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  • C07K16/2827—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
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  • C07K16/2866—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
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  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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  • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
  • C07K2317/565—Complementarity determining region [CDR]
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  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
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  • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
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  • C07K2319/00—Fusion polypeptide
  • C07K2319/32—Fusion polypeptide fusions with soluble part of a cell surface receptor, "decoy receptors"

Definitions

• Bispecific binding molecules are provided that are useful for treating cancer and other diseases.
• Immune checkpoints proteins act as immune system regulators and are a key part of the mechanism of self-tolerance.
• Inhibitory immune checkpoint proteins include:
• PD-1 Programmed cell death protein 1
• PD-L1 programmed cell death ligand 1
• Adenosine A2A receptor A2AR
• B7-H3 CD276
• B7-H4 B and T Lymphocyte Attenuator (BTLA)
• BTLA B7-H4
• BTLA T Lymphocyte Attenuator
• CTL-4 Cytotoxic T-Lymphocyte- Associated protein 4
• IDO Indoleamine 2,3 -di oxygenase
• KIR Killer-cell Immunoglobulin-like Receptor
• Lymphocyte Activation Gene-3 (LAG3); Nicotinamide adenine dinucleotide phosphate NADPH oxidase isoform 2 (NOX2); T-cell Immunoglobulin domain and Mucin domain 3 (TIM-3); V-domain Ig suppressor of T cell activation (VISTA); Sialic acid-binding immunoglobulin-type lectin 7 (SIGLEC7, CD328); and Sialic acid-binding immunoglobulin- type lectin 9 (SIGLEC9, CD329).
• Programmed cell death protein 1 is a receptor protein expressed on T-cells that acts as an immune checkpoint. PD-1 expression is upregulated on activated T cells as part of the mechanism of immune tolerance.
• the ligand for PD-1 is programmed cell death ligand 1 (PD-L1), and binding of PD-L1 to PD-1 transmits an inhibitory signal that reduces the activation and proliferation of antigen-specific T-cells in lymph nodes, and reduces apoptosis in regulatory T cells. Tumor cells often overexpress PD-L1 as a mechanism for avoiding immune surveillance.
• Monoclonal antibodies that inhibit binding between PD-1 and PD-L1 - by binding either the ligand or receptor - have been shown to be effective either as monotherapy or in combination with other agents, in subpopulations of patients with a number of cancers, while being ineffective or refractory in many other cancers.
• Pembrolizumab is a humanized antibody that was first approved by the FDA in 2014 and that is used for treatment of a variety of cancers where the tumor cells express elevated PD-L1.
• Nivolumab is a fully human antibody that was first approved by the FDA in 2014 and also is used for treating a variety of cancers.
• Tumor-associated macrophages are a class of immune cells present in high numbers in the tumor microenvironment (TME), and are associated with cancer-related inflammation. Expression of PD-1 on TAM cells has been shown to decrease macrophage phagocytosis of tumor cells and confers“immunity” on the tumor cells.
• Interleukin- 1b is a pro-inflammatory cytokine that is associated with chronic and acute inflammation and plays an important role in multiple inflammation-associated diseases. Elevated levels of IL-Ib have also been shown to recruit TAM cells and myeloid- derived suppressor cells (MDSC) to the TME and to promote tumor growth and metastasis in breast cancer. Guo et al, Sci. Rep. 6, 36107; doi: 10.1038/srep36107 (2016). In other studies, lung lesions have been shown to be populated with TAM whose pro-tumor activity is up- regulated by activation of the NLRP3 inflammasome and the release of IL-Ib.
• TAM myeloid- derived suppressor cells
• IL-Ib has been shown to promote a pro-tumor phenotype of TAM cells and the level of the cytokine has been correlated to tumor size & stage in renal cell carcinoma (Chittezhath et al, Immunity 41 :815 (2014)).
• mice deficient in IL-Ib fewer animals developed tumors and tumor development was slower.
• Apte, et al European Journal of Cancer, 42:751 (2006). Additionally, it was shown that the lung cancer risk genotype IE-1b-31TT results in increased expression of IL- 1b, providing a microenvironment with elevated inflammatory stimuli and increase lung cancer risk.
• An IL-1 receptor antagonist was shown to suppress metastasis and tumor proliferation by inhibiting angiogenic factors such as VEGF and IL-8. Konishi et al, Oncology 68: 138 (2005); Lewis et al, J. Transl. Med. 4:48 (2006).
• Canakinumab a monoclonal antibody that inhibits IL-Ib activity, has been shown to reduce incident lung cancer and lung cancer mortality. Ridker et al, Lancet, 390:P 1833-1842, (2017).
• Bispecific monoclonal antibodies are non-naturally occurring proteins containing immunoglobulin domains that can simultaneously bind to two different types of antigen.
• Bispecific antibodies can be made in a variety of format and have been used, for example, for cancer immunotherapy and drug delivery. See, for example, Fan et al, J.
• a binding protein may contain a first binding domain and a second binding domain, where the first binding domain specifically binds and inhibits activation of an immune checkpoint protein, and where the second binding domain specifically binds and inhibits the activity of IL-b or IL-1R.
• the immune checkpoint protein may be, for example, PD-1 or PD-L1.
• the first and second binding domains may contain immunoglobulin binding domains, such as human immunoglobulin binding domains.
• These binding protein may further contain a third binding domain that specifically binds and inhibits activation of an immune checkpoint protein, such as PD-1 or PD-L1, and a fourth binding domain that specifically binds and inhibits the activity of IL-b or IL-1R.
• the first and the third binding domains may contain the same CDR regions and the second and the fourth binding domains may contain the same CDR regions.
• the first binding domain may contain a) a heavy chain CDR1 of SEQ ID NO. : 1 ; (b) a heavy chain CDR2 of SEQ ID NO. :2; (c) a heavy chain CDR3 of SEQ ID NO. :3; (d) a light chain CDR1 of SEQ ID NO. :4; (e) a light chain CDR2 of SEQ ID NO.:5; and (f) a light chain CDR3 of SEQ ID NO.:6.
• the first binding domain may contain the heavy chain variable region of SEQ ID NO: 37 and the light chain variable region of SEQ ID NO:38.
• the first binding domain may contain a) a heavy chain CDR1 of SEQ ID NO.:7; (b) a heavy chain CDR2 of SEQ ID NO.8; (c) a heavy chain CDR3 of SEQ ID NO.: 9; (d) a light chain CDR1 of SEQ ID NO.: 10; (e) a light chain CDR2 of SEQ ID NO. : 11 ; and (f) a light chain CDR3 of SEQ ID NO. : 12.
• the first binding domain may contain the heavy chain variable region of SEQ ID NO: 39 and the light chain variable region of SEQ ID NO:40.
• the first binding domain may contain a) a heavy chain CDR1 of SEQ ID NO.: 13; (b) a heavy chain CDR2 of SEQ ID NO.15; (c) a heavy chain CDR3 of SEQ ID NO. : 15; (d) a light chain CDR1 of SEQ ID NO. : 16; (e) a light chain CDR2 of SEQ ID NO.: 17; and (f) a light chain CDR3 of SEQ ID NO.: 18.
• the first binding domain may contain the heavy chain variable region of SEQ ID NO: 41 and the light chain variable region of SEQ ID NO:42.
• the first binding domain may contain (a) a heavy chain CDR1 of SEQ ID NO.: 19; (b) a heavy chain CDR2 of SEQ ID NO.:20; (c) a heavy chain CDR3 of SEQ ID NO.:21; (d) a light chain CDR1 of SEQ ID NO.:22; (e) a light chain CDR2 of SEQ ID NO.:23; and (f) a light chain CDR3 of SEQ ID NO.:24.
• the first binding domain may contain the heavy chain variable region of SEQ ID NO: 43 and the light chain variable region of SEQ ID NO:44.
• the first binding domain may contain (a) a heavy chain CDR1 of SEQ ID NO.:25; (b) a heavy chain CDR2 of SEQ ID NO.:26; (c) a heavy chain CDR3 of SEQ ID NO. :27; (d) a light chain CDR1 of SEQ ID NO. :28; (e) a light chain CDR2 of SEQ ID NO.:29; and (I) a light chain CDR3 of SEQ ID NO.:30.
• the first binding domain may contain the heavy chain variable region of SEQ ID NO: 45 and the light chain variable region of SEQ ID NO:46.
• the first binding domain may contain (a) a heavy chain CDR1 of SEQ ID NO.:31; (b) a heavy chain CDR2 of SEQ ID NO.: 32; (c) a heavy chain CDR3 of SEQ ID NO. :33; (d) a light chain CDR1 of SEQ ID NO. :34; (e) a light chain CDR2 of SEQ ID NO.:35; and (I) a light chain CDR3 of SEQ ID NO.:36.
• the first binding domain may contain the heavy chain variable region of SEQ ID NO: 47 and the light chain variable region of SEQ ID NO:48.
• the second binding domain may contain (a) a heavy chain CDR1 of SEQ ID NO.:49; (b) a heavy chain CDR2 of SEQ ID NO.:5Q; (c) a heavy chain CDR3 of SEQ ID NO.:51; (d) a light chain CDR1 of SEQ ID NO.: 52; (e) a light chain CDR2 of SEQ ID NO.:53; and (f) a light chain CDR3 of SEQ ID NO.:54.
• the first binding domain may contain the heavy chain variable region of SEQ ID NO: 55 and the light chain variable region of SEQ ID NO:56.
• binding proteins where the first binding domain is an antibody binding domain that specifically binds and inhibits activation of PD-1 or PD-L1, and where the second binding domain contains an interleukin- Ib-binding domain from an interleukin- 1 receptor type 1 (IL-1R1) or interleukin-1 receptor type 2 (IL-1R2), optionally coupled to a ligand binding domain from IL-1R accessory protein .
• IL-1R1 interleukin- 1 receptor type 1
• IL-1R2 interleukin-1 receptor type 2
• such a binding protein may contain a first protein chain containing (a) an interleukin- Ib-binding domain of IL-1R1 linked to (b) a VH domain of an immunoglobulin that binds and inhibits PD-1 or PD-L1 linked to (c) an immunoglobulin Fc domain; and a second protein chain containing a VL domain of the immunoglobulin that binds PD-1 or PD-L1.
• the binding protein may contain a first protein chain containing (a) a VH domain of an immunoglobulin that binds that binds and inhibits PD-1 or PD-L1 linked to (b) an interleukin- Ib-binding domain of IL-1R1 linked to (c) an immunoglobulin Fc domain; and a second protein chain containing a VL domain of the immunoglobulin that binds and inhibits PD-1 or PD-L1.
• the binding protein may contain two identical protein chains where each protein chain contains (a) an interleukin- 1 b-binding domain of IL-1R1 linked to (b) an immunoglobulin Fc domain linked to (c) an scFV domain that binds and inhibits activation of PD-1 or PD-L1.
• the binding protein may contain: a first protein chain containing (a) an interleukin- Ib-binding domain of IL-1R1 linked to (b) VL and CL domains of an immunoglobulin that binds and inhibits PD-1 or PD-L1 linked to (c) an
• the binding protein may contain: a first protein chain containing (a) VL and CL domains of an immunoglobulin that binds that binds and inhibits PD-1 or PD-L1 linked to (b) an
• nucleic acid molecules that encode the protein chains described above are provided, together with vectors, including expression vectors that contain these nucleic acid molecules.
• Methods also are provided for the preparation of a binding protein as described above, where the method includes the steps of a) transforming a host cell with vectors that containing nucleic acid molecules encoding the first binding domains and the second binding domain; b) culturing the host cell under conditions that allow synthesis of the binding protein; and c) recovering the binding protein from the culture.
• the host cell may contain vectors containing nucleic acid molecules encoding the first binding domain and second binding domain.
• compositions also are provided that contain a binding protein as described above, together with a pharmaceutically acceptable excipient.
• bispecific binding proteins that contain a first protein chain, a second protein chain and a third protein chain, where the first protein chain contains a heavy chain having VH, CHI, CH2, and CH3 domains, and a first Fab domain (Fabl) at a solvent exposed loop in the CH2 domain, the CH3 domain, or at the interface of the CH2 and CH3 domains; where the second chain contains a second Fab domain and where the third chain contains a third Fab domain.
• the second chain Fab domain associates with the VH and CHI domains of the first protein chain to form a first binding domain
• the third chain Fab domain associates with the first Fab domain at the solvent exposed loop in the first protein to form a second binding domain.
• the solvent exposed loop may contain an amino acid sequence from the CH2 domain, such as ISRTP (SEQ ID NO:57).
• the solvent exposed loop may contain an amino acid sequence from the CH3 domain, such as SNG.
• the solvent exposed loop may contain an amino acid sequence from the interface of the CH2 domain and the CH3 domain, such as AKGQP (SEQ ID . NO: 58).
• the CHI domain may be connected to the CH2 domain via an antibody hinge region.
• the CH2 and CH3 domains may contain an Fc region, such as an Fc region from an IgGl, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD.
• the first protein chain further may contain a first peptide linker between a first terminus of the first Fab domain and the CH2 domain, CH3 domain, or interface of the CH2 and CH3 domains, and/or a second peptide linker between the second terminus of the first Fab domain and the CH2 domain, CH3 domain, or interface of the CH2 and CH3 domains.
• the first and the second peptide linker may be, for example, (G4S)2 (SEQ ID NO: 59), (G4S)3, (SEQ . ID NO: 60), and (G4S)4 (SEQ ID NO:61).
• the first protein chain may contain the following polypeptide domains, from N-terminus to C-terminus: VHl-CHl-hinge-CH2(N- term)-Fabl-CH2(C-term)-CH3, or in a second embodiment may contain the following polypeptide domains, from N-terminus to C-terminus: VHl-CHl-hinge-CH2-CH3(N-term)- Fabl-CH3(C-term). In a third embodiment the first protein chain may contain the following polypeptide domains, from N-terminus to C-terminus: VH1-CH1-CH2-Fabl-CH3.
• the first binding domain may bind specifically to PD-1 or PD-L1 and the second binding domain may bind specifically to IL-Ib or IL-1R, or the first binding domain may bind specifically to IL- 1b or IL-1R and the second binding domain may bind specifically to PD-1 or PD-L1.
• the CDR regions of the first binding domain may be selected from the group consisting of the CDR domains of SEQ ID NO: 1-6: the CDR domains of SEQ ID NO:7-12; the CDR domains of SEQ ID NO: 13-18; the CDR domains of SEQ ID NO: 19-24, the CDR domains of SEQ ID NO:25-30; and the CDR domains of SEQ ID NO:31-36 and the CDR regions of the second binding domain may be the CDR domains of SEQ ID NO:49-54.
• the CDR regions of the first binding domain may be the CDR domains of SEQ ID NO:49-54 and the CDR regions of the second binding domain may be selected from the group consisting of: CDR domains of SEQ ID NO: 1-6: the CDR domains of SEQ ID NO:7-12; the CDR domains of SEQ ID NO: 13-18; the CDR domains of SEQ ID NO: 19-24, the CDR domains of SEQ ID NO:25-30; and the CDR domains of SEQ ID NO:31-36.
• Nucleic acid molecules that encode the FAT binding protein chains described above are provided, together with vectors, including expression vectors that contain these nucleic acid molecules. Also provided are pharmaceutical compositions containing one or more FAT binding proteins and a pharmaceutically acceptable carrier.
• Methods for the preparation of a FAT binding protein include the steps of a) transforming a host cell with vectors that may contain nucleic acid molecules encoding the first, second and third protein chains; b) culturing the host cell under conditions that allow synthesis of the binding protein; and c) recovering the FAT binding protein from the culture.
• the vectors may contain nucleic acid molecules encoding the first, second and protein chains of the FAT protein.
• Methods of treating cancer in a subject include administering a binding protein or pharmaceutical composition as described above to a subject in need thereof. These methods optionally include administering an antitumor agent to the subject in addition to the binding protein.
• the subject may previously have been treated with cancer immune therapy or have been found to be resistant to the therapy.
• the subject may have previously been treated with cancer immune therapy or been found to be refractory to cancer immune therapy.
• the cancer immune therapy may be, for example, treatment with at least one immune checkpoint inhibitor.
• the methods of treating cancer may also include administering an additional anti tumor therapy to the subject, such as chemotherapy, immune therapy, treatment with biologies or small molecules, vaccination, and/or a cell therapy.
• an additional anti tumor therapy such as chemotherapy, immune therapy, treatment with biologies or small molecules, vaccination, and/or a cell therapy.
• the subject may previously have been diagnosed with cancer and be in remission or may previously have been treated for cancer.
• the subject may be considered to be at risk of cancer due to environmental exposure, tobacco use or exposure, genetic mutation, or a family history of cancer.
• the cancer may be lung cancer, such as small cell lung cancer, combined small-cell lung carcinoma, and/or non-small cell lung cancer.
• the non-small cell lung cancer may be, for example, squamous cell lung carcinoma, large cell lung carcinoma, lung adenocarcinoma, pulmonary pleomorphic carcinoma, lung carcinoid tumor, salivary gland carcinoma, or carcinoma NOS (not otherwise specified).
• the cancer may be combined small-cell lung carcinoma, extrapulmonary small-cell carcinoma, extrapulmonary small-cell carcinoma localized in the lymph nodes or small-cell carcinoma of the prostate, or may be a cancer with microsatellite instability.
• Figure 1 shows the chain structure of a BiS2 bispecific antibody.
• Figure 2A shows the chain structure of a BiS3 bispecific antibody.
• Figure 2B shows the chain structure of a binding molecule containing (a) an IL-Ib binding domain derived from IL-1R1 and IL-1R accessory protein and (b) an antibody binding domain.
• Figure 3A shows the chain structure of a FIT-Ig bispecific antibody.
• Figure 3B-3E show the chain structures of four binding molecule containing(a) an IL- 1b binding domain derived from IL-1R1 and IL-1R accessory protein and (b) an antibody binding domain
• Figure 4 shows the chain structure of a FAT-Ig bispecific antibody.
• Figure 5 shows the amino acid sequences of bispecific antibodies that bind IL-Ib and
• Figure 6A and 6B show a table of known antibodies and binding molecules that bind IL-Ib, IL-1R, PD-1 or PD-L1.
• Figure 7 shows binding of bispecific antibodies to cell membrane-bound PD-1 and soluble IL-Ib simultaneously in a multiple-dose sandwich assay, as detected by flow cytometry. Variable binding affinities of ITA series of bispecific antibodies are
• Figure 8 shows binding of bispecific antibodies to cell membrane-bound PD-1 and soluble IL-Ib simultaneously in a multiple-dose sandwich assay, as detected by flow cytometry. Variable binding affinities of ITC, ITD and ITE series of bispecific antibodies are demonstrated, and all the binding affinities are substantially higher than control human IgG.
• Figures 9A-C show binding of bispecific antibodies to cell membrane-bound PD-1 in a multiple-dose sandwich assay, as detected by flow cytometry. Variable binding affinities of ITB and ITF series of bispecific antibodies are demonstrated, and all the binding affinities are substantially higher than control human IgG.
• Figure 10 shows binding of bispecific antibodies to cell membrane-bound PD-1 and soluble IL-Ib simultaneously in a multiple-dose sandwich assay, as detected by flow cytometry. Variable binding affinities of ITB and ITF series of bispecific antibodies are demonstrated. All the binding affinities are substantially higher than control human IgG.
• Figure 11 shows that bispecific antibodies block PD-1 activity in a PD-1/PD-L1 reporter assay. Variable blockade activities are demonstrated for the ITA series of bispecific antibodies. All the blockade activies are substantially higher than control human IgG.
• Figures 12A and 12B show that bispecific antibodies block IL-Ib activity in a IL-Ib functional assay. Variable blockade activities are demonstrated of the ITA series of bispecific antibodies. All the blockade activies are substantially higher than control human IgG.
• Bispecific binding proteins contain at least one first binding domain that specifically binds an immune checkpoint protein and at least one second binding protein that binds IL-Ib.
• the immune checkpoint protein may be, for example, PD-1, PD-L1,
• the checkpoint protein is PD-1 or PD-L1.
• a binding protein simultaneously binds the checkpoint protein PD-1 or PD-L1 and IL-Ib or IL-1R and thereby inhibits binding between PD-1 on CD8 T-cells and PD-L1 on a target cell, such as a tumor cell, and IL-Ib activity. Simultaneous inhibition of IL-Ib activity and PD-1/PD-L1 binding in this fashion provides for improved methods for cancer treatment.
• the first and second binding domains advantageously are human antibody variable domains. Novel bispecific binding protein formats also are provided that allow for specific binding of two antigens including, but not limited to, a checkpoint protein and a cytokine such as IL-Ib. Methods of using the bispecific binding proteins for treating disease, such as cancer, also are provided.
• binding domains that may be used in the binding proteins as described herein may be in any format that specifically binds the target protein.
• the ligand binding domain of the interleukin type I or type II receptor may be used, optionally fused to a sequence from the IL-1 receptor accessory protein, as in Rilonacept.
• the binding domains are derived from the variable domains of human immunoglobulin molecules.
• the binding domains may be derived from an antibody that binds a checkpoint protein and an antibody that binds IL-Ib or IL-1R.
• human antibodies can be selected from large libraries of antibodies displayed on filamentous phage, and the heavy and light chain variable regions of the selected antibodies identified by methods that are well known. See, for example, Winter el al, Annual Review of Immunology 12:433-455 (1994). The nucleic acids encoding these variable regions are then used in constructing the genes encoding the bispecific binding proteins described herein. Alternatively, antibody variable domains from known antibodies may be used.
• human antibodies again IL-Ib, PD-1 and PD-L1 have been approved for use in treating a variety of disease states in humans, and the variable regions from these antibodies can be used to construct the bispecific binding proteins described herein. Examples of suitable antibodies are shown below in Tables 1 and 6.
• the CDR regions from an antibody with known specificity against IL- 1b, IL-1R, PD-1 or PD-L1 can be inserted into known human framework regions using methods of CDR grafting that are well known in the art. See, for example, Williams and Matthews,“Humanising Antibodies by CDR Grafting” in Antibody Engineering
• CDR regions may be derived from the CDR regions shown or Figure 1 or from the CDR regions of the antibodies described in Table 6.
• the skilled artisan will recognize that other antibodies that specifically bind IL-Ib, IL-1R, PD-1 or PD-L1 exist in addition to those described herein, and that the CDR regions of those antibodies may be used in constructing the binding proteins as described herein.
• canakinumab is an FDA-approved human antibody that binds IL-Ib, and the amino acid sequences of the heavy and light chain variable regions are well known. See Rondeau et al, MAbs 7: 1151 (2015).
• the entire heavy and light chain variable regions of canakinumab can be used to construct the bispecific protein proteins as described herein; alternatively, the CDR regions of canakinumab can be inserted into alternative human variable framework sequences using methods of CDR grafting that are well known in the art. See, for example, Winter and Harris, Trends in Pharmacological Sciences 14: 139-143 (1993).
• An alternative IL-Ib binding antibody is the humanized SK48-E26 antibody described in WO1995/01997.
• anakinra is an FDA-approved, recombinant, nonglycosylated form of human interleukin -1 receptor antagonist (IL-IRa). Compared to native human IL- lRa, anakinra contains an extra N-terminal methionine residue. Anakinra competitively inhibits binding of IL-l a and IL-I b to the IL-1 receptor type 1. This IL-1R1 binding domain can be used in construction of bispecific proteins to block binding of IL-1 b to the IL-1 receptor. The sequence of the IL-1R1 binding portion of anakinra, which may be used as a suitable binding domain, is
• pembrolizumab is a humanized antibody that was first approved by the FDA in 2014 and that is used for treatment of a variety of cancers where the tumor cells express elevated PD-1.
• Nivolumab is a fully human antibody that was first approved by the FDA in 2014 and also is used for treating a variety of cancers.
• Cemiplimab is a human antibody that was first approved in 2018 for treatment of metastatic cutaneous squamous cell carcinoma.
• the amino acid sequences for the heavy and light chain variable domains of pembrolizumab, nivolumab, and cemiplimab, are known, as are the sequences of the CDR regions.
• durvalumab is a human antibody that was first approved in 2017 for treatment of metastatic urothelial cancer. Atezolizumab was first approved in 2016 and is used for treating lung cancer.
• the amino acid sequences for the heavy and light chain variable domains of durvalumab and atezolizumab are known, as are the sequences of the CDR regions.
• variable domains and CDR regions of pembrolizumab, nivolumab, cemeplimab, atezolizumab, avelumab, durvalumab and canakinumab are shown in Table 1 below.
• a list of other known antibodies again IL-Ib, PD-1 and PD-L1 is shown in Figure 6.
• the IL-1 Receptor type has a binding domain with the sequence:
• Rilonacept is an immunoglobulin fusion protein, where the binding domain contains the IL-1 receptor accessory protein (IL-1RAP) fused to the type I IL-1 receptor.
• IL-1RAP IL-1 receptor accessory protein
• the appropriate binding domains are selected, they are incorporated into a format that contains at least one binding domain that specifically binds IL-Ib or IL-1R and at least one domain that specifically binds a checkpoint protein.
• the format of the binding protein is that of a bivalent or multivalent bispecific antibody, containing immunoglobulin variable and constant chain domains arranged to contain two different binding domains, as opposed to the naturally occurring homodimeric structure of a bivalent but monospecific human antibody.
• bispecific antibodies are well known in the art and are described in, for example, Brinkmann and Kontermann, inAbs 9: 182 (2017) and Spiess et al., Molecular Immunology, 67: 95-106 (2015).
• the bispecific binding proteins described herein can be in any format known in the art that is stable, suitable for administering to a subject, and contains at least one binding domain the binds IL-Ib or IL-1R and at least one binding domain that binds a checkpoint protein.
• bispecific binding protein formats examples include:
• Fc-less bispecific antibody formats including: two scFv molecules joined by a linker (Kontermann, Acta Pharmacol Sin 26: 1-9 (2005)); bispecific single-domain antibody fusion proteins (Weidle et al, Cancer Genomics Proteomics 10: 155-68 (2013;)) and diabodies (Atwell et al, Mol Immunol 33: 1301-12 (1996)); Fab fusion proteins ( Schoonjans et al, J Immunol, 165:7050-7 (2000); and miniantibodies (Pluckthun and Pack, Immunotechnology 3:83-105 (1997) and Muller et al, FEBS Lett 432:45 49(1998));
• Bispecific IgGs with an asymmetric Fc region e.g. asymmetric Fc regions using the “knobs into holes” method (Ridgway et al, Protein Eng, 9:617-21 (1996); Shatz et al,
• bispecific antibody formats in addition to the specific formats described above can be used to construct a bispecific antibody that binds IL-Ib or IL-1R and a checkpoint protein.
• the bispecific antibody is either a 2+2 scFv-based structure or a 2+2 Fab-based structure as described in more detail below. 2+2 scFv-based structures
• a first 2+2 scFv-based structure is the structure shown in Figure 1, referred to herein as BiS2.
• the BiS2 format contains two protein chains:
• a heavy chain that contains (from N- to C-terminus): a single chain Fv containing a first VH domain and a first VL domain (arranged VH-VL or VL-VH, i.e. the domains can be in either order), where the scFv binds the first target (IL-Ib, IL-1R or the checkpoint protein, respectively); a second VH domain; and CHI, CH2, and CH3 domains, and
• the BiS2 protein assembles via non-covalent homodimeric binding of the CH3 and CH2 domains and heterodimeric binding between the CHI and CL domains on the heavy chain and the second VH and VL domains on the light chain.
• the binding between the CHI and CL domains and the VH and VL domains forms a Fab domain that binds the second target (the checkpoint protein or I L- 1 b/I L- 1 R. respectively).
• disulfide bonds also form between the hinge regions, and between the CHI and CL domains in the same manner as are found in naturally-occurring IgG molecules.
• a second 2+2 sc Fv-based structure is the structure shown in Figure 2A, referred to herein as BiS3.
• the BiS3 format also contains two protein chains:
• a heavy chain that contains (from N- to C-terminus): a first VH domain; CHI, CH2, and CH3 domains; and a single chain Fv containing a second VH domain and a second VL domain (where the VH and VL domains can be in either order), where the scFv binds the first target (IL-Ib or the checkpoint protein, respectively);
• the BiS3 protein also assembles via homodimeric binding of the CH3 and CH2 domains and heterodimeric binding between the CHI and CL domains on the heavy chain and the second VH and VL domains on the light chain.
• the binding between the CHI and CL domains and the VH and VL domains forms a Fab domain that binds the second target (the checkpoint protein or I L- 1 b/I L- 1 R. respectively).
• disulfide bonds also form between the CH2 domains, and between the CHI and CL domains in the same manner as are found in naturally-occurring IgG molecules.
• An alternative binding protein structure is the homodimeric structure shown in Figure 2B, in which the heavy chain VH and CHI domains are replaced by a binding domain derived from the extracellular ligand binding domain of an interleukin- 1 receptor.
• This binding domain contains all or part of the extracellular binding domain from type 1 or type 2 IL-1R, optionally conjugated to the extracellular protein binding domain from interleukin- 1 accessory protein (IL-lRAcP).
• IL-lRAcP is a receptor subunit of the functional IL-1 receptor and forms a receptor heterodimer with IL-1RI.
• the binding protein assembles via non-covalent homodimeric binding of the CH3 and CH2 domains.
• disulfide bonds also form between the CH2 domains, and between the hinge domains in the same manner as are found in naturally-occurring IgG molecules.
• a first 2+2 Fab-based structure is the structure shown in Figure 3A, referred to herein as FIT-Ig (see Gong et al, MABS, 2017, 9: 1118-1128 (2017)).
• the FIT-Ig format contains three protein chains:
• a heavy chain that contains (from N- to C-terminus): a first VL domain; a first CL domain, a first VH domain, a first CHI domain, and CH2 and CH3 domains;
• the first CL domain may be linked to the first VH domain via a peptide linker such as, for example, a flexible hydrophilic linker having the sequence (GGGGS)x, where x is 1-5.
• a linker is absent.
• the FIT-Ig protein assembles via: non-covalent homodimeric binding of the CH3 and CH2 domains; heterodimeric binding between the first VH and CHI domains on the heavy chain and the second VL and CL domains on the light chain; and heterodimeric binding between the first VL and CL domains and the second VH and CHI domains on the Fd chain
• Two identical Fab binding domains are formed by binding of the heavy chain to the light chain, and two distinct but identical Fab domains are formed by binding of the heavy chain to the Fd chain as shown in Figure 3.
• disulfide bonds also form between the CH2 domains, and between the hinge domains in the same manner as are found in naturally - occurring IgG molecules.
• FIG. 3B-3E An alternative 2+2 binding protein is shown in Figures 3B-3E, in which one of the antibody Fab binding domains is replaced by a binding domain derived from the extracellular ligand binding domain of an interleukin- 1 receptor.
• This binding domain contains all or part of the extracellular binding domain from type 1 or type 2 IL-1R, optionally conjugated to the extracellular protein binding domain from interleukin-1 accessory protein (IL-lRAcP).
• IL- lRAcP is a receptor subunit of the functional IL-1 receptor and forms a receptor heterodimer with IL-1RI.
• the binding proteins assemble via non- covalent homodimeric binding of the CH3 and CH2 domains; heterodimeric binding between the VH and CHI domains on one chain and the VL and CL domains on the second protein chain.
• disulfide bonds also form between: the CHI and CL domains, between the CH2 domains, and between the hinge domains in the same manner as are found in naturally-occurring IgG molecules.
• a second 2+2 Fab-based structure is the novel structure shown in Figure 4, referred to herein as FAT-Ig.
• the FAT-Ig format contains three protein chains:
• the first CL and first CL domains are disposed at a solvent-exposed loop in the CH3 domain.
• the first VL and CL disposed at the solvent-exposed loop are connected to the CH2 domain, CH3 domain, or interface of the CH2 and CH3 domains via flexible peptide linkers.
• the FAT-Ig protein assembles as shown in Figure 4 via: non-covalent homodimeric binding of the CH3 and CH2 domains; heterodimeric binding of the heavy chain CHI and VH domains with the light chain CL and VL domains; and heterodimeric binding of the heavy chain CL and VL domains with the Fd chain CHI and VH domains.
• Two identical Fab binding domains are formed by binding of the heavy chain to the light chain, and two distinct but identical Fab domains are formed by binding of the heavy chain to the Fd chain as shown in Figure 4.
• disulfide bonds also form between the hinge domains, and between the CHI and CL domains in the same manner as are found in naturally-occurring IgG molecules.
• the novel FAT-Ig antibody format can be used to bind any two desired antigens, and is not limited to I L- 1 b/I L- 1 R and a checkpoint protein.
• each of the four specific binding proteins described above the binding domains that specifically bind I L- 1 b/I L- 1 R and the checkpoint protein are disposed asymmetrically within the binding protein i.e. the structure of the binding protein is different when the first binding domain binds I L- 1 b/I L- 1 R and the second binding domain binds the checkpoint protein compared to when the first binding domain binds the checkpoint protein and the second binding domain binds I L- 1 b/I L- 1 R. Accordingly, each of the four specific binding proteins can exist in two alternative forms for any given pair of binding domains.
• the domains of the bispecific binding proteins described herein may be joined into contiguous protein chains using linkers.
• the linkers may be used, for example, to connect the variable heavy and light chains of an scFv, or to connect the CL/VL domains into the heavy chain constant domains in the FAT-Ig format.
• Suitable linkers are well known in the art and, when present, advantageously contain at least four amino acids, although longer or shorter linkers may also be used.
• the linkers advantageously are flexible, hydrophilic and have little or no secondary structure of their own. Linkers may be approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or approximately 50 residues in length.
• the linkers may be the same, or have different lengths and/or amino acid sequences.
• the length of the linker may be varied to facilitate protein folding, target binding, and/or expression. For example, different multiples of (Gly-Ser)x units may be used to improve or optimize protein folding, target binding, and/or expression using methods that are well-known in the art.
• bispecific binding proteins such as Fc-less bispecific antibodies, asymmetric IgGs with heavy and light chains from two different antibodies and bispecific IgGs with an asymmetric Fc region
• the particular 2+2 bispecific antibodies described above may each be produced using suitable expression constructs in recombinant host cells.
• Nucleic acids encoding the heavy, light and Fd chains can be prepared synthetically using, for example, a commercial gene synthesis vendor such as Thermo Fisher (Carlsbad, CA).
• the host cells are eukaryotic cells and the gene for each chain advantageously is synthesized with a sequence that encodes an N-terminal signal sequence that causes secretion of the translated protein from the host cell.
• the gene for each chain is inserted into a suitable expression vector, for example, pTT5 vector (Durocher et al, Nucleic Acids Res. 30:E9 (2002)), and the resulting expression constructs are then transfected into a culture of suitable host cells for transient expression.
• a suitable expression vector for example, pTT5 vector (Durocher et al, Nucleic Acids Res. 30:E9 (2002))
• Methods of efficiently transfecting expression vectors into host cells are well known in the art using, for example, cationic lipids. See Feigner el al, Proc. Nat ⁇ Acad. Sci USA 84:7413 (1987).
• Other methods of delivering expression vectors into cells also are well known in the art.
• Methods of making host cells that provide stable expression of a desired protein by integrating expression constructs into the genome of suitable host cells also are well known, as are methods of stable expression using episomal vectors containing a mammalian origin of replication that act as extrachromosomal elements in the nucleus of the host cell.
• the host cells advantageously are eukaryotic cells, for example, a single-celled eukaryote (e.g ., a yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell) or a hybridoma.
• a single-celled eukaryote e.g a yeast or other fungus
• a plant cell e.g., a tobacco or tomato plant cell
• an animal cell e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell
• a hybridoma eukaryotic cells
• host cells examples include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (see Gluzman et al, 1981, Cell 23: 175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or their derivatives such as Veggie CHO and related cell lines which grow in serum-free media (see Rasmussen et al, 1998, Cytotechnology 28:31) or CHO strain DX-B11, which is deficient in DHFR (see Urlaub et al, 1980, Proc. Natl. Acad. Sci.
• COS-7 line of monkey kidney cells ATCC CRL 1651
• L cells C127 cells
• 3T3 cells ATCC CCL 163
• CHO Chinese hamster ovary
• the host cell is a CHO cell, such as CHO-3E7.
• a host cell is a cultured cell that can be transformed or transfected with a polypeptide-encoding nucleic acid, which can then be expressed in the host cell.
• the phrase "recombinant host cell” can be used to denote a host cell that has been transformed or transfected with a nucleic acid to be expressed.
• a host cell also can be a cell that comprises the nucleic acid but does not express it at a desired level unless a regulatory sequence is introduced into the host cell such that it becomes operably linked with the nucleic acid.
• the term“host cell” as used herein refers not only to the particular subject cell but to the progeny or potential progeny of such a cell.
• Suitable cell culture media for growing eukaryotic host cells are well known in the art and are commercially available from, for example, Thermo Fisher (Grand Island, NY).
• the host cells are cultured under suitable conditions to allow assembly of the protein chains in the endoplasmic reticulum of the host cell, followed by secretion of the bispecific binding proteins into the cell culture supernatant.
• the assembly of the correct structure of the binding protein (as opposed to, for example, non-specific pairing of the chains leading to formation of inactive proteins) can be improved by altering the relative ratios of the expression vectors used to transfect the host cells. This variation in the vector ratio also can be used to counteract, say, less efficient production of one of the chains compared to a different chain.
• methods of optimizing the vector ratio(s) are well known in the art.
• the conditioned medium containing the bispecific binding protein is collected, and the bispecific binding protein is purified using methods that are well known in the art.
• the binding protein can be purified using methods that may include ion- exchange chromatography, size-exclusion chromatography, and affinity chromatography, such as protein A affinity chromatography.
• Methods of protein purification are described in, for example, Burgess and Deutscher (Eds)“Guide to Protein Purification, Volume 436 (Methods in Enzymology) 2nd Edition (2009). Purity of the protein can be confirmed using methods well-known in the art, such as RT-HPLC, SDS-PAGE and the like.
• the correct assembly of the multichain binding protein can be shown by, for example, non-denaturing gel electrophoresis, to show that the binding protein has the expected molecular weight.
• SDS-PAGE can be used to show that each of the expected protein chains is present and has the expected molecular weight.
• Western blohing using a suitable anti- Fhiman IgG antibody can further show that the measured proteins are immunoglobulin chains.
• the route of administration of the binding molecule can be, for example, oral, parenteral, by inhalation or topical.
• parenteral as used herein includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration.
• the binding molecules may be delivered directly to the site of the adverse cellular population thereby increasing the exposure of the diseased tissue to the therapeutic agent.
• the binding molecules may be administered in a pharmaceutically effective amount for the treatment of diseases such as certain types of cancers.
• the pharmaceutical compositions can comprise pharmaceutically acceptable carriers, including, for example, water, ion exchangers, proteins, buffer substances, and salts. Preservatives and other additives can also be present.
• the carrier can be a solvent or dispersion medium. Suitable formulations for use in the therapeutic methods disclosed herein are described in Remington's
• sterile injectable solutions can be prepared by incorporating the binding molecule(s) by itself or in combination with other active agents in an effective amount in an appropriate solvent followed by filtered sterilization.
• the preparations may also be packaged and sold in the form of a kit.
• Such articles of manufacture can have labels or package inserts indicating that the associated compositions are useful for treating a subject suffering from, or predisposed to a disease or disorder.
• Parenteral formulations can be a single bolus dose, an infusion or a loading bolus dose followed with a maintenance dose. These compositions can be administered at specific fixed or variable intervals, for example, once a week or monthly, or on an "as needed" basis.
• composition can be administered as a single dose, multiple doses or over an established period of time in an infusion. Dosage regimens also can be adjusted to provide the optimum desired response (for example, a therapeutic or prophylactic response).
• the composition may be used for treatment of cell-mediated diseases such as certain types of cancers including for example, bone cancer, pancreatic cancer, cancer of the head and neck, cutaneous or intraocular melanoma, uterine cancer, cancer of the central nervous system (CNS), ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, colon cancer, breast cancer, melanoma, colorectal cancer, testicular cancer, Hodgkin's disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, sarcomas of soft tissues, cancer of the urethra, cancer of the penis, prostate cancer, chronic or acute leukemia, solid tumors of childhood, lymphocytic lymphomas, cancer of the bladder, cancer of the kidney or ureter, adrenocortical carcinoma, AIDS-related cancers, Childhood Cerebellar Astrocytoma, Childhood Cerebral Astrocytoma, Basal Cell Carcinoma, extrahepati
• pleuropulmonary blastoma pleuropulmonary blastoma, ureter transitional cell cancer, retinoblastoma,
• the cancer may be, for example, esophageal squamous-cell carcinomas (ESCC) or esophageal adenocarcinomas (EAC).
• ESCC esophageal squamous-cell carcinomas
• EAC esophageal adenocarcinomas
• pancreatic cancer the cancer may be, for example, exocrine cancer, pancreatic
• the cancer may be, for example, hepatocellular carcinoma (HCC), hepatoblastoma, cholangiocarcinoma, cholangiocellular cystadenocarcinoma, angiosarcoma, or leiomyosarcoma.
• HCC hepatocellular carcinoma
• cholangiocarcinoma cholangiocellular cystadenocarcinoma
• angiosarcoma or leiomyosarcoma.
• the cancer may be adenocarcinoma, carcinoid tumors, gastrointestinal stromal tumors (GIST), lymphoma, sarcomas, adenosquamous carcinoma (Ad-SCC) or squamous carcinoma (SCC).
• the cancer may be In situ, ductal carcinoma in situ (DCIS), invasive, invasive ductal carcinoma (IDC), invasive lobular carcinoma (ILC), triple-negative breast cancer, inflammatory breast cancer, angiosarcoma, or Paget disease of the breast.
• the cancer may be epithelial tumors, benign epithelial ovarian tumors, borderline epithelial ovarian tumors, malignant epithelial ovarian tumors, germ cell tumors, teratoma, dysgerminoma, endodermal sinus tumor and choriocarcinoma, primary peritoneal carcinoma, fallopian tube cancer or ovarian stromal tumors.
• the cancer may be light chain myeloma, non-secretory myeloma, solitary plasmacytoma, extramedullary plasmacytoma, monoclonal gammopathy of undetermined significance (MGUS), smoldering multiple myeloma (SMM), Immunoglobulin D (IgD) myeloma or Immunoglobulin E (IgE) myeloma.
• MGUS monoclonal gammopathy of undetermined significance
• SMM smoldering multiple myeloma
• IgD Immunoglobulin D
• IgE Immunoglobulin E
• compositions for treating these diseases vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic.
• the patient is a human, but non-human mammals including transgenic mammals can also be treated.
• Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.
• the amount of at least one binding molecule to be administered can be readily determined by one of ordinary skill in the art without undue experimentation. Factors influencing the mode of administration and the respective amount of at least one binding molecule include, but are not limited to, the severity of the disease, the history of the disease, and the age, height, weight, health, and physical condition of the individual undergoing therapy. Similarly, the amount of binding molecule, to be administered will depend upon the mode of administration and whether the subject will undergo a single dose or multiple doses of this agent.
• the binding molecule may also be used in the manufacture of a medicament for treating a type of cancer, including, for example, the cancers listed above.
• the subject treated with the compositions described herein may be treatment naive or may be pretreated with one or more other therapies (for example, at least one other anti cancer therapy) prior to receiving the medicament comprising the binding molecule. It is not necessary that the subject was a responder to pretreatment with the prior therapy or therapies. Thus, the subject that receives the medicament comprising the binding molecule, could have responded, responded poorly, responded initially but subsequently failed to respond, or could have failed to respond to pretreatment with the prior therapy, or to one or more of the prior therapies where pretreatment comprised multiple therapies. Accordingly, the present disclosure provides methods to treat patients that are poor responders or non-responders to other therapies comprising administering a binding molecule as described herein. Also provided are methods to overcome or prevent resistance to cancer therapies or to prevent or delay relapse, comprising administering a binding molecule as disclosed herein, or a composition as described herein.
• a person skilled in the art can determine whether a person showed no response or was refractory to that medicament.
• a non-response to an anti-cancer medicament may be reflected in an increased suffering from cancer, such as an increased growth of a cancer/tumor and/or increase in the size of a tumor, in the formation of (or increase in) metastases or an increase in the number or size of metastases.
• a non-response may also be the development of a tumor or metastases, for example after resection of a tumor, in the shortening of time to disease progression, or in the increase in the size of (a) tumor(s) and/or (a) metastases, for example in neoadjuvant therapy.
• a patient group can be identified that does not respond to treatment with anti-cancer medicaments and this group of patients may then be treated with the binding molecules described herein.
• the binding molecules and compositions containing the binding molecules may also be used to treat patients that are, for example, poor-responders or non-responders to another therapy.
• non-responder as used herein can refer to an individual/patient/subject that is less likely to respond to a treatment using an anti-cancer medicament.
• Less likely to respond as used herein refers to a decreased likeliness that a pathological complete response will occur in a patient treated with an anti-cancer medicament.
• a patient can be initially a good responder, and resistance to treatment can develop during treatment with such an anti-cancer medicament, leading to poor or no-response to the treatment.
• good responder refers to an individual whose tumor does not demonstrate growth, metastases, increase in number or size of metastases, etc. during or after treatment using an anti-cancer medicament, for example based on serial imaging studies, an individual that does not experience tumor growth, metastases, increase in number or size of metastases, etc. over a period of time (for example, about 1 year following initial diagnosis), and/or an individual that experiences a certain life span (for example, about 2 years or more following initial diagnosis).
• pool responder refers to an individual whose tumor grows or metastasizes during or shortly thereafter standard therapy, for example using an anti cancer medicament, or who experiences adverse clinical effects attributable to the tumor.
• “poor responder” also includes indivduals who transitioned from“good responder” to‘poor responder” during treatment with an anti-cancer medicament.
• the subject could be treated with the binding molecules disclosed herein.
• binding molecule as described herein and at least one other therapy.
• the binding molecule and the at least one other therapy can be co-administered together in a single composition or can be co administered together at the same time or overlapping times in separate compositions.
• a binding molecule can be used as an adjuvant therapy.
• the binding molecule may also be used in the manufacture of a medicament for treating a subject suffering from a cancer, where the binding molecule is administered before a subject has been treated with at least one other therapy.
• the binding molecules can also be used in methods of preventing or reducing the risk of cancer in a subject by administering to the subject an effective amount of a binding protein or composition containing the binding protein.
• the cancer may be lung cancer and the patient may be in remission from a previously diagnosed and/or previously treated cancer.
• the patient may be considered to be at risk of cancer due to environmental exposure, tobacco use or exposure, genetic mutation, or a family history of cancer. Examples:
• binding domains are based on heavy and light chain variable regions from human antibodies that have received regulatory approval for use in humans.
• “O,”“E” and“K” indicate domains that bind IL-Ib
• “M” and“B” indicate binding domains that bind PD-1.
• the amino acid sequences of the chains of the binding proteins are shown in Figure 5
• Target DNA sequences encoding the binding proteins were synthesized and subcloned into the pTT5 vector (Durocher et al, Nucleic Acids Res. 30:E9 (2002) for expression in CHO-3E7 cells.
• the amino acid sequences of the coding sequences are shown in Figure 5.
• CHO-3E7 cells were grown in serum-free FreeStyleTM CHO Expression Medium (Life Technologies, Carlsbad, CA, USA). The cells were maintained in Erlenmeyer Flasks (Coming Inc., Acton, MA) at 37°C with 5% CCh on an orbital shaker (VWR Scientific, Chester, PA). One day before transfection, the cells were seeded in Coming Erlenmeyer Flasks. On the day of transfection, DNA and transfection reagent were mixed and then added into the cells culture, during which the recombinant plasmids encoding target antibody were transiently transfected into CHO-3E7 cells. The cell culture supernatant collected on day 6 was used for purification. Table 2 lists the heavy chain, light chain and Fd chain combination of each antibody and each of the plasmid ratios that were screened.
• Each of the expressed proteins was analyzed by SDS-PAGE under reducing and non reducing conditions and Western blot analysis (using Goat Anti-Human IgG-HRP
• Figures 8-12 show additional binding data for additional binding proteins.
• Figure 8 shows binding curves for binding of bispecific antibodies to cell membrane-bound PD-1 and soluble IL-Ib simultaneously in a multiple-dose sandwich assay, as detected by flow cytometry. Variable binding affinities of the ITC, ITD and ITE series of bispecific antibodies are shown. All the binding affinities are substantially higher than control human IgG.
• Figure 9 shows binding curves for binding to cell membrane-bound PD-1 in a multiple-dose sandwich assay, as detected by flow cytometry. Variable binding affinities of the ITB and ITF series of bispecific antibodies are shown. All the binding affinities are substantially higher than control human IgG.
• Figure 10 shows binding curves for binding to cell membrane-bound PD-1 and soluble IL-Ib simultaneously in a multiple-dose sandwich assay, as detected by flow cytometry. Variable binding affinities of ITB and ITF series of bispecific antibodies are shown. All the binding affinities are substantially higher than control human IgG.
• FIG 11 shows that bispecific antibodies block PD-1 activity in a PD-1/PD-L1 reporter assay. Variable blockade activities are shown for the ITA series of bispecific antibodies. All the blockade activies are substantially higher than control human IgG.
• Figure 12 shows that bispecific antibodies block IL-Ib activity in a IL-Ib functional assay. Variable blockade activities are demonstrated of the ITA series of bispecific antibodies. All the blockade activies are substantially higher than control human IgG.

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