 expression has been identified as a marker for depleted virus-specific CD8 ⁺ T cells. For example, PD-1 ⁺ CD8 ⁺ T cells exhibit impaired effector function and PD-1-related T cell depletion that can be restored by blocking PD-1 / PD-L1 interactions. This results in the restoration of virus-specific CD8 ⁺ T cell-mediated immunity and shows that blocking PD-1 signaling with antagonistic antibodies restores T cell effector function. Immunotherapy based on blockade of PD-1 / PD-L1 not only results in disruption of T cell resistance to tumor antigens, but also a strategy to re-activate virus-specific effector T cells and eradicate pathogens in chronic viral infections Also provide. Thus, the antibodies and hybrid proteins of the invention are useful for the treatment of chronic viral infections including, but not limited to, HCV and HIV, and lymphocytic choriomeningitis virus (LCMV).

  Bispecific binding proteins, such as bispecific antibodies, can be advantageously administered with a second agent to a patient in need thereof. For example, in some embodiments, a bispecific binding protein, such as a bispecific antibody, is administered to a subject with an antineoplastic agent. In some embodiments, a bispecific binding protein, such as a bispecific antibody, is administered to a subject with an angiogenesis inhibitor. In some embodiments, a bispecific binding protein, such as a bispecific antibody, is administered with an anti-inflammatory or immunosuppressive agent.

  Anti-tumor agents include cytotoxic chemotherapeutic agents, targeted small and biological molecules, and radiation. Non-limiting examples of chemotherapeutic agents include cisplatin, dacarbazine (DTIC), dactinomycin, irinotecan, mechloretamine (nitrogen mustard), streptozocin, cyclophosphamide, carmustine (BCNU), lomustine (CCNU), Doxorubicin (adriamycin), daunorubicin, procarbazine, mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil, vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel (taxotere), aldesleukin, asparaginine, prsulfandin, busulfantin, busulfandin Floxuridine, fludarabine, hydroxyurea, ifosfamide, interfero Alpha, leuprolide, megestrol, melphalan, mercaptopurine, prikamycin, mitotane, pegasparagase, pentostatin, pipobroman, pricamycin, streptozocin, tamoxifen, teniposide, test lactone, thioguanine, thiotepa, uracil mustard, vinorelbine, Chlorambucil, taxol, and combinations thereof.

  Targeted small and biological molecules include, but are not limited to, inhibitors of signal transduction pathway components such as modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and tumor-specific antigens. Drugs that bind to. Non-limiting examples of growth factor receptors involved in tumorigenesis include platelet derived growth factor (PDGFR), insulin-like growth factor (IGFR), nerve growth factor (NGFR), and fibroblast growth factor (FGFR). Receptors, as well as receptors of the epidermal growth factor receptor family, examples are EGFR (erbB1), HER2 (erbB2), erbB3, and erbB4.

  EGFR antagonists include antibodies that bind to EGFR or an EGFR ligand and inhibit ligand binding and / or receptor activation. For example, an agent may block the formation of receptor dimers or heterodimers with other EGFR family members. Examples of the EGFR ligand include EGF, TGF-α amphiregulin, heparin-binding EGF (HB-EGF), and beta-regulin. An EGFR antagonist can bind externally to the extracellular portion of EGFR (which may or may not inhibit ligand binding) or it can bind internally to a tyrosine kinase domain. EGFR antagonists further include agents that inhibit EGFR-dependent signaling, for example, by inhibiting the function of components of the EGFR signaling pathway. Examples of EGFR antagonists that bind to EGFR include, but are not limited to, biological molecules such as antibodies specific to EGFR (and functional equivalents thereof), and small molecules such as the cytoplasmic domain of EGFR. Synthetic kinase inhibitors that act directly on

  Small molecules and biological inhibitors include inhibitors of epidermal growth factor receptor (EGFR), such as gefitinib, erlotinib and cetuximab, inhibitors of HER2 (eg, trastuzumab, trastuzumab emtansine (trastuzumab-DM1; T-DM1) and pertuzumab), anti-VEGF antibodies and fragments (eg, bevacizumab), antibodies that inhibit CD20 (eg, rituximab, ibritumomab), anti-VEGFR antibodies (eg, ramcilmab (IMC-1121B), IMC-1C11, and CDP791), anti-PDGFR antibodies, and imatinib. Small molecule kinase inhibitors can be specific for a particular tyrosine kinase or can be inhibitors of more than one kinase. For example, the compound N- (3,4-dichloro-2-fluorophenyl) -7-({[(3aR, 6aS) -2-methyloctahydrocyclopenta [c] pyrrol-5-yl] methyl} oxy)- 6- (Methyloxy) quinazolin-4-amine (also known as XL647, EXEL-7647 and KD-019) has several receptor-type tyrosines including EGFR, EphB4, KDR (VEGFR), Flt4 (VEGFR3) and ErbB2. It is an in vitro inhibitor of kinase (RTK) and also an inhibitor of SRC kinase involved in pathways that cause tumor unresponsiveness to certain TKIs. In one embodiment of the invention, treatment of a subject in need comprises administration of a rho-kinase inhibitor of formula I and administration of KD-019.

  Dasatinib (BMS-354825, Bristol-Myers Squibb, New York) is another ATP site-competing Src inhibitor that is orally bioavailable. Dasatinib is also a Bcr-Abl (FDA approved for use in patients with chronic myelogenous leukemia (CML) or Philadelphia chromosome positive (Ph +) acute lymphoblastic leukemia (ALL)), and c-Kit , PDGFR, c-FMS, EphA2, and SFK. Two other oral tyrosine kinase inhibitors of Src and Bcr-Abl are Bostinib (SKI-606) and Saracatinib (AZD0530).

  In one embodiment, bispecific binding proteins such as bispecific antibodies are used in combination with antiviral agents to treat chronic viral infections. For example, for HCV, the following drugs can be used. HCV protease inhibitors include, but are not limited to, boceprevir, telaprevir (VX-950), ITMN-191, SCH-900518, TMC-435, BI-201335, MK-7909, VX-500, VX-813 , BMS790052, BMS650032 and VBY376. HCV nonstructural protein 4B (NS4B) inhibitors include, but are not limited to, clemizole and other NS4B-RNA binding inhibitors, such as, but not limited to, benzimidazole RBI (B-RBI) And indazole RBI (I-RBI). HCV nonstructural protein 5A (NS5A) inhibitors include, but are not limited to, BMS-790052, A-689, A-831, EDP239, GS5885, and PP1461. HCV polymerase (NS5B) inhibitors include, but are not limited to, nucleoside analogs (eg, Valopicitabine, R1479, R1626, R7128), nucleotide analogs (eg, IDX184, PSI-7801, PSI-7777 and non-nucleosides). Analogs (eg, firibvir, HCV-796, VCH-759, VCH-916, ANA598, VCH-222 (VX-222), BI-207127, MK-3281, ABT-072, ABT-333, GS9190, BMS791325) Ribavirin or ribavirin analogs such as Taribabirin (Viramidine, ICN3142), Mizoribine, Merimepodiv (VX-4) 7), it can be used mycophenolate mofetil and mycophenolic acid salts.

  When the bispecific antibody of the invention is administered with a second agent, the first and second agents can be administered sequentially or simultaneously. Each drug can be administered in one or more doses, which can be any schedule including, but not limited to, twice a day, daily, weekly, biweekly, and monthly. Can be administered.

  The invention also includes adjuvant administration of bispecific antibodies. Supplementary administration means that a second agent is administered to the patient in addition to the first agent already administered to treat the disease or symptoms of the disease. In some embodiments, the adjunct administration comprises administering the second agent to a patient whose administration of the first agent did not treat or sufficiently treated the disease or disease symptoms. In other embodiments, the adjunct administration comprises administering a second agent to a patient whose disease has been effectively treated by administration of the first agent.

  In certain embodiments, the dose of a bispecific binding protein, such as a bispecific antibody, is administered daily, every other day, every other day, every third day, once a week, twice a week, three times a week, or 2 Administer once per week. In other embodiments, two, three or four doses of a bispecific binding protein, such as a bispecific antibody, are administered daily, every two days, every third day, once a week or once every two weeks, Administer to subjects. In some embodiments, the dose (s) of bispecific binding protein, such as a bispecific antibody, is administered for 2, 3, 5, 7, 14, or 21 days. In certain embodiments, the dose of a bispecific binding protein, such as a bispecific antibody, is 1 month, 1.5 months, 2 months, 2.5 months, 3 months, 4 months, 5 months, 6 It is administered for months or longer.

  Methods of administration include, but are not limited to, parenteral, intradermal, intravitreal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, buccal, sublingual, intranasal, brain Methods of administration may be internal, intravaginal, transdermal, transmucosal, rectal, by inhalation or topically, especially to the ear, nose, eyes or skin. The mode of administration is left to the practitioner's discretion. In most cases, administration results in the release of a bispecific binding protein, such as a bispecific antibody, into the bloodstream. For the treatment of ocular diseases, intravitreal administration of bispecific binding proteins such as bispecific antibodies is preferred.

  In certain embodiments, it may be desirable to administer a bispecific binding protein, such as a bispecific antibody, locally. This may be achieved by, for example, but not limited to, local infusion, topical application, injection, catheter, or implant, said implant being porous, non-porous or, for example, a silastic membrane A gelatinous material comprising a film such as In such cases, administration can selectively target local tissue without substantially releasing a bispecific binding protein, such as a bispecific antibody, into the bloodstream.

  Pulmonary administration can also be used, for example, through the use of an inhaler or nebulizer, and the use of a formulation with an aerosolizing agent, or perfusion of a fluorocarbon or synthetic pulmonary surfactant. In certain embodiments, bispecific binding proteins, such as bispecific antibodies, are formulated as suppositories, with traditional binders and vehicles such as triglycerides.

  In another embodiment, bispecific binding proteins, such as bispecific antibodies, are delivered to vesicles, particularly liposomes (Langer, 1990, Science 249: 1527-1533, Treat et al., Liposomes in the. Therapeutic of Infectious Disease and Bacterial Infection, Lopez-Berstein and Fiddler (eds.), Liss, New York, pp. 353-365 (1989), Lopez Berstein, 27. See the same book).

  In another embodiment, a bispecific binding protein, such as a bispecific antibody, is delivered in a controlled release system (see, eg, Goodson, Medical Applications of Controlled Release, supra, vol. 2, pp. 115- 138 (1984)). Examples of controlled release systems are discussed in the review by Langer, 1990, Science 249: 1527-1533. In one embodiment, a pump may be used (Langer, supra, Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14: 201, Buchwald et al., 1980, Surgery 88: 507, Saudek et al. 1989, N. Engl. J. Med. 321: 574). In another embodiment, a polymeric material may be used (Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974), Controlled Drug Drabi BioDr. , Smolen and Ball (eds.), Wiley, New York (1984), Ranger and Peppas, 1983, J. Macromol. Sci. Rev. Macromol. Chem. 23:61, and Levy et al., 1985, Science 2:28. 190, During et al., 1989, Ann. Neurol. 25: 351, Howard et al., 1989, J. Neurosurg. 71: 105).

  The above administration schedules are provided for illustration only and should not be considered limiting. One skilled in the art will readily understand what doses and dosing schedules are appropriate.

  It should be understood and expected that variations of the principles disclosed herein may be made by those skilled in the art and that such modifications are intended to be included within the scope of the present disclosure.

  Throughout this application various publications are referenced. These publications are hereby incorporated by reference in their entirety to more fully describe the state of the art to which this disclosure relates. The following examples further illustrate the present disclosure, but should in no way be construed as limiting the scope of the present disclosure.

  US provisional patent applications 61 / 710,420, 62 / 061,097, 61 / 927,907 and international patent application numbers PCT / US2013 / 063754, PCT / US2015 / 011657, and PCT / US2015 / 0554569 No. is hereby incorporated by reference in its entirety.

Example 1 Expression and physical characterization of bispecific antibodies to VEGFR2 and PDL1 This example describes the transient expression of bispecific antibodies to VEGFR2 and PDL1 in HEK293 and purification, aggregation analysis by protein A (SEC). -By UPLC), serum stability test and thermal stability test (Tm measurement using DSC).

Summary The antibody λR3B1 (against VEGFR2) was isolated directly from the Dyax Fab 310 phagemid library by panning on immobilized VEGFR2. A light chain shuffling library was constructed by pairing the light chain pool with the heavy chain of λR3B1. After screening with both human VEGFR2 and mouse VEGFR2, B1C4 capable of binding to both human VEGFR2 and mouse VEGFR2 was isolated. Further modifications in its light chain CDR3 and heavy chain CDR1 and CDR2 were made by soft mutagenesis, where only 10% of the residues were mutated to the other 19 amino acids (except cysteine). B1C4A7, a higher affinity and more stable antibody, was identified by screening a library that was softly mutated under extremely stringent conditions.

  Anti-PDL1 antibodies tccR3D7 and tccR3A11 were also identified from the Dyax Fab 310 phagemid library by panning on PDL1. From the tccR3D7_HCDR1 library, D7A8, an antibody with higher affinity and lower oxidative sensitivity, was isolated, in which the three amino acids of methionine located in tccR3D7 heavy chain CDR1 were randomly mutated.

To construct a BsAb, the anti-PDL1 antibody D7A8 (or A11) is first reformatted into a single chain variable fragment (scFv) in the VH- (SG ₄ ) ₅ -VL orientation, and then the scFv is Connected by (FIG. 1) and added to the C-terminus of the conventional antibody B1A1 or B1C4A7 (against VEGFR2). To reduce effector function, leucine at positions 234 and 235 (IgG1-CH2 domain) of B1A1 or B1C4A7 was mutated to alanine.

  The genes for bispecific antibodies (A1-A8) and (A7-A8) were inserted into the mammalian expression vector pBh1 and transiently expressed in the HEK293 cell line. The supernatant was collected and loaded onto a protein A column to purify the bispecific antibody.

  The purity and stability of the bispecific antibodies (A1-A8) and (A7-A8) were analyzed using SDS-PAGE, SEC-UPLC and DSC. Serum stability of these two bispecific antibodies was examined by incubating BsAb in mouse serum after Western blotting.

Materials and Methods Monospecific and bispecific antibodies expressed and tested

Expression and purification Materials for expression, purification and SDS-PAGE Materials for expression, purification and SDS-PAGE are listed in the table below.

Methods for expression, purification and SDS-PAGE Mammalian expression vectors carrying bispecific antibodies were transfected into free HEK293 cells by using Polyjet transfection reagent according to the manufacturer's instructions. The 6-day culture was collected and the filtered supernatant was loaded onto affinity column protein A connected to an AKTAxpress protein purification system. The purified antibody was buffer exchanged into PBS and filtered. The antibody concentration was obtained by measuring absorbance at 280 nm with a Nanodrop photometer and calculated by using theoretical coefficients.

  Antibody samples were prepared in reduced and non-reduced states by adding loading dye with or without reducing agent. Sample tubes were heated at 100 ° C. for 10 minutes and reduced and non-reduced samples along with molecular weight standards were loaded onto 4-12% NUPAGE® NOVEX bis-Tris gel. The gel was run for about 50 minutes at a constant voltage of 200V. The gel was then gel stained with protein dye and destained in QH2O until a protein band was observed. A photograph of the gel is shown in FIG.

UPLC and DSC
UPLC and DSC materials

UPLC and DSC Methods Method for UPLC The machine was programmed according to manufacturing instructions. The sample was first loaded into a UPLC small volume sample vial, 10 μg sample was loaded and injected onto a BEH200SEC column. The antibody was separated in pH = 7.4 PBS buffer at a flow rate of 0.4 ml / min for 10 minutes, and the antibody eluted from the column was detected by TDA at a wavelength of 280 nM. At least two injections were made for each sample. The area percentage of each peak was analyzed with the software Empower 3 provided by Waters.

DSC Method A sample at a concentration of 1 mg / ml was loaded into a 96 deep well plate, followed by DSC analysis using a Nano DSC. In order to obtain a sufficient sample blank, several buffer (PBS) injections were made. A 15 minute pre-scan was performed before running the sample to ensure the correct starting temperature. A temperature increase of 60 ° C./hour was performed while monitoring from 25 ° C. to 100 ° C. Prior to analysis, a thermogram of a buffer-only sample was subtracted from each antibody, and Tm was calculated after deconvolution using Nano DSC software.

Stability in mouse serum Materials for mouse serum stability

Method for mouse serum stability 200 ng of bispecific or monospecific antibody, respectively, was added to 90% mouse serum and incubated at 37 ° C. for 5 days. Heat treated antibodies in mouse serum were loaded on a 4-12% Bis-Tris protein gel under reducing conditions and electrophoresed. Separated proteins were transferred to PVDF membrane according to manufacturing instructions. After blocking with 3% PBS milk, the membrane was incubated with 1/2000 HRP-anti-human IgG (H + L) antibody. After washing 3 times with PBST, the membrane was first reacted with ECL. Pictures were taken with ImageQuant LAS4000 at various exposure time points to obtain high quality images.

Results Expression and purification Mammalian expression vectors carrying bispecific antibodies were transfected into free HEK293 cells. The bispecific antibody was purified with protein A and the buffer was replaced with PBS. Antibody concentration was measured at 280 nm with a Nanodrop photometer and calculated using theoretical coefficients. The results are shown in the table below.

  This indicates that bispecific antibodies A1-A8 and A7-A8 can be transiently expressed in HEK-293 cells. More than 10 mg of BsAb (A1-A8) and BsAb (A7-A8) were recovered from 1 L of supernatant collected on the 6th day after protein A purification and buffer exchange.

SDS-PAGE analysis 5 μg of each antibody was loaded on 4-12% NuPAGE gels in reduced or non-reduced state. The gel was photographed after staining with protein dye and destaining with H ₂ O. The result is shown in FIG. This shows that the purity of both BsAbs is estimated by SDS-PAGE and is over 95%.

SEC-UPLC analysis SEC-UPLC analysis was performed using a column: ACQUITY UPLC BEH200SEC, 1.7 μm, 4.6 × 150 mm. UPLC system: Waters ACQUITY UPLC with TDA detector, eluent: PBS, pH = 7.4, flow rate: 0.4 mL / min, injection: 10 μg; wavelength 280 nm; percentage of major peaks listed by each curve To do. Two injections were made for each sample. The results of the size exclusion chromatogram (UV trace at 280 nm) of protein A purified bispecific antibody in the UPLC system are shown in FIG. The percentage of peak area versus retention time is summarized in the table. The average peak area of two measurements is also listed in the table.

  SEC-HPLC analysis of BsAb (A1-A8) and BsAb (A7-A8) revealed that less than 2% and less than 1% aggregation was detected, respectively. It was also found that the aggregation of BsAb (A7-A8) increased with higher concentration but was still less than 3%.

Measurement of Tm by DSC The results of DSC scanning of bispecific antibodies in PBS buffer are shown in FIG. 21 and the table below.

  As a result, the Tm1 of BsAb (A7-A8) -LALA was about 60 ° C. as measured by DSC scanning and was lower than the lowest Tm1 of the parent antibodies B1C4A7 and D7A8-LALA (both about 69 ° C.). The lowest Tm1 of BsAb (A7-A8) was contributed by scFv D7A8 since it was reported that the scFv antibody fragment was not as stable as IgG. By using a longer linker between the heavy and light chain variable domains of D7A8, or by further manipulation of the scFv, such as the addition of a disulfide bond between the two domains, its thermal stability can be improved.

Serum stability FIG. 22 shows the results of Western blotting of a bispecific antibody treated in mouse serum at 37 ° C. for 5 days. As shown in the figure, after treating BsAb (A1-A8) -LALA and BsAb (A7-A8) -LALA at 37 ° C. for 5 days, no short heavy and light chain bands were observed in the reduced state. . Therefore, the results indicate that both BsAb (A1-A8) -LALA and BsAb (A7-A8) -LALA were stable in mouse serum at 37 ° C. for up to 5 days.

Example 2 In Vitro Characterization of BI Specific Antibody (A7-A8) This example demonstrates in vitro characterization of bispecific antibody A7-A8 and its parent antibodies B1C4A7 (against VEGFR2) and dsD7A8- Describes the efficacy comparison with LALA (against PDL1).

Overview The previous example was obtained by transient expression in HEK293 and purified by protein A, pure bispecific antibody A7-A8 was detected by 1% as detected by SEC-UPLC analysis. It was reported to have a higher molecular weight aggregation of less than. BsAb (A7-A8) was also stable in mouse serum at 37 ° C. for up to 5 days.

  The bispecific antibody A7-A8, which can cross-react with the parent antibodies B1C4A7 (to VEGFR2) and dsD7A8-LALA (to PDL1) alone with human and mouse species, soluble and cell-expressed VEGFR2 and PDL1 (human and mouse Binding ability to both species), ability to inhibit ligands (VEGF and PDL1) binding to reactive receptors (VEGFR2 and PD1), ability to block phosphorylation of VEGFR2 / downstream molecules, and ability to stimulate cytokine IL2 and INFγ secretion Tested.

Materials and Methods Antibodies to be tested

Dose-responsive binding ELISA
Materials for dose-responsive binding ELISA

Dose-responsive binding ELISA method Antigens VEGFR2-Fc (human and mouse) and PDL1-Fc (human and mouse) were coated on Immulon 2HB plates at 1 μg / ml at 4 ° C. overnight. The plate was blocked with 3% PBSM. Serially diluted bispecific and monospecific antibodies were added to the blocked plates and incubated at room temperature (RT) for about 1 hour. After washing the plate, HRP-conjugated anti-human IgG Fab specific antibody (HRP anti-hFab) antibody was added (1/5000 dilution in 3% PBSM). The plate was washed again and then developed by adding TMB reaction buffer. After stopping the reaction with 1N H ₂ SO ₄ , the plate was read at OD450 with a plate reader.

Dose response competition ELISA
Materials for dose response competitive ELISA

Dose Response Competition ELISA Method Both human and mouse PDL1-Fc were labeled with biotin according to the manufacturer's instructions.

Ligand VEGF (human and mouse) and PDL1 (human and mouse) were coated overnight at 4 ° C. at 1 μg / ml on Immulon 2HB plates. The plate was blocked with 3% PBSM. Serially diluted antibodies are mixed with human or mouse VEGFR2 (final concentration 0.5 μg / ml) or biotinylated human and mouse PDL1 in round bottom plates blocked with 96 well PBSM and incubated for 1 hour at RT did. Each antibody-receptor mixture was transferred to a blocked ligand-coated plate and incubated for an additional hour at RT. After washing the plate, HRP-anti-human IgG Fc specific antibody (for VEGF-VEGFR2) or strep-HRP (for PD1-PDL1) was added (diluted 1/5000 with 3% PBSM), respectively. The plate was washed again and then developed with TMB reaction buffer. After stopping the reaction with 1N H ₂ SO ₄ , the plate was read at OD450 with a plate reader.

Dynamic analysis by Biacore Materials for Biacore

Method for Biacore Surface preparation Binding affinity was evaluated using surface plasmon resonance (SPR) on a Biacore T200 instrument (GE Healthcare). The CM5 chip was equilibrated in 10 μl / ml running buffer HBSEP (running buffer). Two flow cells of the CM5 chip were activated with 1: 1 NHS / EDC injection for 5 minutes. The second flow cell was fixed with 5 μg / ml hVEGFR2 diluted with 10 mM sodium acetate buffer at pH 5 to reach 40-100 RU of immobilized protein. The surface was subsequently blocked with a 5 minute injection of ethanolamine.

Sample runs were performed in HBSEP running buffer at 30 μl / min. Samples were serially diluted in running buffer at concentrations ranging from 1.5 to 100 nM. Samples were injected onto the two flow cells with a 3 minute association time and a 10 minute dissociation time. Regeneration was performed after each binding cycle using a 30 second injection of 20 mM HCL. Sensorgrams were obtained for each concentration and the resulting curve was fit to a 1: 1 Langmuir binding model with a blank flow cell subtraction using Bia evaluation software.

Cross-linked ELISA
Materials for cross-linked ELISA

Cross-linking ELISA Method Human PDL1-Fc was labeled with a Thermo-Fisher biotin labeling kit according to the manufacturer's instructions.

Human VEGFR2 was coated on Immulon 2HB plates at 1 μg / ml at 4 ° C. overnight. The plate was blocked with 3% PBSM. 1 nM bispecific or monospecific antibody was mixed with biotin-labeled hPDL1 in a circular 96-well plate blocked with PBSM and incubated for 1 hour at RT. The mixture was transferred to a PBSM blocked hVEGFR2 coated plate and incubated for an additional hour at room temperature. After washing the plate, HRP-strep was added (1/5000 dilution with 3% PBSM). The plate was washed again and developed by adding TMB reaction buffer. After stopping the reaction with 1N H ₂ SO ₄ , the plate was read at OD450 with a plate reader.

Cell binding assays Materials for cell binding

Methods for cell binding PAE-KDR cells (in DMEM containing glutamine supplemented with 10% heat inactive FBS), EOMA cells (in DMEM containing glutamine supplemented with 10% heat inactive FBS), MDA-MB- 231 cells (in RPMI 1640 supplemented with 10% heat inactive FBS containing glutamine) and B16-F10 cells (in RPMI 1640 supplemented with 10% heat inactive FBS containing glutamine) were grown to 90% confluence. Cells were harvested, washed first and then resuspended at 5 × 10 ⁵ cells / ml in ice-cold FACS buffer (PBS, 1% BSA). 100 μl of cell suspension was added to each well of a 96 well round bottom microliter plate and spun down (1200 rpm, 5 minutes). Serially diluted bispecific and monospecific antibodies were added to the cells and incubated with the cells for 1 hour at 4 ° C. Cells were washed three times by centrifugation at 1500 rpm for 5 minutes in 200 μl ice-cold FACS buffer. The cells were resuspended in 100 μl PE-anti-human antibody (1/100) and incubated at 4 ° C. for at least 30 minutes in the dark. Cells were washed by resuspending 3 times in 200 μl PBS and reading fluorescence.

Phosphorylation assay Material for phosphorylation assay

Solution for phosphorylation assay Lysis buffer: 50 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton × 100, 1 mM EDTA, proteinase inhibitor cocktail (1: 500): phosphatase inhibitor cocktail 2 (1: 200) and phosphatase inhibitor cocktail 3 (1: 200)
2. Stripping buffer: 100 mM 2-mercaptoethanol, 2% sodium dodecyl sulfate and 62.5 mM Tris-HCl pH 6.7
3. Loading buffer: 4 ml 100% glycerol, 2.4 ml 1M Tris / HCl pH 6.8, 0.8 g SDS, 4 mg bromophenol blue, 0.5 ml 2-mercaptoethanol and 3.1 ml ddH ₂ O.

Phosphorylation Assay Method PAE / KDR (0.4 × 10 ⁶ ) cells in 1 ml 10% FBS / DMEM or EOMA (1 × 10 ⁶ ) cells in 1.5 ml 10% FBS / DMEM culture medium Added to well plate, incubated for 4 hours at 37 ° C., incubated with 5% CO ₂ until cells attached. The medium was removed and serum free DMEM was added and the cells were left overnight. The next day, serially diluted antibodies are added to the cells, first incubated at 37 ° C., 5% CO ₂ for 20 minutes, then VEGF (PAE / KDR and EOMA, final concentrations 30 ng / ml and 50 ng / ml, respectively) are added to the cells. Added to. The cell mixture was incubated for an additional 10 minutes at 37 ° C., 5% CO ₂ . The cells were washed once with serum-free medium. Then 100 μl of lysis buffer was added and the cells were detached from the dish and placed in a 1.5 ml Eppendorf tube and incubated on ice for about 2 hours. The floating sample was centrifuged at 10,000 rpm for 10 minutes at 4 ° C., and the supernatant was collected. Protein concentration was measured with a Bio-Rad protein assay kit. 30 μg of protein was then mixed with 8 μl of loading buffer and boiled for 5 minutes. The mixture was loaded onto a 4-12% NUPAGE gel and the gel was run to separate the proteins. The protein and marker were transferred together onto the PVDF membrane overnight. The next day, the membrane was blocked with 3% PBS milk and then anti-phospho-VEGFR2 (1/1000) or anti-phospho-MAPK was added to the membrane. The membrane was incubated overnight at 4 ° C. After the membrane was washed twice with 0.1% T-PBS, 1: 1500 anti-rabbit antibody conjugated HRP was added and incubated with the membrane for 1 hour at RT. The membrane was washed 3 times with PBST, ECL was added and photographed with ImageQuant LAS4000 (for phospho-protein).

  Pre-added secondary antibody was removed by incubating the membrane with stripping buffer at 50 ° C. for 45 minutes. Membranes were washed twice with PBST and then incubated with rabbit anti-KDR antibody or anti-MAPK antibody (1-400 dilution in 10 ml 3% PBSM). The membrane was incubated with rabbit antibody at 4 ° C. overnight. The membrane was washed again and HRP-anti-rabbit antibody (diluted 1-1500) was added to the membrane. After 1 hour incubation at RT, the membrane was washed 3 times before adding ECL and taking luminescence images with ImageQuant LA4000 (for total protein).

Cytokine secretion assay Materials for phosphorylation assay

Phosphorylation assay Using Histopaque-1077 according to the manufacturer's instructions, PBMCs from LeukoPak (enriched leukocyte export containing high concentrations of blood cells including monocytes, lymphocytes, platelets, plasma, and erythrocytes). Isolated. PBMC were cultured at 2 × 10 ⁴ cells / well in 96 well plates containing IMDM (supplemented with 2 mM glutamine, 25 mM HEPES, 3.024 g / L sodium bicarbonate) and 10% FBS and serially diluted. Activated with 0.01 μg / mL SEB for 5-7 days in the presence of bispecific antibody or its parent antibody. On day 6 or 7, supernatants were collected for IL-2 and IFNγ measurements using human IFN-γ and human IL-2 Quantikine ELISA kits according to the manufacturer's instructions.

Results Dose response binding to human and mouse VEGFR2 and PDL1 BsAb (A7-A8) -LALA serially diluted with parental monospecific antibodies B1C4A7 (against VEGFR2) and dsD7A8-LALA (against PDL1) , Alone, in 96-well plates coated with hVEGFR2, or mVEGFR2, or hPDL1 and mPDL1, respectively, and incubated for 1 hour at RT. The plate was then incubated with HRP-conjugated anti-human IgG Fab specific antibody (goat). Plates were washed and peroxidase substrate was added and A450 nm read. Data points were mean of duplicate determinations ± standard deviation. The EC50 for various antigens is calculated using a program called Graphpad Prism “log (agonist) vs. response-variable slope (4 parameters)” and listed at the top of each graph. The results are shown in FIG.

  As a result, it is shown that BsAb (A7-A8) -LALA was able to bind strongly to recombinant human VEGFR2, mouse VEGFR2, human PDL1 and mouse PDL1. The EC50 was 0.128 nM for hVEGFR2, 0.598 nM for mVEGFR2, 0.079 nM for hPDL1, and 0.137 nM for mPDL1, respectively. The relevant EC50 for B1C4A7 for hVEGFR2 and mVEGFR2 was 0.107 nM and 0.466 nM, respectively. Regarding dsD7A8 for hPDL1 and mPDL1, they were 0.121 nM and 0.172 nM, respectively.

Dose response assay to determine IC50 BsAb serially diluted with parental monospecific antibodies B1C4A7 (against VEGFR2) and dsD7A8-LALA (against PDL1) for blocking of VEGF-VEGFR2 interaction (A7-A8) -LALA alone was incubated with a fixed amount of either human or mouse VEGFR2-Fc (final 0.5 μg / ml) in solution for 1 hour at RT, after which the mixture was Transfer to 96-well plates coated with either VEGF or mouse VEGF and incubate for an additional hour. The amount of h / mVEGFR2 bound to immobilized h / mVEGF was quantified by incubation of the plate with HRP-anti-human IgG Fc specific antibody.

  For blocking PD1-PDL1 interaction, serially diluted BsAb (A7-A8) -LALA with parental monospecific antibodies B1C4A7 (for VEGFR2) and dsD7A8-LALA (for PDL1) Alone with a fixed amount of biotin labeled with either human or mouse PDL1-Fc (final 0.5 μg / ml) in solution for 1 hour at RT, after which the mixture is either human PDL1 or mouse PDL1 Were transferred to 96-well plates coated with either of the above and incubated for an additional hour. The amount of h / mPDL bound to immobilized h / mPD1 was quantified by incubation of the plate with HRP-strep.

  The result is shown in FIG. Data points are the mean ± standard deviation of duplicate determinations. Using the program called “Log (Inhibitor) vs. Response-Variable Slope (4 parameters)” from GraphPad Prism, the IC 50 of the different interactions is calculated and listed at the top of each graph.

  This result indicates that BsAb (A7-A8) was able to strongly block the binding of ligand VEGF or PDL1 to the respective receptors VEGFR2 and PD1. The IC50 for the hVEGF-hVEGFR2 interaction was 1.29 nM for BsAb (A7-A8) -LALA versus 1.72 nM for B1C4A7. The IC50 for the mVEGF-mVEGFR2 interaction was 0.801 nM for BsAb (A7-A8) -LALA, compared to 0.973 nM for B1C4A7. The IC50 for hPDL1-hPD1 interaction was 2.51 nM for BsAb (A7-A8) -LALA, compared to 3.04 nM for dsD7A8-LALA. The IC50 for mPDL1-mPD1 interaction was 5.59 nM for BsAb (A7-A8) -LALA, compared to 2.80 nM for dsD7A8-LALA.

Binding to VEGFR2 and PDL1 expressed by cells Along with parent antibodies B1C4A7 (against VEGFR2) and dsD7A8 (against PDL1), serially diluted BsAb (A7-A8) -LALA alone, Added: KDR-PAE (hVEGFR2), EOMA (mVEGFR2), MDA-MB-231 (hPDL1) and B16-F10 (mPDL1), then incubated at 4 ° C. for 1 hour. Cells were then incubated with PE-labeled anti-human IgG Fc specific antibody. Cells were washed and fluorescence was read. The fluorescence intensity of the antibody on the cell surface antigen was calculated and plotted against the antibody concentration. The results are shown in FIG. EC50's for different antigens are calculated using GraphPad Prism's program called “log (agonist) vs. response-variable slope (4 parameters)” and listed at the top of each graph.

  This result indicates that BsAb (A7-A8) -LALA was also able to bind to cell-expressed human VEGFR2, mouse VEGFR2, human PDL1 and mouse PDL1. The EC50 values of BsAb (A7-A8) -LALA for KDR / PAE, MDA-MB-231 and B16-F10 were 0.533 nM, 0.223 nM and 0.122 nM, respectively. The relevant EC50 for KDR / PAE for B1C4A7 was 0.522 nM, and for dsD7A8 for MDA-MB-231 and B16-F10, the values were 0.159 nM and 0.042 nM, respectively. The EC50 determination of BsAb (A7-A8) -LALA against EOMA-expressed mVEGFR2 was prevented by binding of EOMA expression to PDL1 (FIG. 25).

  The EC50 and IC50 for the bispecific antibody A7-A8 and its parent antibodies B1C4A7 and dsD7A8-LALA are listed in the table below. Due to the very close EC50 and IC50 between BsAb (A7-A8) -LALA and its related parent antibody B1A1 or dsD7A8-LALA, BsAb (A7-A8) -LALA is either B1A1 or dsD7A8-LALA. It is shown to be equally powerful.

Kinetic analysis hVEGFR-Fc or hPDL1-Fc was immobilized at pH 5 on a series S CM5 sensor chip using standard amine coupling chemistry. Antibodies were injected at 30 μl / min at concentrations ranging from 1.5-100 nM on the immobilized surface using 1 × HBSEP as the running buffer. The contact time (meeting stage) was 3 minutes. The dissociation time was 6-10 minutes. Regeneration was performed after each binding cycle using an injection of 20 mM HCL for 30 seconds at a flow rate of 30 μl / min. Sensorgrams were obtained at each concentration and the resulting curve was fitted to a 1: 1 Langmuir binding model using Biavaluation software. The results are shown in FIG. The association rate (on) (ka), dissociation rate (kd) and calculated KD are listed at the top of each graph.

  This kinetic analysis by Biacore shows that sAb (A1-A8) -LALA rapidly associates with either VEGFR2 or PDL1 and slowly dissociates from either VEGFR2 or PDL1. The very slow dissociation rate from VEGFR2 and PDL1 was outside the measuring device range. The KD values for BsAb (A7-A8) were approximately 47 pM and 1.2 pM for hVEGFR2 and hPDL1, respectively, comparable to 52 pM for B1C4A7 and 7.7 pM for dsD7A8-LALA.

Cross-binding ELISA for two targets
Scheme 1 shown in FIG. 27 was used for the cross-linked ELISA. As shown in the figure, complexes 2 and 3 can be found on the surface of immobilized hVEGFR2, but only complex 3 can be detected by strep-HRP. Complex 1 that can be found in solution is washed away during processing.

  A single dilution of BsAb (A7-A8) -LALA in series with the parent monospecific antibodies B1C4A7 (for VEGFR2) and dsD7A8-LALA (for PDL1) alone, a fixed amount of biotin-labeled human PDL1 -Fc (final 0.5 μg / ml) was incubated in solution for 1 hour at RT, after which the mixture was transferred to a 96-well plate coated with hVEGFR2 and incubated for an additional hour. Bound BsAb / biotin-PDL1 complex could be detected by HRP-strep (shown as in Scheme 1). The results are shown in FIG. Data points are the mean of 6 determinations ± standard deviation.

  As shown in the figure, the bispecific antibody A7-A8 was able to bind to VEGFR2 and PDL1 simultaneously, as examined by cross-binding ELISA. This shows that the conformation of the bispecific antibody does not change after binding to the first target. Thus, the first target binding bispecific antibody was able to bind to the second target.

Phosphorylation assay Serum starved cells are incubated with varying amounts of antibody for 30 minutes at room temperature followed by 30 ng / ml hVEGF (for KDR-PAE) or 50 ng / ml mVEGF (for EOMA). For another 15 minutes. Cells were lysed and protein concentration was measured. 30 μg of protein was separated by SDS PAGE and subjected to immunoblotting analysis using an anti-phosphotyrosine antibody against the receptor and MAPK. The signal was detected using sensitive chemiluminescence. The results are shown in FIG.

  The efficacy of BsAb (A7-A8) -LALA was examined by testing its ability to block phosphorylation of human or mouse VEGFR2 and downstream molecules MAPK. FIG. 29 shows that the phosphoprotein bands of human VEGFR2, mVEGFR2, and MAPK are much weaker in the presence of 100 or 10 nM BsAb (A7-A8) -LALA and B1C4A7. The difference in band density when treated with BsAb (A7-A8) and B1C4A7 was negligible and could not be distinguished by the eye.

Cytokine secretion assay Serially diluted BsAb (A7-A8) -LALA alone with parent antibodies B1C4A7 (against VEGFR2) and dsD7A8 (against PDL1) PBMC activated by 0.01 mg / mL SEB (From donor BG). On day 6, supernatants were collected for IL-2 and IFNγ measurements by using the Duoset kit according to the manufacturing instructions. The results are shown in FIG. Data points are the mean ± standard deviation of triplicate determinations, calculated by the following formula:
(Sample Read-Unstimulated Read) / (SEB Stimulus Read-Unstimulated Read) ^* 100

  Results show that in the presence of BsAb (A7-A8) -LALA and dsD7A8, secretion of cytokines IL2 and INFγ in PBMC when stimulated by SEB was significantly higher than anti-VEGFR2 antibody B1C4A7 (FIG. 30). Indicated. There was no difference between treatment with BsAb (A7-A8) -LALA and dsD7A8.

  Taken together, the above results demonstrate that BsAb (A7-A8) -LALA retains the potency of its parent antibodies B1C4A7 (to VEGFR2) and dsD7A8-LALA (to PDL1).

Example 3 In Vitro Characterization of Bispecific Antibody (A1-A8) This example demonstrates in vitro characterization of bispecific antibody A1-A8 and its parent antibodies B1A1 (against VEGFR2) and dsD7A8 Describes efficacy comparison with LALA (against PDL1).

  As reported in the previous example, pure bispecific antibody A1-A8 obtained by transient expression in HEK293 and purified by protein A was less than 1% detected by SEC-UPLC analysis. Of higher molecular weight aggregation. BsAb (A1-A8) was also very stable in mouse serum after 5 days incubation at 37 ° C.

  The bispecific antibody A1-A8, which can cross-react with human and mouse species, alone with its parent antibodies B1A1 (against VEGFR2) and dsD7A8-LALA (against PDL1), soluble and cell-expressed VEGFR2 and PDL1 It was tested for its ability to bind to, ligand (VEGF and PDL1), ability to bind to receptors (VEGFR2 and PD1), ability to block phosphorylation of VEGFR2 / downstream molecules, and ability to stimulate cytokine IL2 and INFγ secretion .

Materials and Methods The materials and methods herein are the same as those described in Example 2 above, except that the following antibodies, including the bispecific antibody A1-A8, were used.

Results Dose response binding to human and mouse VEGFR2 and PDL1 Dose response to human and mouse VEGFR2 and PDL1 was measured in the same manner as above. The results are shown in FIG.

  As a result, it has been shown that BsAb (A1-A8) -LALA can bind strongly to recombinant human VEGFR2, human PDL1, and mouse PDL1. EC50 is 0.128 nM for hVEGFR2, 0.088 nM for hPDL1, and 0.160 nM for mPDL1, respectively, and the associated EC50 for hVEGFR2 for B1A1 is 0.127 nM, for hPDL1 and mPDL1 for dsD7A8, They are 0.121 nM and 0.172 nM, respectively.

Dose response assay to determine IC50 The assay was performed as described above. The result is shown in FIG. Data points are the mean ± standard deviation of duplicate determinations. IC50s for the various interactions are calculated using a program called Graphpad Prism “log (inhibitor) vs. response-variable slope (4 parameters)” and are listed at the top of each graph.

  As a result, it is shown that BsAb (A1-A8) was able to strongly block the binding to VEGFR2 and PD1, which are receptors of ligand VEGF or PDL1, respectively. The IC50 for the hVEGF-hVEGFR2 interaction was 1.19 nM for BsAb (A1-A8) -LALA versus 1.60 nM for B1A1. The IC50 for hPDL1-hPD1 interaction was 2.51 nM for BsAb (A1-A8) -LALA versus 3.04 nM for dsD7A8-LALA. The IC50 for mPDL1-mPD1 interaction was 3.20 nM for BsAb (A1-A8) -LALA versus 2.80 nM for dsD7A8-LALA.

Binding to cell-expressed VEGFR2 and PDL1 Binding assays were performed as described above. The results are shown in FIG.

  The results indicate that BsAb (A1-A8) -LALA was also able to bind to cell-expressed human VEGFR2, human PDL1 and mouse PDL1. EC50 values of BsAb (A1-A8) -LALA for KDR / PAE, MDA-MB-231 and B16-F10 were 0.779 nM, 0.402 nM and 0.113 nM, respectively. The related EC50 value for KDR / PAE for B1A1 was 0.177 nM, and the related EC50 values for dsD7A8 for MDA-MB-231 and B16-F10 were 0.159 nM and 0.042 nM, respectively. (FIG. 33).

  The EC50 and IC50 of the bispecific antibody A1-A8 and its parent antibodies B1A1 and dsD7A8-LALA are listed in the table below. BsAb (A1-A8) -LALA is either B1A1 or dsD7A8-LALA due to the very close EC50 and IC50 between BsAb (A1-A8) -LALA and its related parent antibody B1A1 or dsD7A8-LALA. It is shown to be as powerful as

Kinetic analysis The kinetic assay was performed as described above. The results are shown in FIG. The association rate (on-rate) (ka), dissociation rate (off-rate) (kd) and calculated KD are listed at the top of each graph.

  Kinetic analysis by Biacore (FIG. 34) shows that BsAb (A1-A8) -LALA rapidly associates with either VEGFR2 or PDL1 and stays longer with either VEGFR2 or PDL1. The very slow dissociation rate of binding to VEGFR2 and PDL1 was outside the instrument's measurement range. The KD values for BsAb (A1-A8) were about 200 pM and 3.2 pM for hVEGFR2 and hPDL1, respectively, comparable to 164 pM for B1A1 and 7.7 pM for dsD7A8-LALA.

Cross-binding ELISA for two targets
The binding assay was performed as described above. The result is shown in FIG. The data point is the mean ± standard deviation of 6 determinations.

  As shown in the figure, the bispecific antibody A18 was able to bind to VEGFR2 and PDL1 when examined by cross-binding ELISA (FIG. 36). This result indicates that the conformation of the bispecific antibody did not change after binding to the first target. Thus, the first target binding bispecific antibody was able to bind to the second target.

Phosphorylation assay The assay was performed as described above. The result is shown in FIG.

  The efficacy of BsAb (A1-A8) -LALA was examined by testing its ability to block phosphorylation of human VEGFR2 and the downstream molecule MAPK. FIG. 36 shows that in the presence of 1.5 μg / ml (7.5 nM) of BsAb (A1-A8) -LALA and BsAb (A7-A8) -LALA, both VEGFR2 and MAPK phosphoprotein bands were antibody treated. It is shown that it was considerably weaker than the band without. In this experiment, the parent antibody concentration was 1.5 μg / ml (10 nM). Therefore, more monospecific antibodies were used in this experiment than bispecific antibodies.

Cytokine secretion assay The cytokine secretion assay was performed as described above. Serially diluted BsAb (A1-A8) -LALA and BsAb (A7-A8) were added to PBMC (derived from donor BG) activated with 0.01 μg / mL SEB. On day 6, supernatants were collected using the Duoset kit according to the manufacturer's instructions for the measurement of IL-2 and IFNγ. Data points were mean ± standard deviation of duplicate determinations. The result is shown in FIG.

  As a result, in the presence of BsAb (A1-A8) -LALA and BsAb (A7-A8) -LALA, secretion of cytokines IL2 and INFγ in PBMC when stimulated by SEB is higher than control antibodies B1A1 and B1C4A7 (FIG. 37).

  Taken together, the above results demonstrate that BsAb (A1-A8) -LALA retains the potency of its parent antibodies B1A1 (to VEGFR2) and dsD7A8-LALA (to PDL1).

Example 4 Antitumor effect I
In this example, the BsAb (A1-A8) was tested for its effect on CT26 mouse colon cancer cells.

More specifically, 0.1 mL of CT26 mouse colon carcinoma cells (0.3 × 10 ⁶ cells / mouse) in serum-free RPMI-1640 medium was injected subcutaneously into the right lower abdomen of mice for tumor development. . When the average tumor size reached approximately 75-125 mm ³ , mice were randomly assigned to 5 experimental groups according to tumor size. Each group consisted of 13 mice. Mice were treated with antibodies as shown in the table below. Each dose was adjusted to 200 μl with PBS and mice were injected intraperitoneally twice a week for up to 3 weeks. Body weight and tumor volume were measured twice weekly. The results are shown in the table below.

  The results show that B1C4A7, D7A8, combinations thereof, and BsAb (A7-A8) reduced tumor volume. The combination of the two antibodies (B1C4A7 and D7A8) and BsAb (A7-A8) was found to be more potent than the two individual antibodies alone in reducing tumor volume in this CT26 model. Since all antibodies were fully human antibodies, immunogenicity occurred for treatments over 2 weeks. Therefore, results up to 3 weeks after treatment were reported. The results also show that no significant weight loss was seen in any treatment group (data not shown).

Example 5 Antitumor effect II
In this example, the BsAb (A1-A8) was tested for its effect on MC38 mouse colon cancer cells.

Each mouse was inoculated subcutaneously with MC38 mouse colon carcinoma cells (1 × 10 ⁶ cells / mouse) in the lower right abdomen for tumor development. When the average tumor size reached approximately 90 mm ³ , mice were randomly assigned to 9 experimental groups according to their tumor size. Each group consisted of 13 mice. Mice were treated with antibodies as shown in FIGS. 39A and 39B. Each dose was adjusted to 200 μl with PBS and animals were injected twice a week for up to 3 weeks intraperitoneally. Body weight and tumor volume were measured twice weekly. The results are shown in FIGS. 39A and 39B.

  As shown in the figure, B1C4A7, an antibody against VEGFR2, showed only moderate efficacy. In contrast, anti-PDL1 antibodies D7A8, BsAb (A7-A8), and the combination of B1C4A7 and D7A8 completely inhibited tumor growth and no significant differences were observed between the three groups. Since all antibodies are fully human antibodies, immunogenicity has occurred for treatments longer than 2 weeks. Results were reported up to 3 weeks after treatment. Furthermore, no significant weight loss was observed for all mice at 3 weeks (data not shown). All antibodies were constructed in the normal IgG1 version (FIG. 39A) and LALA version (FIG. 39B), and CH2 leucines 234 and 235 were mutated to alanine to reduce effector function. No significant difference was observed between these two versions.

Example 6 In Vitro Characterization of Additional Bispecific Antibodies In this example, additional examples of bispecific antibodies shown in FIG. 1A (ie, HC-C terminal fusions) were generated and examined. For this purpose, different orientations of bispecific antibodies against VEGFR2 and PDL1 were generated using anti-PDL1 antibody A11 or D7A8 and anti-VEGFR2 antibody B1A1 or B1C4A7. These bispecific antibodies, related components, and binding specificities are listed in the table below. For bispecific antibodies, the following two linkers were used:
15GS: GGGGSGGGGSGGGGS (SEQ ID NO: 68)
30GS: GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 69).
In addition, mutations were introduced into the scFv to add a disulfide bond between the two variable domains. Mutations were also introduced into the CH2 domain to reduce effector function (LALA version IgG), where the LL residue of CH2 (eg, bold in SEQ ID NOs: 28, 30 and 32) was changed to AA.

  The purity and stability of these bispecific antibodies were analyzed by SDS-PAGE and SEC-UPLC as described above. The results are shown in FIGS.

  As shown in FIG. 40, several degraded bands were observed in the BsAb (B1A1-A11) and BsAb (B1C4A7-A11) variants.

  In contrast, no resolved bands were observed after orientation changes like BsAb (A11-B1A1) and BsAb (D7A8-B1A1) variants (FIG. 41).

  SEC-UPLC results showed that bispecific antibodies are more stable when disulfide bonds are added. For example, in the case of BsAb (A11-B1A1) _30cc, the monomer is over 95.5%, but for A11 IgG, BsAb (A11-B1A1), and BsAb (A11-B1A1) _30, the monomer is each 97.8%, 94.3%, and more than 93.8%. Similarly, among the BsAb (D7A8-B1A1) variants, for BsAb (D7A8-B1A1) _30cc, the monomer is over 97.3%, while D7A8 IgG, BsAb (D7A8-B1A1) and BsAb For (D7A8-B1A1) _30, the monomers were greater than 99%, 86.6%, and 90.2%, respectively.

  The binding of these bispecific antibodies to PDL1 and VEGR2 was analyzed in the same manner as above. The result is shown in FIG. These bispecific antibodies against PDL1 and VEGR2 were found to be as potent as the parent antibody.

  Blocking the interaction between the ligand and the receptor was also examined by the above method. The result is shown in FIG. These bispecific antibodies have been found to be as potent as the parent antibody.

Biacore analysis was performed as described above. The results are shown in the following table.

Claims (22)

  A bispecific binding protein comprising a first region that binds to human PDL1 and a second region that binds to human KDR.   The bispecific binding protein of claim 1 which is a bispecific antibody.   2. The bispecific binding protein of claim 1 which is a fusion protein.   Bispecific antibodies comprising IgG, IgA, IgE, or IgD, and scFv.   The bispecific antibody of claim 4, comprising an IgG that binds to PD-L1 and an scFv that binds to KDR.   5. The bispecific antibody of claim 4, comprising an IgG that binds KDR and an scFv that binds PD-L1.   It comprises a heavy chain variable domain that binds to human PD-L1 and has three complementarity determining regions (CDRs), and the amino acid sequences of CDR1, CDR2, and CDR3 are SEQ ID NOs: 10-12, or SEQ ID NOs: 58-60, respectively. The bispecific antibody according to claim 5 or 6, wherein   6. A light chain variable domain that binds to human PD-L1 and has three CDRs, wherein the amino acid sequences of CDR1, CDR2, and CDR3 are SEQ ID NO: 13-15, or SEQ ID NO: 61-63, respectively. Or the bispecific antibody according to 6.   The bispecific antibody according to claim 5 or 6, comprising a heavy chain variable domain that binds to human KDR and has three CDRs, wherein the amino acid sequences of CDR1, CDR2, and CDR3 are SEQ ID NOs: 16 to 18, respectively. .   The bispecific antibody according to claim 5 or 6, comprising a light chain variable domain that binds to human KDR and has three CDRs, wherein the amino acid sequences CDR1, CDR2, and CDR3 are SEQ ID NOs: 19 to 21, respectively.   The bispecific antibody according to claim 5 or 6, comprising a heavy chain variable domain that binds to human KDR and has three CDRs, wherein the amino acid sequences CDR1, CDR2, and CDR3 are SEQ ID NOs: 22-24, respectively.   The bispecific antibody according to claim 5 or 6, comprising a light chain variable domain that binds to human KDR and has three CDRs, wherein the amino acid sequences CDR1, CDR2, and CDR3 are SEQ ID NOs: 25-27, respectively.   Comprising heavy and light chains, said heavy and light chains comprising SEQ ID NOs: 34 and 31, SEQ ID NOs 30 and 35, SEQ ID NOs 36 and 31, SEQ ID NOs 30 and 37, SEQ ID NOs 38 and 33, 39, SEQ ID NO: 40 and 33, SEQ ID NO: 32 and 41, SEQ ID NO: 42 and 29, SEQ ID NO: 28 and 43, SEQ ID NO: 44 and 29, SEQ ID NO: 28 and 45, SEQ ID NO: 46 and 29, SEQ ID NO: 28 and 47 SEQ ID NOs: 48 and 29, SEQ ID NOs: 28 and 49, SEQ ID NOs: 51 and 50, SEQ ID NOs: 52 and 50, SEQ ID NOs: 53 and 50, SEQ ID NOs: 54 and 29, SEQ ID NOs: 55 and 29, and SEQ ID NOs: 56 and 29. 7. The bispecific antibody of claim 5 or 6, comprising the sequence of each heavy / light chain pair selected from the group consisting of:   A method of treating a patient in need of reduced immunosuppression or reduced angiogenesis, wherein the patient in need of such reduction in immunosuppression or angiogenesis is defined in any one of claims 1-3. 14. The method of treatment comprising administering the bispecific binding protein of claim 1 or the bispecific antibody of any one of claims 4-13.   14. A method for treating cancer, wherein the bispecific binding protein according to any one of claims 1 to 3 or any one of claims 4 to 13 is provided to a patient in need thereof. Administering said bispecific antibody.   The cancer is selected from the group consisting of lung cancer, colorectal cancer, renal cell cancer, glioblastoma, ovarian cancer, bladder cancer, gastric cancer, multiple myeloma, non-small cell lung cancer and pancreatic cancer. 15. The method according to 15.   14. The bispecific binding protein according to any one of claims 1 to 3, the bispecific antibody according to any one of claims 4 to 13, or an isolated polypeptide encoding the polypeptide chain thereof. Nucleic acid molecules.   A vector comprising the nucleic acid molecule of claim 17.   A cultured host cell comprising the vector of claim 18.   20. A method for producing a polypeptide comprising culturing the host cell of claim 19 under conditions that allow expression of the nucleic acid molecule.   14. The bispecific binding protein according to any one of claims 1 to 3 or the conjugate of the bispecific antibody according to any one of claims 4 to 13 wherein the bispecificity is the same. The conjugate, wherein the binding protein or the bispecific antibody is conjugated to an agent selected from the group consisting of a contrast agent, a therapeutic agent and a cytotoxic agent. A pharmaceutical composition comprising:
The bispecific binding protein according to any one of claims 1 to 3, the bispecific antibody according to any one of claims 4 to 13, or the conjugate according to claim 21, and Said pharmaceutical composition comprising a pharmaceutically acceptable carrier.