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WO2018083087A2 - Protéines de liaison - Google Patents

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WO2018083087A2
WO2018083087A2 PCT/EP2017/077891 EP2017077891W WO2018083087A2 WO 2018083087 A2 WO2018083087 A2 WO 2018083087A2 EP 2017077891 W EP2017077891 W EP 2017077891W WO 2018083087 A2 WO2018083087 A2 WO 2018083087A2
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Prior art keywords
seq
binding protein
present
lag
cdrh2
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PCT/EP2017/077891
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WO2018083087A3 (fr
Inventor
Milloni CHHABRA
Yuliya DEMYDCHUK
Carolyn Enever
David FAVRE
Shari Gordon
David Granger
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Glaxosmithkline Intellectual Property (No.2) Limited
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Publication of WO2018083087A2 publication Critical patent/WO2018083087A2/fr
Publication of WO2018083087A3 publication Critical patent/WO2018083087A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/18—Antivirals for RNA viruses for HIV
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], 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
    • C07K16/2818—Immunoglobulins [IGs], 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 CD28 or CD152
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/567—Framework region [FR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • the present invention relates to a binding protein specific for at least one of the immune checkpoint receptors PD-1 and LAG-3.
  • the invention relates to a binding protein specific for both PD-1 and LAG-3.
  • the invention also provides methods of preparing the binding proteins, pharmaceutical compositions containing the binding proteins and medical uses of the binding proteins.
  • T-cell co-stimulatory and co-inhibitory molecules play a crucial role in regulating T-cell activation, subset differentiation, effector function and survival (Chen et al 2013, Nature Rev. Immunol. 13: 227-242).
  • co-signalling receptors co-localize with T-cell receptors at the immune synapse, where they synergize with TCR signalling to promote or inhibit T-cell activation and function (Flies et al 2011, Yale J. Biol. Med. 84: 409-421).
  • immune checkpoints The ultimate immune response is regulated by a balance between co-stimulatory and co-inhibitory signals (“immune checkpoints”) (Pardoll 2012, Nature 12: 252-264).
  • PD-1 Programmed death-1
  • LAG-3 Lymphocyte Activation Gene 3
  • PD-1 (also called CD279) is a 288 amino acid protein receptor expressed on activated T-cells and B-cells, natural killer cells and monocytes.
  • PD-1 is a member of the CD28/CTLA-4 (cytotoxic T lymphocyte antigen)/ICOS (inducible co-stimulator) family of T-cell co-inhibitory receptors (Chen et al 2013, Nat. Rev. Immunol. 13: 227-242).
  • CTLA-4 cytotoxic T lymphocyte antigen
  • ICOS inducible co-stimulator family of T-cell co-inhibitory receptors
  • the primary function of PD-1 is to attenuate the immune response (Riley 2009, Immunol. Rev. 229: 114-125).
  • PD-1 has two ligands, PD-ligandl (PD-L1) and PD-L2.
  • PD-L1 (also called CD274, B7H1) is expressed widely on both lymphoid and non-lymphoid tissues such as CD4 + and CD8 + T-cells, macrophage lineage cells, peripheral tissues as well as on tumor cells, virally-infected cells and autoimmune tissue cells.
  • PD-L2 (CD273, B7-DC) has a more restricted expression than PD-L1, being expressed on activated dendritic cells and macrophages (Dong et al 1999, Nature Med.).
  • PD-L1 is expressed in most human cancers, including melanoma, glioma, non-small cell lung cancer, squamous cell carcinoma of head and neck, leukemia, pancreatic cancer, renal cell carcinoma, and hepatocellular carcinoma, and may be inducible in nearly all cancer types (Zou and Chen 2008, Nat. Rev. Immunol. 8: 467-77).
  • PD-1 binding to its ligands results in decreased T-cell function including decreased activation, decreased proliferation and altered cytokine secretion. This ability is exploited by chronic viral infections and tumors to evade immune response.
  • Blockade of PD-1 binding to reverse immunosuppression has been studied in autoimmune, viral and tumor immunotherapy (Ribas 2012, NEJM 366: 2517-2519; Watanabe et al 2012, Clin. Dev. Immunol. Volume 2012, Article ID: 269756; Wang et al 2013, J. Viral Hep. 20: 27- 39).
  • Blocking PD-1 with antagonists, including monoclonal antibodies, has been studied clinically in treatments of cancer and chronic viral infections (Sheridan 2012, Nature Biotechnology 30: 729-730).
  • T-cell exhaustion a loss of function is referred to as T-cell exhaustion and is characterized by reduced proliferative potential, cytokine production, cytotoxic function and cell survival (Freeman 2008, PNAS 105: 10275-10276). Studies suggest that blockade of PD- 1 may reverse T cell exhaustion in bacterial infections, parasitic infections, viral infections and sepsis (secondary to infection).
  • PD-1 deficient mice have been shown to be resistant to bacterial infection with both Listeria monocytogenes (Yao et al., Blood, 2009, 113(23): 5811-8) and Streptococcus pneumonia (McKay et al., J Immunol, 2015, 194(5): 2289-99). Also, studies have shown that PD-1 blockade increased T cell effector functions in cells derived from tuberculosis patients (Singh et al., J Infect Dis, 2013, 208(4): 603-15; Jurado et al., J Immunol. 2008, 181(1): 116-25).
  • PD-1 blockade is also thought to have utility in the treatment of other chronic viral infections.
  • PD-1 blockade with an anti-PDl fully human monoclonal antibody BMS-936558
  • BMS-936558 fully human monoclonal antibody
  • Ye and colleagues reviewed the role of PD-1/PD-L1 pathway in hepatitis B infection and concluded that this was likely to help reverse T cell exhaustion, although suggested that other immune checkpoint inhibitors may also be important.
  • anti-PD-Ll antibodies were shown to increase proliferation and cytokine production by CMV (cytomegalovirus) specific T cells and to increase the response of HSV (herpes simplex virus) specific CD8 cells to an HSV peptide in an animal model of HSV infection.
  • CMV cytomegalovirus
  • HSV herpes simplex virus
  • Treatment of mice injected with EBV (epstein barr virus) infected cord blood with anti-PD-1 antibodies reduced EBV induced lymphoma growth. HIV infection is characterized by exhausted CD8 T cells that are unable to proliferate, produce cytokines and perform cytotoxic functions. This results in the immune system being unable to mount an effective antiviral response.
  • PD-1 blockade can enhance antiviral responses to eliminate cells expressing viral antigens resulting in reduced immune stimulation, inflammation and non-AIDS morbidities.
  • Latent HIV genomes have been shown to be concentrated within CD4+ memory T cells.
  • PD-1 blockade in the presence of suboptimal T cell stimulation increased HIV reactivation from CD4 T cells.
  • PD-1 is also highly expressed on memory CD8 cells during HIV infection (Yamamoto et al., Blood, 117:4805-4815, 2011).
  • CD8 T cells are potentially cytotoxic and kill infected cells.
  • PD-1 blockade has been tested in animal models of HIV-1 infection and it improved antiviral responses. More specifically, treatment of SIV- infected viremic macaques with an anti-PD-1 antibody improved the polyfunctionality of SIV-specific CD8+ T and B cells, reduced viremia and improved overall survival.
  • PD-Ll is expressed on a wide variety of tumors and studies on animal models have shown that PD-Ll on tumors inhibits T-cell activation and lysis of tumor cells and may lead to increased death of tumor-specific T-cells.
  • the PD-1: PD-Ll system also plays an important role in induced T-regulatory (Treg) cell development and in sustaining Treg function (Francisco et al 2010, Immunol. Rev. 236: 219-242).
  • PD-1 antibodies and methods of using in treatment of disease are described in US Patent Nos.: US 7,595,048; US 8,168,179; US 8,728,474; US 7,722,868; US 8,008,449; US 7,488,802; US 7,521,051; US 8,088,905; US 8,168,757; US 8,354,509; and US Publication Nos.
  • OPDIVO/nivolumab is a fully human monoclonal antibody marketed by Bristol Myers Squibb directed against the negative immunoregulatory human cell surface receptor PD-lwith immunopotentiation activity.
  • Nivolumab binds to and blocks the activation of PD-1, an Ig superfamily transmembrane protein, by its ligands PD-L1 and PD-L2, resulting in the activation of T-cells and cell-mediated immune responses against tumor cells or pathogens.
  • Activated PD-1 negatively regulates T-cell activation and effector function through the suppression of P13k/Akt pathway activation.
  • Other names for nivolumab include: BMS- 936558, MDX-1106, and ONO-4538.
  • nivolumab The amino acid sequence for nivolumab and methods of using and making it are disclosed in US Patent No. US 8,008,449.
  • Nivolumab has also been used to treat 12 HIV+ patients with non- small cell lunch cancers. Whilst no changes were observed in plasma HIV viral load, CD4 or CD8 cell counts, there was a drastic decrease in the total HIV-DNA levels in one patient suggesting a possible effect on the reservoir (Guihot et al., IAS Abstract A-854-0121-02601).
  • KEYTRUDA/pembrolizumab is an anti-PD-1 antibody marketed for the treatment of lung cancer by Merck.
  • the amino acid sequence of pembrolizumab and methods of using are disclosed in US Patent No. 8,168,757.
  • a multicenter study to evaluate pembrolizumab in patients with HIV and relapsed/refactory cancers has been proposed (Uldrick, Clinical Trial Design Considerations: Leveraging Cancer Immunotherapy Studies to Evaluate HIV
  • the anti-PD-Ll antibody BMS-936559 also targets the PD1-PDL1 axis.
  • a clinical study of 8 HIV infected participants receiving one infusion of BMS-936559 there was a trend toward increased HIV-1 Gag specific CD8+ T cell responses over 28 days post infusion, including increased CD107a expression which is consistent with reversal of CD8+ T cell exhaustion (Eron et al., Safety, Immunologic and Virologic Activity of Anti-PD-Ll in HIV-1 participants on ART, Abstract No. 25, Conference on Retroviruses and Opportunistic Infections, February 22- 25 2016, Boston).
  • PD-1 blockade to reduce immunosuppression or reverse T-cell exhaustion has led to the suggestion that it may be useful as adjunctive therapy in treatment of cancers and infectious diseases. For example, several clinical trials testing combination therapy with a PD-1 antibody and a cancer vaccine are underway or completed. In these vaccines, the PD-1 antibody could be viewed as an adjuvant.
  • LAG-3 (also known as CD223), is a member of the immunoglobulin supergene family and is a membrane protein structurally and genetically related to CD4.
  • LAG-3 is expressed on activated CD4 + and CD8 + T cells, killer (NK) cells, plasmacytoid dendritic cells (DCs) and tumor-infiltrating lymphocytes, e.g., infiltrating lymphocytes in head and neck squamous cell carcinoma (HNSCC).
  • LAG-3 is also expressed on highly suppressive induced and natural Tregs.
  • highly suppressive FoxP3+nTregs and FoxP3-iTregs are LAG-3 positive in melanoma and colorectal cancer (Camisaschi et al. (2010) J. Immunol. 184(11):6545-6551; Scurr et al. (2014) Mucosal.
  • Ligands for LAG-3 include, e.g., MHC Class II and L-SECtin. Blockade of LAG-3 can restore activities of effector cells and diminish suppressor activity of Tregs.
  • Blockade of LAG-3 can restore activities of effector cells and diminish suppressor activity of Tregs.
  • in vitro studies of antigen-specific T cell responses show that the addition of anti-LAG-3 antibodies leads to increased T cell proliferation, higher expression of activation antigens such as CD25, and higher concentrations of cytokines such as interferon- ⁇ and interleukin-4 suggesting that LAG-3 blockade down-regulates antigen-dependent stimulation of CD4 + T lymphocytes (Huard et al. (1994) Eur. J. Immunol. 24:3216-3221).
  • LAG-3 blockade has also been shown to reinvigorate CD8 + lymphocytes in both tumor, self-antigen (Gross et al. (2007) J Clin Invest. 117:3383-3392) and viral models (Blackburn et al. (2009) Nat. Immunol. 10:29-37).
  • CD4 + CD25 + regulatory T cells have been shown to express LAG-3 upon activation and antibodies to LAG-3 inhibit suppression by induced Treg cells, both in vitro and in vivo, suggesting that LAG-3 contributes to the suppressor activity of Treg cells (Huang, C. et al. (2004) Immunity 21:503-513).
  • Anti-LSECtin has been shown to inhibit B16 melanoma cell growth (Xu et al. (2014) Cancer Res. 74(13):3418-3428).
  • LAG-3 negatively regulates T cell signalling and functions and is believed to contribute to T cell exhaustion during chronic viral infections (including HIV), parasitic infections, sepsis and cancer. Viral infections
  • LAG-3 is typically though not exclusively co-expressed on PD-1 + cells. Although co- expression of LAG-3 and PD-1 can be found on functional T cells, co-expression is a hallmark of compromised function in cases of chronic infection and cancer (Nguyen and Ohashi, (2015) Nat. Rev. I mmunol. 15:45-56). For example, latent HIV genomes have been shown to be concentrated within CD4+ memory T cells that express PD1 and LAG3 (Fromentin et.al PLOS Pathogens 2016). Specifically, in relation to HIV, the levels of LAG 3 and PD1 expression during the acute phase of HIV infection was associated with clinical disease progression (Hoffman M PLOS Pathogens 2016 Jul 14;12(7)).
  • the degree of CD8 + T cell exhaustion correlates with the number of inhibitory receptors expressed (Blackburn et al. (2009) Nat. I mmunol. 10(1): 29-37).
  • both PD-1 and LAG-3 associate with the T cell receptor complex upon engagement with their cognate ligands (Yokosuka et al., (2012) J. Exp. Med. 209:1201-1217; Hannier and Triebel, (1999), I nternational Immunology 11:1745- 1752).
  • High PD-l/LAG-3 expression correlates with T cell infiltration in melanoma.
  • Co- blockade of LAG-3 with anti-PD-1 or PD-L1 can result in tumor suppressive activities in preclinical models.
  • anti-LAG-3 and anti-PD-1 blockade show efficacy in SaIN fibrosarcoma and MC38 colon carcinoma models (Woo et al. (2012) Cancer Res. 72(4):917- 27).
  • PD-l/LAG-3 blockade is also efficacious in a lymphocytic choriomeningitis virus (LCMV) model.
  • LCMV lymphocytic choriomeningitis virus
  • PD-Ll plus LAG-3 blockade during chronic LCMV infection enhances antiviral CD8+ T cell responses (Blackburn et al. (2009) Nat. Immunol. 10(1): 29-37).
  • antagonizing LAG-3 and PD-1 restored CD4+, CD8+ and Tfh function and resulted in the clearance of
  • TESARO Immunomodulatory Activity for Cancer Treatment, Poster 3217, presented at the 2016 American Association for Cancer Research Annual Meeting, April 16-20, 2016, New La, Louisiana).
  • TESARO are also reportedly in preclinical development with a bispecific anti- LAG-3/PD-1 antibody for cancer.
  • Inhibitory pathways have different mechanisms of suppression, thus targeting multiple immune checkpoints should increase the frequency of patients responding to therapy.
  • Tumor-specific CD8 T cells express high levels of PD-1, but also co-express CTLA-4 and other inhibitory receptors (Ahmadzadeh et al., Blood, 2009, 114:1537-1544). Accordingly, a clinical trial combining blockade of PD-1 and CTLA-4 obtained higher response rates than previously reported for either monotherapy in patients with melanoma (Wolchok et al., N Engl J Med., 2013, 369: 122-133).
  • the invention provides a binding protein specific to human LAG-3 and human PD-1, which comprises an antibody specific for human LAG-3 attached by a linker to one or more epitope binding domains specific to human PD-1, wherein the antibody specific for human LAG-3 comprises one or more of CDRHl, CDRH2 and CDRH3, wherein CDRHl is selected from the group consisting of: CDRHl as present in SEQ.
  • CDRH2 is selected from the group consisting of: CDRH2 as present in SEQ ID NO:l and CDRH2 that differs from the CDRH2 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids
  • CDRH3 is selected from the group consisting of: CDRH3 as present in SEQ ID NO: 1 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids
  • the antibody specific for human LAG-3 comprises one or more of CDRL1, CDRL2 and CDRL3, wherein CDRL1 is selected from the group consisting of: CDRL1 as present in SEQ ID NO:2 and CDRL1 that differs from the CDRL1 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3
  • the binding protein specific to human LAG-3 and human PD-1 exhibits >50% inhibition of LAG3-M HCII interaction in competition flow cytometry assay, and an IC50 of less than or equal to 5 nM in the PD-l/PDL-1 competition assay.
  • the invention provides a binding protein specific to human LAG- 3 and human PD-1 having the general formula (I):
  • H(LAG-3) is an antibody heavy chain of IgG class comprising CDRH l, CDRH2 and CDRH3, wherein said CDRHl is selected from : CDRHl present in SEQ I D NO:l, and CDRHl that differs from the CDRHl present in SEQ I D NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids; wherein CDRH2 is selected from : CDRH2 present in SEQ I D NO:l and CDRH2 that differs from the CDRH2 present in SEQ I D NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRH3 is selected from : CDRH3 as present in SEQ I D NO: 1 and CDRH3 that differs from the CDRH3 present in SEQ I D NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids; L(LAG-3) is an antibody light chain of the IgG class comprising CDRL1 and CDRL2, wherein said CDRL
  • A is a bond or a peptide linker
  • VH(PD-I) is an antibody heavy chain variable domain having CDRHl, CDRH2 and CDRH3, wherein said CDRHl is selected from: CDRHl present in SEQ ID NO:3, and CDRHl that differs from the CDRHl present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids; wherein CDRH2 is selected from: CDRH2 present in SEQ ID NO:3 and CDRH2 that differs from the CDRH2 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRH3 is selected from: CDRH3 as present in SEQ ID NO: 3 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids.
  • the binding protein specific to human LAG-3 and human PD-1 of formula (I) exhibits >50% inhibition of LAG3-MHCII interaction in competition flow cytometry assay, and an IC50 of less than or equal to 5 nM in the PD-l/PDL-1 competition assay.
  • the invention provides a binding protein specific to human PD-1 that comprises one or more of CDRHl, CDRH2 and CDRH3, wherein CDRHl is selected from the group consisting of: CDRHl as present in SEQ ID NO:3 and CDRHl that differs from the CDRHl present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids, wherein CDRH2 is selected from the group consisting of: CDRH2 as present in SEQ ID NO:3 and CDRH2 that differs from the CDRH2 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids, and wherein CDRH3 is selected from the group consisting of: CDRH3 as present in SEQ ID NO: 3 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids.
  • the binding protein exhibits an IC50 of less than or equal to 5 nM in the PD-l/PDL-1 competition assay.
  • the binding protein specific to human PD-l is additionally capable of neutralising a human checkpoint inhibitor other than PD-1.
  • a human checkpoint inhibitor is a human protein that limits the function of a human immune cell by inhibiting signalling cascades that modulate the activation, proliferation, cytokine production or function of said immune cell following stimulation. Neutralisation of a human checkpoint inhibitor blocks the biological activity of the said human checkpoint inhibitor.
  • the invention provides a binding protein specific to human LAG-3, which comprises: one or more of CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: CDRH1 as present in SEQ. ID NO:l and CDRH1 that differs from the CDRH1 present in SEQ.
  • CDRH2 is selected from the group consisting of: CDRH2 as present in SEQ ID NO:l and CDRH2 that differs from the CDRH2 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids
  • CDRH3 is selected from the group consisting of: CDRH3 as present in SEQ ID NO: 1 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids
  • CDRL1, CDRL2 and CDRL3 wherein CDRL1 is selected from the group consisting of: CDRL1 as present in SEQ ID NO:2 and CDRL1 that differs from the CDRL1 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids
  • CDRL2 is selected from the group consisting of: CDRL2 as present in SEQ ID NO:2 and CDRL
  • the binding protein specific to human LAG-3 exhibits >50% inhibition of LAG3-MHCII interaction in a competition flow cytometry assay.
  • the binding protein specific to human LAG-3 is additionally capable of neutralising a human checkpoint inhibitor other than LAG-3.
  • a human checkpoint inhibitor is a human protein that limits the function of a human immune cell by inhibiting signalling cascades that modulate the activation, proliferation, cytokine production or function of said immune cell following stimulation. Neutralisation of a human checkpoint inhibitor blocks the biological activity of the said human checkpoint inhibitor.
  • the invention provides isolated nucleic acids encoding a binding protein of the invention (i.e. a binding protein specific to human PD-1 and human LAG-3, a binding protein specific to human PD-1 (and optionally another checkpoint inhibitor) and a binding protein specific to human LAG-3 (and optionally another checkpoint inhibitor)), vectors containing isolated nucleic acids encoding these binding proteins and host cells containing the vectors.
  • a binding protein of the invention i.e. a binding protein specific to human PD-1 and human LAG-3, a binding protein specific to human PD-1 (and optionally another checkpoint inhibitor) and a binding protein specific to human LAG-3 (and optionally another checkpoint inhibitor)
  • vectors containing isolated nucleic acids encoding these binding proteins and host cells containing the vectors i.e. a binding protein specific to human PD-1 and human LAG-3, a binding protein specific to human PD-1 (and optionally another checkpoint inhibitor) and a binding protein specific to human LAG-3 (and optionally another check
  • the invention provides a pharmaceutical composition comprising a binding protein of the invention and a pharmaceutically acceptable excipient.
  • the invention also provides methods of treating diseases using a binding protein of the invention or a pharmaceutical composition of the invention.
  • the invention provides methods of treating cancer and methods of treating and curing HIV.
  • Figure 1 is a bar graph showing the mean IFNy produced in the mixed lymphocyte reaction assay after 5 days of co-culture with isolated CD4 T cells, monocyte derived dendritic cells(MDDC) and either the LAG3/PD1 bispecific 57E02x51A09-188001, LAG 3 or PD1 monovalent antagonists or combinations thereof.
  • the bar graph shows the mean and standard deviation from duplicate assays, with technical triplicates per assay. Antibodies were tested at concentrations ranging from 0.195-200nM.
  • the LAG3/PD1 bispecific 57E02x51A09-188001 resulted in greater IFNy production from CD4+ T cells compared to LAG3 or PD1 monovalent antagonists or combinations thereof.
  • Figure 2 is a bar graph showing the fraction of HIV specific CD8 T cells that proliferated (CFSEdim) during 6 days of culture in the presence of the LAG3/PD1 mAbdAb 57E02-51A09- 188001 (black bars) or the control antibody (grey bars).
  • the bar graph shows the mean and standard deviation from 6 replicates with the Bonferroni adjusted p value from the pairwise comparison shown above the bars.
  • Figure 2 refers to the LAG3/PD1 mAbdAb incorrectly as 57E02-51A09-188 (the correct name is 57E02-51A09-188001).
  • Figure 3 is a scatterplot graph showing the fraction of cells producing certain cytokines derived from 19 HIV infected stably ART treated donors stimulated with SEB & SEA or left unstimulated in the presence or absence of the LAG3/PD1 mAbdAb 57E02-51A09-188001 or the control antibody as follows: A) the fraction of CD4 memory &/ effector T cells that produce IFNy, IL2 and TNFa, B) The fraction of CD8 memory &/ effector T cells that produce IFNy, IL2 and TNFa, C) The fraction of CD4 memory &/effector T cells that dually produce IL2 and TNFa and D) The fraction of CD8 memory &/effector T cells that dually express IFNy, and CD107. In each case statistical significance was determined via a mixed effects model followed by pairwise comparisons. Bonferroni adjusted p values are presented in the graph. It should be noted that data was analyzed on the Data 0,2 transformed scale to make variances more homogene
  • Figure 4 is a scatterplot graph showing the levels of HIV Gag RNA per million CD4 T cells from five HIV infected stably treated (ST) donors.
  • CD4 T cells were cultured alone (CD4 circles & triangles) or in the presence of monocyte derive dendritic cells (mDDC- asterisk & squares) in the presence of the LAG3/PD1 bispecific antibody 57E02-51A09-188001 or a control antibody VHDUM.
  • P-values presented in the graph were obtained via pairwise comparisons of antibodies, after a Bonferroni adjustment for multiple comparisons. Data was analysed on the logio transformed scale, and a separate analysis was done for each donor.
  • the invention provides a binding protein specific to human PD-1 and human LAG-3, a binding protein specific to human PD-1 (and optionally another checkpoint inhibitor) and a binding protein specific to human LAG-3 (and optionally another checkpoint inhibitor).
  • the invention provides a binding protein, which comprises an antibody specific for human LAG-3 attached by a linker to one or more epitope binding domains specific to human PD-1, wherein the antibody specific for human LAG-3 comprises one or more of CDRHl, CDRH2 and CDRH3, wherein CDRHl is selected from the group consisting of: CDRHl as present in SEQ ID NO:l and CDRHl that differs from the CDRHl present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids, wherein CDRH2 is selected from the group consisting of: CDRH2 as present in SEQ ID NO:l and CDRH2 that differs from the CDRH2 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids, and wherein CDRH3 is selected from the group consisting of: CDRH3 as present in SEQ ID NO: 1 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:
  • the binding protein i.e. the binding protein specific to human PD-1 and human LAG-3.
  • the binding protein exhibits >50% inhibition of LAG3-MHCII interaction in competition flow cytometry assay, and an IC50 of less than or equal to 5 nM in the PD-l/PDL-1 competition assay.
  • an antibody is a heterotetrameric glycoprotein of the IgA or IgG classes that is composed of two heavy chains and two light chains, wherein the heavy chains comprise the VH and CHI domains, and the light chains comprise the VL and CL domains. More particularly, the antibody is of the IgG class. In one embodiment the antibody is selected from the IgGl, lgG2, lgG3, lgG4 and lgG4PE subclasses. In a more particular embodiment, the antibody is selected from the IgGl subclass, even more particularly, the antibody has an IgGl disabled isotype (LAGA).
  • LAGA IgGl disabled isotype
  • an antibody is a glycoprotein of the IgA or IgG classes that is composed of two heavy chains and two light chains, wherein the heavy chains comprise the VH and CHI domains, and the light chains comprise the VL and CL domains. More particularly, the antibody is of the IgG class. In one embodiment the antibody is selected from the IgGl, lgG2, lgG3, lgG4 and lgG4PE subclasses. In a more particular embodiment, the antibody is selected from the IgGl subclass, even more particularly, the antibody has an IgGl disabled isotype (LAGA).
  • LAGA IgGl disabled isotype
  • the heavy chains comprise the CHI, CH2, and VH domains and the light chains comprise the CL and VL domains.
  • the heavy chains contain the CHI, CH2, CH3 and VH domains and the light chains contain the CL and VL domains.
  • the CHI, CH2, CH3, VH, CL and VL domains referred to above may be complete domains or modified domains which have been truncated or contain N or C-terminal extensions, or in which one or more loops have been replaced by sequences which are not characteristic of the domain in question.
  • Other modifications are also included, for example, the addition of other post-translational modifications such as phosphorylation, deamidation, oxidation, disulphide bond scrambling, isomerisation, C-terminal lysine clipping and N-terminal glutamine cyclisation or the inclusion of one or more non-natural amino acids.
  • antibodies are glycoproteins and the domains above may be glycosylated.
  • the term epitope binding domain specific to human PD1 refers to a folded protein structure which retains its tertiary structure independent of the rest of the binding protein.
  • the epitope binding domain specific to human PD1 has CDRs permitting the binding protein to bind human PD-1.
  • the epitope binding domain specific to human PD1 has CDRs permitting the binding protein to exhibit an IC50 of less than or equal to 5 nM in the PD- 1/PDL-l competition assay.
  • the epitope binding domain specific to human PD1 is a single variable domain of an antibody (in other words the protein scaffold is an immunoglobulin scaffold). This single variable domain may be capable of binding human PD1 independently of a different variable region or domain.
  • the single variable domain may be a complete antibody variable domain such as VH, VHH and VL or a modified domain which has been truncated or contains N or C-terminal extensions, or in which one or more loops have been replaced by sequences which are not characteristic of the domain in question.
  • the single variable domain may be a VH domain or a modified VH domain which has been truncated or contains N or C-terminal extensions, or in which one or more loops have been replaced by sequences which are not characteristic of the domain in question.
  • the single variable domain may be a VH domain.
  • the epitope binding domain specific to human PD1 has a non- immunoglobulin scaffold having loops connecting elements of secondary structure which can be engineered to include CDR regions.
  • Non immunoglobulin scaffolds include CTLA-4 (Evibodies; Journal Immunological Methods 248(1-2): 31-45, 2001), lipocalin, Protein A derived molecules such as Z-domain of Protein A (Affibodies, Protein Eng Des Sel 17: 455- 462, 2004 and EP1641818), A-domain (Avimer/Maxibody), heat shock proteins such as GroEI and GroES, transferrin (trans-body), ankyrin repeat protein (DARPin), peptide aptamer, C- type lectin domain (Tetranectin), human ⁇ -crystallin and human ubiquitin (affilins), PDZ domains, scorpion toxin kunitz-type domains of human protease inhibitors, and fibronectin/adnectin.
  • the antibody specific for LAG-3 (that forms part of the binding protein specific to human PD-1 and human LAG-3) comprises CDRH1, CDRH2 and CDRH3, wherein said CDRH1 is selected from: CDRH1 present in SEQ ID NO:l, and CDRH1 that differs from the CDRH1 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids; wherein CDRH2 is selected from: CDRH2 present in SEQ ID NO:l and CDRH2 that differs from the CDRH2 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRH3 is selected from: CDRH3 as present in SEQ ID NO: 1 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids.
  • the antibody specific for LAG-3 (that forms part of the binding protein specific to human PD-1 and human LAG-3) comprises CDRLl and CDRL2, wherein said CDRLl is selected from: CDRLl present in SEQ ID NO:2 and CDRLl that differs from the CDRLl present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRL2 is selected from: CDRL2 present in SEQ ID NO:2 and CDRL2 that differs from the CDRL2 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids.
  • the antibody specific for LAG-3 (that forms part of the binding protein specific to human PD-1 and human LAG-3) comprises CDRLl and CDRL2, wherein said CDRLl is selected from: CDRLl present in SEQ ID NO:2 and CDRLl that differs from the CDRLl present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRL2 is as present in SEQ ID NO:2.
  • the antibody specific for LAG-3 (that forms part of the binding protein specific to human PD-1 and human LAG-3) comprises CDRLl, CDRL2 and CDRL3, wherein said CDRLl is selected from: CDRLl present in SEQ ID NO:2 and CDRLl that differs from the CDRLl present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids; wherein CDRL2 is selected from: CDRL2 present in SEQ ID NO:2 and CDRL2 that differs from the CDRL2 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRL3 is selected from: CDRL3 as present in SEQ ID NO:2, and CDRL3 that differs from the CDRL3 present in SEQ ID N0:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids.
  • the antibody specific for LAG-3 (that forms part of the binding protein specific to human PD-1 and human LAG-3) comprises CDRL1, CDRL2 and CDRL3, wherein said CDRL1 is selected from: CDRL1 present in SEQ ID NO:2 and CDRL1 that differs from the CDRL1 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids; wherein CDRL2 is as present in SEQ ID NO:2; and wherein CDRL3 is selected from: CDRL3 as present in SEQ ID NO:2, and CDRL3 that differs from the CDRL3 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids.
  • the one or more epitope binding domains specific to PD1 comprise CDRH1, CDRH2 and CDRH3, wherein said CDRH1 is selected from: CDRH1 present in SEQ ID NO:3, and CDRH1 that differs from the CDRH1 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids; wherein CDRH2 is selected from: CDRH2 present in SEQ ID NO:3 and CDRH2 that differs from the CDRH2 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRH3 is selected from: CDRH3 as present in SEQ ID NO: 3 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids.
  • the one or more epitope binding domains specific to PD1 comprise CDRH1, CDRH2 and CDRH3, wherein said CDRH1 is selected from: CDRH1 present in SEQ ID NO:3, and CDRH1 that differs from the CDRH1 present in SEQ ID NO:3 by the addition or deletion or substitution of 1 amino acid; wherein CDRH2 is selected from: CDRH2 present in SEQ ID NO:3 and CDRH2 that differs from the CDRH2 present in SEQ ID NO:3 by the addition or deletion or substitution of 1 or 2 amino acids; and wherein CDRH3 is selected from: CDRH3 as present in SEQ ID NO: 3 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:3 by the addition or deletion or substitution of 1 or 2 amino acids.
  • the complementarity determining regions or CDRs for the antibody specific to LAG-3 and the one or more epitope binding domains specific to PD-1 can be defined by any numbering convention, for example the Kabat, Chothia, AbM and contact conventions.
  • the CDR regions for SEQ ID NO.l, SEQ ID NO. 2 and SEQ ID NO 3 defined by each method are set out in Table 1. Table 1:
  • each CDR may be modified by one, two or three amino acid substitutions, deletions or additions to form CDR variants.
  • the binding protein specific to human PD-1 and LAG-3 contains one or more CDR variants
  • the binding protein retains biological activity, defined as exhibiting >50% inhibition of LAG3- MHCII interaction in competition flow cytometry assay, and an IC50 of less than or equal to 5 nM in the PD-l/PDL-1 competition assay.
  • a single CDR variant may contain substitutions, additions or deletions, in any combination, compared to the amino acid sequence of the unmodified CDR.
  • the modification is a substitution.
  • a CDR is modified by the substitution of 1, 2 or 3 amino acids. More particularly, the modification is a conservative substitution, where amino acids with side chains of similar properties are substituted.
  • amino acids can be classified as being hydrophobic, neutral hydrophilic, acidic, basic, residues that influence chain orientation or aromatic as shown in Table 2 below.
  • a conservative substitution is a substitution of one amino acid residue for another residue in the same group.
  • the antibody specific for LAG-3 (that forms part of the binding protein specific to human PD-1 and human LAG-3) comprises CDRL1 having the sequence
  • RASQX1ISSX2LX3 (SEQ ID NO: 56), wherein Xi is G or S, X 2 is W, F or Y, and X 3 is A or N.
  • the one or more epitope binding domains specific to PD1 have CDRH1, CDRH2 and CDRH3, wherein CDRH1 has the sequence THYMX 4 (SEQ ID NO: 57), wherein X 4 is V or A, wherein CDRH2 has the sequence FIGPAGDX 5 TYYADSVX 6 G (SEQ ID NO: 58) wherein X 5 is T, F or S and X 6 is K or E, and wherein CDRH3 is YTX 7 TSX 8 X 9 DXi 0 YDV (SEQ I D NO : 59), wherein X 7 is A or E, Xs is G, S or D, Xg is V, F or Y, and Xio is T or S.
  • THYMX 4 SEQ ID NO: 57
  • CDRH2 has the sequence FIGPAGDX 5 TYYADSVX 6 G (SEQ ID NO: 58) wherein X 5 is T, F or S and X 6 is
  • the antibody specific for LAG-3 (that forms part of the binding protein specific to human PD-1 and human LAG-3) comprises CDRH1, CDRH2 and CDRH3 as present in SEQ. ID NO. 1.
  • the CDRs may be defined by any numbering convention. In a more particular embodiment, the CDRs are defined by the Kabat numbering convention such that CDRH1 has the sequence defined as SEQ ID NO: 4, CDRH2 has the sequence defined as SEQ ID NO: 5, and CDRH3 has the sequence defined as SEQ ID NO:6.
  • the antibody specific for LAG comprises CDRLl and CDRL2 as present in SEQ ID NO. 2.
  • the CDRs may be defined by any numbering convention. In a more particular embodiment, the CDRs are defined by the Kabat numbering convention such that CDRLl has the sequence defined as SEQID NO: 7, CDRL2 has the sequence defined as SEQ ID NO: 8 and CDRL3 has the sequence defined as SEQ ID NO:9.
  • the antibody specific for LAG comprises CDRLl, CDRL2 and CDRL3 as present in SEQ ID NO. 2.
  • the CDRs may be defined by any numbering convention. In a more particular embodiment, the CDRs are defined by the Kabat numbering convention such that CDRLl has the sequence defined as SEQID NO: 7, CDRL2 has the sequence defined as SEQ ID NO: 8 and CDRL3 has the sequence defined as SEQ ID NO:9.
  • the one or more epitope binding domains specific to PD1 (that form part of the binding protein specific to human PD-1 and human LAG-3) have CDRH1, CDRH2 and CDRH3 as present in SEQ ID NO. 3.
  • the CDRs may be defined by any numbering convention. In a more particular embodiment, the CDRs are defined by the Kabat numbering convention such that CDRH1 has the sequence defined as SEQ ID NO: 10, CDRH2 has the sequence defined as SEQ ID NO: 11 and CDRH3 has the sequence defined as SEQ ID NO:12.
  • the antibody specific for human LAG-3 (that forms part of the binding protein specific to human PD-1 and human LAG-3) comprises two identical heavy chains comprising the sequence defined as SEQ ID NO.l or a variant of SEQ ID NO. 1 that differs in having up to 10 amino acid additions, deletions or substitutions.
  • the heavy chain CDRs are defined as described in any of the above embodiments, and the up to 10 amino acid additions, deletions or substitutions are not within the CDR regions.
  • the antibody specific for human LAG-3 (that forms part of the binding protein specific to human PD-1 and human LAG-3) comprises two identical light chains comprising the sequence defined as SEQ. ID NO.2 or a variant of SEQ ID NO. 2 that differs in having up to 10 amino acid additions, deletions or substitutions.
  • the light chain CDRs are defined as described in any of the above embodiments and the up to 10 amino acid additions, deletions or substitutions are not within the CDR regions.
  • the one or more epitope binding domains specific to human PD1 comprises the sequence defined as SEQ ID NO.3 or a variant of SEQ ID NO. 3 that differs in having up to 10 amino acid additions, deletions or substitutions.
  • the CDRs are defined as described in any of the above embodiments and the up to 10 amino acid additions, deletions or substitutions are not within the CDR regions.
  • the variant light chain, variant heavy chain or epitope binding domain sequences may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions or deletions, in any combination.
  • the modification is a substitution.
  • the sequences are modified by the substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
  • the modification is a conservative substitution (a substitution of one amino acid residue for another residue in the same group of Table 2).
  • the antibody specific for human LAG-3 (that forms part of the binding protein specific to human PD-1 and human LAG-3) comprises two identical heavy chains comprising the sequence defined as SEQ ID NO.l or a sequence that has at least 90% sequence identity to the sequence of SEQ ID NO. 1.
  • the antibody specific for human LAG-3 (that forms part of the binding protein specific to human PD-1 and human LAG-3) comprises two identical light chains comprising the sequence defined as SEQ ID NO.2 or a sequence that has at least 90% sequence identity to the sequence of SEQ ID NO. 2.
  • the one or more epitope binding domains specific to human PD1 (that form part of the binding protein specific to human PD-1 and human LAG-3) comprises the sequence defined as SEQ. ID NO.3 or a sequence that has at least 90% sequence identity to the sequence of SEQ ID NO. 3.
  • Percent identity between a query amino acid sequence and a subject amino acid sequence is the identities value expressed as a percentage, that is calculated by the BLASTP algorithm when a subject amino acid sequence has a 100% query coverage with a query amino acid sequence after a pair wise BLASTP alignment is performed.
  • Such pair-wise BLASTP alignments between a query amino acid sequence and a subject amino acid sequence are performed by using the default settings of the BLASTP algorithm available on the National Center for Biotechnology Institute's website with the filter for low complexity regions turned off.
  • the percentage identity may be determined across the entire length of the query sequence including the CDRs.
  • the percentage identity may exclude the CDRs, for example the CDRs are 100% identity to the subject sequence and the percentage identity variation is in the remaining portion of the query sequence so that the CDR sequence is fixed/intact.
  • the antibody specific for human LAG-3 (that forms part of the binding protein specific to human PD-1 and human LAG-3) comprises two identical heavy chains comprising the sequence defined as SEQ ID NO.l.
  • the antibody specific for human LAG-3 (that forms part of the binding protein specific to human PD-1 and human LAG-3) comprises two identical light chains comprising the sequence defined as SEQ ID NO.2.
  • the one or more epitope binding domains specific to human PD1 (that form part of the binding protein specific to human PD-1 and human LAG-3) comprise the sequence defined as SEQ ID NO.3.
  • the one or more epitope binding domains specific to human PD-1 has a reduced ability to bind to pre-existing antibodies (ADAs) as compared to the equivalent unmodified molecule.
  • reduced ability to bind it is meant that the modified molecule binds with a reduced affinity or reduced avidity to a pre-existing ADA.
  • Said one or more modified epitope binding domains specific to human PD-1 comprise one or more modifications selected from: (a) a C-terminal addition, extension, deletion or tag, and/or (b) one or more amino acid framework substitutions.
  • the one or more modified epitope binding domains specific to human PD-1 comprise: a) a C-terminal sequence consisting of the sequence VTVS(S)nXn (embodiments of this generic sequence are represented as SEQ. ID NO: 63-SEQ ID NO: 78); and also optionally b) one or more amino acid substitutions at positions 14, 41, 108, 110, or 112 compared to a human germline framework sequence wherein: n represents an integer independently selected from 0 or 1;
  • Xii may be present or absent, and if present represents an amino acid extension of 1 to 8 amino acids residues.
  • X is absent.
  • X is present, and is an extension of 1 to 8 amino acids, in particular an extension of 1 to 8 amino acids which comprises an alanine residue, for example a single alanine extension, or an AS, AST, ASTK, ASTKG, ASTKGP extension.
  • X is present, and is an extension of 1 to 8 amino acids, in particular an extension of 1 to 8 amino acids which comprises an A, AAA or T extension.
  • the one or more modified epitope binding domains specific to human PD-1 comprise one or more amino acid substitutions wherein said one or more amino acid substitutions are selected from the group consisting of a P14A substitution, a P41A substitution, a L108A substitution, a T110A substitution, a S112A substitution, a P14K substitution, a P14Q substitution, and a P14T substitution.
  • the one or more epitope binding domains are attached by a linker to the C terminus of the heavy chain of the antibody specific to human LAG-3.
  • the linker may be a bond (such as a peptide bond) or a peptide linker.
  • the linker is a peptide linker from 1 to 100 amino acids in length, more particularly, from 1 to 50 or from 1 to 10 amino acids in length. Suitable linkers are described in published PCT patent application WO2010/136483. I n a more particular embodiment, the linker has the sequence set out as SEQ. I D NO. 30 (STGLDSPT).
  • STGLDSPT SEQ. I D NO. 30
  • the invention provides a binding protein having the general formula (I):
  • H(LAG-3) is an antibody heavy chain of the IgG class comprising CDRHl, CDRH2 and CDRH3, wherein said CDRHl is selected from : CDRHl present in SEQ I D NO:l, and CDRHl that differs from the CDRHl present in SEQ I D NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids; wherein CDRH2 is selected from : CDRH2 present in SEQ ID NO:l and CDRH2 that differs from the CDRH2 present in SEQ I D NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRH3 is selected from : CDRH3 as present in SEQ I D NO: 1 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids; L(LAG-3) is an antibody light chain of the IgG class comprising CDRL1 and CDRL2, wherein said CDRL1 is
  • A is a bond or a peptide linker
  • VH(PD-I) is an antibody heavy chain variable domain having CDRHl, CDRH2 and CDRH3, wherein said CDRHl is selected from: CDRHl present in SEQ ID NO:3, and CDRHl that differs from the CDRHl present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids; wherein CDRH2 is selected from: CDRH2 present in SEQ ID NO:3 and CDRH2 that differs from the CDRH2 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRH3 is selected from: CDRH3 as present in SEQ ID NO: 3 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids.
  • the binding protein of formula (I) is a binding protein specific to human PD-1 and human LAG -3.
  • the binding protein of formula (I) exhibits >50% inhibition of LAG3- MHCII interaction in competition flow cytometry assay, and an IC50 of less than or equal to 5 nM in the PD-l/PDL-1 competition assay.
  • H comprises the VH and CHI domains and L comprises the CL and VL domains.
  • H comprises the CHI, CH2, and VH domains and L comprises the CL and VL domains.
  • H contains the CHI, CH2, CH3 and VH domains and L contains the CL and VL domains.
  • the CHI, CH2, CH3, VH, CL and VL domains referred to above may be complete domains or modified domains which have been truncated or contain N or C-terminal extensions, or in which one or more loops have been replaced by sequences which are not characteristic of the domain in question.
  • Other modifications are also included, for example, the addition of other post-translational modifications such as phosphorylation, deamidation, oxidation, disulphide bond scrambling, isomerisation, C-terminal lysine clipping and N-terminal glutamine cyclisation or the inclusion of one or more non-natural amino acids.
  • antibodies are glycoproteins and the domains above may be
  • H is a heavy chain of the IgGl, lgG2, lgG3, lgG4 or lgG4PE subclasses.
  • n is 2.
  • n is 4.
  • H is of the lgG3 subclass
  • n is 11.
  • the antibody is selected from the IgGl subclass, even more particularly, the antibody has an IgGl disabled isotype (LAGA).
  • VH(PD-I) may be a complete antibody variable domain or a modified domain which has been truncated or contains N or C-terminal extensions, or in which one or more loops have been replaced by sequences which are not characteristic of the domain in question. Other modifications are also included, for example, the addition of other post-translational modifications such as phosphorylation, deamidation, oxidation, disulphide bond scrambling, isomerisation, C-terminal lysine clipping and N-terminal glutamine cyclisation or the inclusion of one or more non-natural amino acids.
  • VH(PD-I) may retain its tertiary structure independent of the rest of the binding protein of formula (I) and/or be capable of binding human PD1 independently of a different variable region or domain.
  • the CDRs for H, L and VH(PD-I) can be defined by any numbering convention, for example the Kabat, Chothia, AbM and contact conventions.
  • the CDR regions for SEQ. ID NO.l, SEQ ID NO. 2 and SEQ ID NO 3 defined by each method are set out above in Table 1.
  • each CDR may be modified by one, two or three amino acid
  • the binding protein of formula (I) containing one or more CDR variant retains biological activity, defined as exhibiting >50% inhibition of LAG3-MHCII interaction in competition flow cytometry assay, and an IC50 of less than or equal to 5 nM in the PD-l/PDL-1 competition assay. It will be appreciated by one of skill in the art that a single CDR variant may contain substitutions, additions or deletions, in any combination, compared to the amino acid sequence of the unmodified CDR.
  • H(LAG-3) comprises CDRHl, CDRH2 and CDRH3, wherein said CDRHl is selected from: CDRHl present in SEQ ID NO:l, and CDRHl that differs from the CDRHl present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids; wherein CDRH2 is selected from: CDRH2 present in SEQ ID NO:l and CDRH2 that differs from the CDRH2 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRH3 is selected from:
  • L(LAG-3) comprises CDRL1 and CDRL2, wherein said CDRL1 is selected from: CDRL1 present in SEQ ID NO:2 and CDRL1 that differs from the CDRL1 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRL2 is selected from: CDRL2 present in SEQ ID NO:2 and CDRL2 that differs from the CDRL2 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids.
  • L(LAG-3) comprises CDRL1 and CDRL2, wherein said CDRL1 is selected from: CDRL1 present in SEQ ID NO:2 and CDRL1 that differs from the CDRL1 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRL2 is as present in SEQ ID NO:2.
  • L(LAG-3) comprises CDRL1, CDRL2 and CDRL3, wherein said CDRL1 is selected from: CDRL1 present in SEQ ID NO:2 and CDRL1 that differs from the CDRL1 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids; wherein CDRL2 is selected from: CDRL2 present in SEQ ID NO:2 and CDRL2 that differs from the CDRL2 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRL3 is selected from: CDRL3 as present in SEQ ID NO:2, and CDRL3 that differs from the CDRL3 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids.
  • L(LAG-3) comprises CDRL1, CDRL2 and CDRL3, wherein said CDRL1 is selected from: CDRL1 present in SEQ ID NO:2 and CDRL1 that differs from the CDRL1 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids; wherein CDRL2 is as present in SEQ ID NO:2; and wherein CDRL3 is selected from: CDRL3 as present in SEQ ID NO:2, and CDRL3 that differs from the CDRL3 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids.
  • L(LAG-3) comprises CDRL1 having the sequence RASQX1ISSX2LX3 (SEQ ID NO: 56), wherein Xi is G or S, X 2 is W, F or Y, and X 3 is A or N.
  • V H (PD-1) comprises CDRH1, CDRH2 and CDRH3, wherein said CDRH1 is selected from: CDRH1 present in SEQ ID NO:3, and CDRH1 that differs from the CDRH1 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids; wherein CDRH2 is selected from: CDRH2 present in SEQ ID NO:3 and CDRH2 that differs from the CDRH2 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRH3 is selected from: CDRH3 as present in SEQ ID NO: 3 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids.
  • VH(PD-I) comprises CDRH1, CDRH2 and CDRH3, wherein said CDRH1 is selected from: CDRH1 present in SEQ ID NO:3, and CDRH1 that differs from the CDRH1 present in SEQ ID NO:3 by the addition or deletion or substitution of 1 amino acid; wherein CDRH2 is selected from: CDRH2 present in SEQ ID NO:3 and CDRH2 that differs from the CDRH2 present in SEQ ID NO:3 by the addition or deletion or substitution of 1 or 2 amino acids; and wherein CDRH3 is selected from: CDRH3 as present in SEQ ID NO: 3 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:3 by the addition or deletion or substitution of 1 or 2 amino acids.
  • V H (PD-1) comprises CDRH1, CDRH2 and CDRH3, wherein CDRH1 has the sequence THYMX 4 (SEQ I D NO: 57), wherein X 4 is V or A, wherein CDRH2 has the sequence FIGPAGDX 5 TYYADSVX 6 G (SEQ I D NO: 58) wherein X 5 is T, F or S and X 6 is K or E, and wherein CDRH3 is YTX 7 TSX 8 X 9 DXi 0 YDV (SEQ I D NO: 59), wherein X 7 is A or E, X 8 is G, S or D, Xg is V, F or Y, and Xio is T or S.
  • THYMX 4 SEQ I D NO: 57
  • CDRH2 has the sequence FIGPAGDX 5 TYYADSVX 6 G (SEQ I D NO: 58) wherein X 5 is T, F or S and X 6 is K or E
  • H(LAG-3) comprises CDRH1, CDRH2 and CDRH3 as present in SEQ ID NO. 1.
  • the CDRs may be defined by any numbering convention.
  • the CDRs are defined by the Kabat numbering convention such that CDRH1 has the sequence defined as SEQ ID NO: 4, CDRH2 has the sequence defined as SEQ ID NO: 5 and CDRH3 has the sequence defined as SEQ ID NO:6.
  • L(LAG-3) comprises CDRLl and CDRL2 as present in SEQ ID NO. 2.
  • the CDRs may be defined by any numbering convention.
  • the CDRs are defined by the Kabat numbering convention such that CDRLl has the sequence defined as SEQ ID NO: 7 and CDRL2 has the sequence defined as SEQ ID NO: 8.
  • L(LAG-3) comprises CDRLl, CDRL2 and CDRL3 as present in SEQ ID NO. 2.
  • the CDRs may be defined by any numbering convention. In a more particular embodiment, the CDRs are defined by the Kabat numbering convention such that CDRLl has the sequence defined as SEQ ID NO: 7 and CDRL2 has the sequence defined as SEQ ID NO: 8 and CDRL3 has the sequence defined as SEQ ID NO:9.
  • V H (PD-1) has CDRH1, CDRH2 and CDRH3 as present in SEQ ID NO. 3.
  • the CDRs may be defined by any numbering convention. In a more particular embodiment, the CDRs are defined by the Kabat numbering convention such that CDRH1 has the sequence defined as SEQ ID NO: 10, CDRH2 has the sequence defined as SEQ ID NO: 11 and CDRH3 has the sequence defined as SEQ ID NO:12.
  • VH has the sequence defined as SEQ ID NO.l or a variant of SEQ ID NO. 1 that differs in having up to 10 amino acid additions, deletions or substitutions.
  • the CDRs are defined as described in any of the above
  • the up to 10 amino acid additions, deletions or substitutions are not within the CDR regions.
  • VL has the sequence defined as SEQ ID NO.2 or a variant of SEQ ID NO. 2 that differs in having up to 10 amino acid additions, deletions or substitutions.
  • the CDRs are defined as described in any of the above embodiments and the up to 10 amino acid additions, deletions or substitutions are not within the CDR regions.
  • VH(PD-I) has the sequence defined as SEQ ID NO.3 or a variant of SEQ ID NO. 3 that differs in having up to 10 amino acid additions, deletions or substitutions.
  • the CDRs are defined as described in any of the above embodiments and the up to 10 amino acid additions, deletions or substitutions are not within the CDR regions.
  • VH, VL and VH(PD-I) may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions or deletions, in any combination.
  • the modification is a substitution.
  • the sequences are modified by the substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.
  • the modification is a conservative substitution (a substitution of one amino acid residue for another residue in the same group of Table 2).
  • VH has the sequence defined as SEQ ID NO.l or a sequence that has at least 90% sequence identity to the sequence of SEQ. ID NO. 1.
  • VL has the sequence defined as SEQ ID NO.2 or a sequence that has at least 90% sequence identity to the sequence of SEQ ID NO. 2.
  • VH(PD-I) has the sequence defined as SEQ ID NO.3 or a sequence that has at least 90% sequence identity to the sequence of SEQ ID NO. 3.
  • Percent identity between a query amino acid sequence and a subject amino acid sequence is the identities value expressed as a percentage, that is calculated by the BLASTP algorithm when a subject amino acid sequence has a 100% query coverage with a query amino acid sequence after a pair wise BLASTP alignment is performed.
  • Such pair-wise BLASTP alignments between a query amino acid sequence and a subject amino acid sequence are performed by using the default settings of the BLASTP algorithm available on the National Center for Biotechnology Institute's website with the filter for low complexity regions turned off.
  • the percentage identity may be determined across the entire length of the query sequence including the CDRs.
  • the percentage identity may exclude the CDRs, for example the CDRs are 100% identity to the subject sequence and the percentage identity variation is in the remaining portion of the query sequence so that the CDR sequence is fixed/intact.
  • VH, VL and VH(PD-I) have CDR sequences as defined in any of the above embodiments and a framework that is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical to the framework of SEQ ID NO.l (for V H ), SEQ ID NO. 2 (for V L ) and SEQ ID NO.3 (for V H (PD-l)).
  • VH has the sequence defined as SEQ ID NO.l.
  • VL has the sequence defined as SEQ ID NO.2.
  • VH(PD-I) has the sequence defined as SEQ ID NO.3.
  • A is a peptide bond or a peptide linker from 1 to 100 amino acids in length.
  • A is a peptide linker from 1 to 100 amino acids in length, more particularly, from 1 to 50 or from 1 to 10 amino acids in length.
  • Suitable linkers are described in published PCT patent application WO2010/136483.
  • I n a more particular embodiment, the linker has the sequence set out as SEQ. I D NO. 30 (STGLDSPT).
  • STGLDSPT SEQ. I D NO. 30
  • the 57E02x51A09-188001 mAbdAb described in the Examples is a binding protein of formula (I) that is specific to human PD-1 and human LAG-3. It inhibits the negative signals from PD-1 and LAG-3 receptors by preventing the interaction with their respective ligands. I n so doing, it facilitates T cell activation demonstrated by NFAT signalling in reporter gene assays, and cytokine production in mixed lymphocyte reaction assays.
  • the 57E02x51A09- 188001 mAbdAb has been shown to promote T cell activation and HIV RNA production in CD4 T cells from HIV donors, and the proliferative and functional capacity of CD8 T cells from HIV donors, supporting its use in the treatment of HIV.
  • the Examples disclose 16 PD-1 dAbs (in mAbdAb format) with CDR sequences that are related to the PD-1 dAb 51A09-188001 present in the exemplified mAbdAb. These variant dAbs also block PD-1 signalling as determined by a reporter gene assays. It is reasonable to expect that these and other variants with related CDRs would be capable of blocking both PD-1 signalling when formatted as a mAbdAb with a LAG-3 mAb, and hence have utility in the indications disclosed in the medical uses section.
  • the Examples disclose 4 LAG-3 mAbs with CDR sequences that are related to the LAG-3 mAb 57E02 present in the exemplified mAbdAb. These variant mAbs also block LAG-3 signalling as determined by a reporter gene assay. It is reasonable to expect that these and other variants with related CDRs would be capable of blocking both LAG-3 signalling when formatted as a mAbdAb with a PD-1 dAb, and hence have utility in the indications disclosed in the medical uses section.
  • the specific 57E02x51A09-188001 mAbdAb exemplified advantageously showed greater improvements in T cell activation (as measured by cytokine production in the MLR assay) than the combination of the corresponding LAG-3 mAb (57E02) and PD-1 dAb (in mAbdAb format with an anti-RSV mAb; RSV-51A09-188001).
  • Other PD-l/LAG-3 mAbdAbs were made, some of which shared the same PD-1 dAb epitope binding domain.
  • the 57E02x51A09-188001 mAbdAb (which showed greater improvements in T cell activation that the combination of the component binding proteins) exhibited the best pharmacokinetics (lowest clearance) in macaques before anti-drug antibodies developed, subsequently resulting in rapid clearance (the anti-drug antibodies were expected as the antibody administered to the macaque is a human antibody that would be recognised as foreign by the macaque).
  • the invention provides a binding protein specific to human PD-1 that comprises one or more of CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: CDRH1 as present in SEO ID NO:3 and CDRH1 that differs from the CDRH1 present in SEO ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids, wherein CDRH2 is selected from the group consisting of: CDRH2 as present in SEO ID NO:3 and CDRH2 that differs from the CDRH2 present in SEO ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids, and wherein CDRH3 is selected from the group consisting of: CDRH3 as present in SEO ID NO: 3 and CDRH3 that differs from the CDRH3 present in SEO ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids.
  • the binding protein specific to human PD-1 exhibits an IC50 of less than or equal to 5
  • the binding protein specific to human PD1 is a single variable domain of an antibody which retains its tertiary structure in the absence of other antibody domains (in other words the protein scaffold is an immunoglobulin scaffold).
  • This single variable domain may be capable of binding human PD1 independently of a different variable region or domain.
  • Single variable domains that retain their tertiary structure and ability to bind human PD-1 in the absence of other antibody domains are also referred to as domain antibodies or dAbs.
  • the single variable domain may be a complete antibody variable domain such as VH, VHH and VL or a modified domain which has been truncated or contains N or C-terminal extensions, or in which one or more loops have been replaced by sequences which are not characteristic of the domain in question.
  • the single variable domain may be a VH domain or a modified VH domain which has been truncated or contains N or C- terminal extensions, or in which one or more loops have been replaced by sequences which are not characteristic of the domain in question.
  • the binding protein specific to human PD-1 has a non- immunoglobulin scaffold having loops connecting elements of secondary structure which can be engineered to include CDR regions.
  • Non immunoglobulin scaffolds include CTLA-4 (Evibodies; Journal Immunological Methods 248(1-2): 31-45, 2001), lipocalin, Protein A derived molecules such as Z-domain of Protein A (Affibodies, Protein Eng Des Sel 17: 455- 462, 2004 and EP1641818), A-domain (Avimer/Maxibody), heat shock proteins such as GroEI and GroES, transferrin (trans-body), ankyrin repeat protein (DARPin), peptide aptamer, C- type lectin domain (Tetranectin), human ⁇ -crystallin and human ubiquitin (affilins), PDZ domains, scorpion toxin kunitz-type domains of human protease inhibitors, and
  • fibronectin/adnectin These scaffolds are subjected to protein engineering to arrange the CDRs in a functional manner (for a summary of alternative antibody formats see Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No 9, 1126-1136).
  • the binding protein specific to human PD-1 has the CDR sequences listed above for any particular embodiment of the epitope binding domain specific for PD-1.
  • the binding protein specific to human PD-1 contains one or more CDR variants
  • the binding protein specific to human PD-1 containing one or more CDR variants retains biological activity, defined as an IC50 of less than or equal to 5 nM in the PD- 1/PDL-l competition assay.
  • the binding protein specific to human PD-1 comprises the sequence defined as SEQ ID: NO.
  • SEQ ID NO:3 or a variant of SEQ ID NO:3 that differs in having up to ten amino acid additions, deletions or substitutions, or a sequence that has at least 90% sequence identity to the sequence of SEQ. ID NO: 3, as described above in relation to the corresponding embodiments of the epitope binding domain specific to human PD-1.
  • the binding protein specific to human PD-1 is additionally capable of neutralising a human checkpoint inhibitor other than PD-1 such that the binding protein is specific for human PD-1 and another checkpoint inhibitor.
  • neutralisation of a human checkpoint inhibitor refers to blockade of the biological activity of the said human checkpoint inhibitor. Because the biological activity of a human checkpoint inhibitor is to inhibit signalling cascades that modulate the activation, proliferation or cytokine production of an immune cell following stimulation, neutralisation of the human checkpoint inhibitor increases or restores the biological activity of the immune cell following
  • Cytokine production post stimulation of an immune cell is complicated, with the expression of certain cytokines being increased and others decreased, in a manner that depends upon the type of immune cell and the nature of the stimulation.
  • the effects of particular stimulants on cytokine production in particular cell types is well documented and known in the art (e.g. it is known that IL2, IFNy and TNFa are produced by CD4+ and CD8+ T cells following stimulation by bacterial toxins such as the staphylococcal enterotoxins SEB/SEA).
  • Neutralisation of a checkpoint inhibitor would lead to those cytokines normally upregulated upon stimulation being produced, and to reduced expression of those cytokines normally downregulated upon stimulation.
  • cytokine production may be measured by ELISA, ELISPOT or by intracellular staining of cells and acquisition on a flow cytometer. These are standard techniques and some suitable protocols are outlines in Chapter 24, HIV Protocols, Second Ed., vol. 485, 2009. ELISAs can be used to measure the levels of cytokines released from immune cells. They are often performed on blood samples or the supernatants from cultured cells. ELISPOT assays can be performed on mononuclear cells or CD4+ or CD8+ T cell populations whilst cytokine flow cytometry is of course capable of providing separate information on CD4+ and CD8+ cells when mixed cell populations (e.g. peripheral blood mononuclear cells) are used.
  • mixed cell populations e.g. peripheral blood mononuclear cells
  • an increase in cytokine production refers to the situation where the mean level of the measured cytokine determined from at least three samples following stimulation is higher in the presence of the binding protein than in the absence of the binding protein.
  • an increase in cytokine production refers to the situation where the mean level of the measured cytokine determined from at least three samples following stimulation in the presence of binding protein is greater than or equal to the mean level of the measured cytokine determined from at least three samples in the absence of binding protein plus one standard deviation (using the larger of the standard deviations taken in the presence or absence of binding protein).
  • an increase in cytokine production refers to the situation where the mean level of the measured cytokine determined from at least three samples following stimulation in the presence of binding protein is greater than or equal to the mean level of the measured cytokine determined from at least three samples in the absence of binding protein plus two standard deviations (using the larger of the standard deviations taken in the presence or absence of binding protein).
  • an increase in cytokine production refers to the situation where the mean level of the measured cytokine determined from at least three samples following stimulation in the presence of binding protein is greater than or equal to the mean level of the measured cytokine determined from at least three samples in the absence of binding protein plus three standard deviations (using the larger of the standard deviations taken in the presence or absence of binding protein).
  • a reduction in cytokine production refers to the situation where the mean level of the measured cytokine determined from at least three samples following stimulation in the presence of binding protein is 3, 20 or 50 fold higher than the mean level of the measured cytokine determined from at least three samples in the absence of binding protein.
  • a reduction in cytokine production refers to the situation where the mean level of the measured cytokine determined from at least three samples following stimulation is lower in the presence of the binding protein than in the absence of the binding protein.
  • a reduction in cytokine production refers to the situation where the mean level of the measured cytokine determined from at least three samples following stimulation in the presence of binding protein is less than or equal to the mean level of the measured cytokine determined from at least three samples in the absence of binding protein minus one standard deviation (using the larger of the standard deviations taken in the presence or absence of binding protein).
  • a reduction in cytokine production refers to the situation where the mean level of the measured cytokine determined from at least three samples following stimulation in the presence of binding protein is less than or equal to the mean level of the measured cytokine determined from at least three samples in the absence of binding protein minus two standard deviations (using the larger of the standard deviations taken in the presence or absence of binding protein).
  • a reduction in cytokine production refers to the situation where the mean level of the measured cytokine determined from at least three samples following stimulation in the presence of binding protein is less than or equal to the mean level of the measured cytokine determined from at least three samples in the absence of binding protein minus three standard deviations (using the larger of the standard deviations taken in the presence or absence of binding protein).
  • a reduction in cytokine production refers to the situation where the mean level of the measured cytokine determined from at least three samples following stimulation in the presence of binding protein is 10%, 20% or 50% lower than the mean level of the measured cytokine determined from at least three samples in the absence of binding protein.
  • blockade of a human checkpoint inhibitor should increase proliferation of immune cells.
  • Proliferation following stimulation in vitro may be measured by a technique that uses carboxy fluorescein diacetate succinimidyl ester (CSFE), a cell permeable dye which allows monitoring of cell division by flow cytometry essentially as described in Chapter 19, Methods in Cell Biology, Vol. 75, 2004.
  • CSFE carboxy fluorescein diacetate succinimidyl ester
  • an increase in proliferation refers to the situation where the mean percentage of immune cells that had divided (calculated by determining the percentage that had diluted CSFE after subtracting background division, namely the percentage of cells that divided in the negative control) determined from at least three samples is higher in the presence of binding protein than in the absence of binding protein.
  • an increase in proliferation refers to the situation where the mean percentage of cells that had divided determined from at least three samples in the presence of binding protein is equal to or higher than the mean percentage of cells that had divided determined from at least three samples in the absence of binding protein plus one standard deviation (the larger of the standard deviations taken in the presence or absence of binding protein being used).
  • an increase in proliferation refers to the situation where the mean percentage of cells that had divided determined from at least three samples in the presence of binding protein is equal to or higher than the mean percentage of cells that had divided determined from at least three samples in the absence of binding protein plus two standard deviations (the larger of the standard deviations taken in the presence or absence of binding protein being used).
  • an increase in proliferation refers to the situation where the mean percentage of cells that had divided determined from at least three samples in the presence of binding protein is equal to or higher than the mean percentage of cells that had divided determined from at least three samples in the absence of binding protein plus three standard deviations (the larger of the standard deviations taken in the presence or absence of binding protein being used).
  • an increase in proliferation refers to the situation where the mean percentage of cells that had divided determined from at least three samples in the presence of binding protein is 1.2, 1.5, 5, or 20-fold greater than the mean percentage of cells that had divided in the absence of the binding protein determined from at least three samples at the beginning of the treatment period.
  • Activation of immune cells is normally inhibited by human checkpoint inhibitors.
  • blockade of a human checkpoint inhibitor should result in increased activation.
  • the activation of immune cells is routinely monitored by measuring changes in transcription factors or the upregulation of cell surface proteins. It will be appreciated that the profile of transcription factors and cell surface proteins following activation will differ dependent upon the nature of the immune cell. However, changes in transcription factors and cell surface protein expression upon activation in particular cell types are well documented and known in the art.
  • Changes in transcription factors can be monitored in vitro (following stimulation known to induce activation) or in vivo. Changes to transcription factors such as AKT, NFAT or NFkB can be monitored using standard protein techniques such as a western blot or using reporter gene systems. Luciferase reporter systems are commonly used where T, B or monocyte cell lines are engineered with luciferase expression under the control of the relevant transcription factor promoter. In the Examples, the ability of binding protein to produce a dose dependent increase in NFAT driven luciferase expression was measured in PD-1 expressing Jurkat T cells.
  • an increase in activation refers to the situation where the mean level of the transcription factor, its activity or downstream reporter gene product determined from at least three samples is higher in the presence of the binding protein than in the absence of the binding protein. In one embodiment, an increase in activation refers to the situation where the mean level of the transcription factor, its activity or downstream reporter gene product determined from at least three samples in the presence of binding protein is greater than or equal to the corresponding value determined from at least three samples in the absence of binding protein plus one standard deviation (using the larger of the standard deviations taken in the presence or absence of binding protein).
  • an increase in activation refers to the situation where the mean level of the transcription factor, its activity or downstream reporter gene product determined from at least three samples in the presence of binding protein is greater than or equal to the corresponding value determined from at least three samples in the absence of binding protein plus two standard deviations (using the larger of the standard deviations taken in the presence or absence of binding protein).
  • an increase in activation refers to the situation where the mean level of the transcription factor, its activity or downstream reporter gene product determined from at least three samples in the presence of binding protein is greater than or equal to the corresponding value determined from at least three samples in the absence of binding protein plus three standard deviations (using the larger of the standard deviations taken in the presence or absence of binding protein).
  • an increase in activation refers to the situation where the mean level of the transcription factor or reporter gene product determined from at least three samples in the presence of binding protein is 1.5, 15 or 50 fold higher than the corresponding value determined from at least three samples in the absence of binding protein.
  • a decrease in activation refers to the situation where the mean level of the transcription factor, its activity or downstream reporter gene product determined from at least three samples is lower in the presence of the binding protein than in the absence of the binding protein. In one embodiment, a decrease in activation refers to the situation where the mean level of the transcription factor, its activity or downstream reporter gene product determined from at least three samples in the presence of binding protein is less than or equal to the corresponding value determined from at least three samples in the absence of binding protein minus one standard deviation (using the larger of the standard deviations taken in the presence or absence of binding protein).
  • a decrease in activation refers to the situation where the mean level of the transcription factor, its activity or downstream reporter gene product determined from at least three samples in the presence of binding protein is less than or equal to the corresponding value determined from at least three samples in the absence of binding protein minus two standard deviations (using the larger of the standard deviations taken in the presence or absence of binding protein). Even more particularly, a decrease in activation refers to the situation where the mean level of the transcription factor, its activity or downstream reporter gene product determined from at least three samples in the presence of binding protein is less than or equal to the corresponding value determined from at least three samples in the absence of binding protein minus three standard deviations (using the larger of the standard deviations taken in the presence or absence of binding protein). In one embodiment a decrease in activation refers to the situation where the mean level of the transcription factor or reporter gene product determined from at least three samples in the presence of binding protein is 10%, 20% or 50% lower than the corresponding value determined from at least three samples in the absence of binding protein.
  • activation is measured by measuring changes in production, activation or phosphorylation status of particular transcription factors, it is apparent that how activation is assessed depends upon whether the transcriptional factor in question is normally expressed in activated or non-activated immune cells.
  • cell surface proteins are upregulated on activated immune cells; these include CD69 on T cells, B cells and NK cells, CD80 and CD86 on antigen presenting cells, as well as HLADR and CD38 on T cells. Expression of these cell surface proteins (or activation markers) on mononuclear cells may be monitored using flow cytometry.
  • an increase in immune cell activation refers to the situation where either mean frequency of immune cells expressing particular activation markers or levels of expression of said markers determined from at least three samples is increased in the presence of the binding protein than in the absence of the binding protein
  • an increase in activation refers to the situation where either the mean frequency of immune cells expressing particular activation markers from at least three samples in the presence of binding protein, or where levels of expression of said markers determined from at least three samples in the presence of binding protein is increased compared to the corresponding value determined from at least three samples in the absence of binding protein plus one standard deviation (the larger of the standard deviations taken in the presence or absence of binding protein being used).
  • an increase in activation refers to the situation where either the mean frequency of immune cells expressing particular activation markers determined from at least three samples in the presence of binding protein, or where levels of expression of said markers determined from at least three samples in the presence of binding protein is equal to or higher than the corresponding value determined from at least three samples in the absence of binding protein plus one standard deviation (the larger of the standard deviations taken in the presence or absence of binding protein being used).
  • an increase in activation or differentiation refers to the situation where either the mean frequency of immune cells expressing particular activation markers determined from at least three samples in the presence of binding protein, or where levels of expression of said markers determined from at least three samples in the presence of binding protein is equal to or higher than the corresponding value determined from at least three samples in the absence of binding protein plus two standard deviations (the larger of the standard deviations taken in the presence or absence of binding protein being used).
  • an increase in activation refers to the situation where either the mean frequency of immune cells expressing particular activation markers determined from at least three samples in the presence of binding protein, or where levels of expression of said markers determined from at least three samples in the presence of binding protein is equal to or higher than the corresponding value determined from at least three samples in the absence of binding protein plus three standard deviations (the larger of the standard deviations taken in the presence or absence of binding protein being used).
  • an increase in activation refers to the situation where either the mean frequency of immune cells expressing particular activation markers determined from at least three samples in the presence of binding protein, or where levels of expression of said markers determined from at least three samples in the presence of binding protein is increased by 1.5 fold, 5 fold, 10 fold, 20 fold or 50 fold compared to the corresponding value determined from at least three samples in the absence of binding protein.
  • the human checkpoint inhibitor other than PD-1 is selected from the group consisting of CTLA-4, TIM-3, CD160 and TIGIT.
  • the binding protein comprises a domain specific for binding human PD-1 (which may be that described in any of the foregoing embodiments) attached by a linker to one or more domains specific for a human checkpoint inhibitor other than PD-1.
  • a domain specific for binding human PD-1 is attached by a linker to the C terminus of the heavy chain of an antibody specific for a human checkpoint inhibitor other than PD-1 (in this context an antibody is defined as an immunoglobulin comprising two heavy and two light chains, wherein each heavy chain comprises a variable region and one or more constant regions and where each light chain comprises a variable region and a constant region).
  • an antibody is defined as an immunoglobulin comprising two heavy and two light chains, wherein each heavy chain comprises a variable region and one or more constant regions and where each light chain comprises a variable region and a constant region).
  • there are two binding domains specific for PD-1 one attached to the C-terminus of each of the two heavy chains of the antibody specific for a human checkpoint inhibitor other than PD-1.
  • the linker may be a bond (such as a peptide bond) or a peptide linker.
  • the linker is a peptide linker from 1 to 100 amino acids in length, more particularly, from 1 to 50 or from 1 to 10 amino acids in length. Suitable linkers are described in published PCT patent application WO2010/136483.
  • the linker has the sequence set out as SEQ. ID NO. 30 (STGLDSPT).
  • STGLDSPT SEQ. ID NO. 30
  • the invention provides a binding protein having the general formula (II):
  • H(CPI) is an antibody heavy chain of the IgG class and L(CPI) is an antibody light chain of the IgG class; such that H(CPI) and L(CPI) together form an antibody specific for a human checkpoint inhibitor other than PD-1; n is an integer selected from 2, 4 and 11; A is a bond or a peptide linker; and
  • VH(PD-I) is an antibody heavy chain variable domain having CDRHl, CDRH2 and CDRH3, wherein said CDRHl is selected from: CDRHl present in SEQ ID NO:3, and CDRHl that differs from the CDRHl present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids; wherein CDRH2 is selected from: CDRH2 present in SEQ ID NO:3 and CDRH2 that differs from the CDRH2 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRH3 is selected from: CDRH3 as present in SEQ ID NO: 3 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids.
  • the binding protein of formula (II) is a binding protein specific for human PD-1 and another checkpoint inhibitor.
  • the binding protein of formula (II) exhibits an IC50 of less than or equal to 5 nM in the PD-l/PDL-1 competition assay and exhibits neutralisation of the human checkpoint inhibitor other than PD-1.
  • H(CPI) comprises the VH and CHI domains and L(CPI) comprises the CL and VL domains.
  • H(CPI) comprises the CHI, CH2, and VH domains and L(CPI) comprises the CL and VL domains.
  • H(CPI) contains the CHI, CH2, CH3 and VH domains and L(CPI) contains the CL and VL domains.
  • the CHI, CH2, CH3, VH, CL and VL domains referred to above may be complete domains or modified domains which have been truncated or contain N or C-terminal extensions, or in which one or more loops have been replaced by sequences which are not characteristic of the domain in question.
  • Other modifications are also included, for example, the addition of other post-translational modifications such as phosphorylation, deamidation, oxidation, disulphide bond scrambling, isomerisation, C-terminal lysine clipping and N-terminal glutamine cyclisation or the inclusion of one or more non-natural amino acids.
  • antibodies are glycoproteins and the domains above may be
  • H(CPI) is a heavy chain of the IgGl, lgG2, lgG3, lgG4 or lgG4PE subclasses.
  • n is 2.
  • H(CPI) is of the lgG2 subclass
  • n is 4.
  • H(CPI) is of the lgG3 subclass
  • n is 11.
  • the antibody is selected from the IgGl(PE) subclass, even more particularly, the antibody has an IgGl disabled isotype (LAGA).
  • VH(PD-I) is as defined above in any of the embodiments relating to a binding protein of formula (I).
  • the binding protein of formula (II) containing one or more CDR variants in VH(PD-I) retains biological activity, defined as an IC50 of less than or equal to 5 nM in the PD-l/PDL-1 competition assay.
  • A is as defined above in any of the embodiments relating to a binding protein of formula (I).
  • A is a peptide bond or a peptide linker from 1 to 100 amino acids in length.
  • A is a peptide linker from 1 to 100 amino acids in length, more particularly, from 1 to 50 or from 1 to 10 amino acids in length.
  • Suitable linkers are described in published PCT patent application WO2010/136483.
  • the linker has the sequence set out as SEQ. ID NO. 30 (STG LDSPT).
  • STG LDSPT SEQ. ID NO. 30
  • This CTLA4/PD1 bispecific also facilitates cytokine production in the mixed lymphocyte reaction assay.
  • 16 variant dAbs having related CDRs to 51A09-188001 also block PD-1 signalling when formatted as a mAbdAb with an anti-RSV mAb, as determined by a reporter gene assay. It is reasonable to expect that these and other variants with related CDRs would be capable of blocking both PD-1 signalling when formatted as a mAbdAb of formula (II) with other checkpoint inhibitors. Changes to the framework regions would also be expected to be tolerated.
  • the invention provides a binding protein specific to human LAG-3, which comprises: one or more of CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: CDRH1 as present in SEQ ID NO:l and CDRH1 that differs from the CDRH1 present in SEQ.
  • CDRH2 is selected from the group consisting of: CDRH2 as present in SEQ ID NO:l and CDRH2 that differs from the CDRH2 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids
  • CDRH3 is selected from the group consisting of: CDRH3 as present in SEQ ID NO: 1 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids
  • CDRL1, CDRL2 and CDRL3 wherein CDRL1 is selected from the group consisting of: CDRL1 as present in SEQ ID NO:2 and CDRL1 that differs from the CDRL1 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids
  • CDRL2 is selected from the group consisting of: CDRL2 as present in SEQ ID NO:2 and CDRL
  • the binding protein specific to human LAG-3 exhibits >50% inhibition of LAG3-MHCII interaction in a competition flow cytometry assay.
  • the binding protein specific to human LAG-3 is an antibody.
  • an antibody is a heterotetrameric glycoprotein of the IgA or IgG classes that is composed of two heavy chains and two light chains, wherein the heavy chains comprise the VH and CHI domains, and the light chains comprise the VL and CL domains. More particularly, the antibody is of the IgG class.
  • the antibody is selected from the IgGl, lgG2, lgG3, lgG4 and lgG4PE subclasses. In a more particular embodiment, the antibody is selected from the IgGl subclass, even more particularly, the antibody has an IgGl disabled isotype (LAGA).
  • an antibody is a glycoprotein of the IgA or IgG classes that is composed of two heavy chains and two light chains, wherein the heavy chains comprise the VH and CHI domains, and the light chains comprise the VL and CL domains. More particularly, the antibody is of the IgG class. In one embodiment, the antibody is selected from the IgGl, lgG2, lgG3, lgG4 and lgG4PE subclasses. In a more particular embodiment, the antibody is selected from the IgGl subclass, even more particularly, the antibody has an IgGl disabled isotype (LAGA).
  • LAGA IgGl disabled isotype
  • the heavy chains comprise the CHI, CH2, and VH domains and the light chains comprise the CL and VL domains.
  • the heavy chains contain the CHI, CH2, CH3 and VH domains and the light chains contain the CL and VL domains.
  • the CHI, CH2, CH3, VH, CL and VL domains referred to above may be complete domains or modified domains which have been truncated or contain N or C-terminal extensions, or in which one or more loops have been replaced by sequences which are not characteristic of the domain in question.
  • Other modifications are also included, for example, the addition of other post-translational modifications such as phosphorylation, deamidation, oxidation, disulphide bond scrambling, isomerisation, C-terminal lysine clipping and N-terminal glutamine cyclisation or the inclusion of one or more non-natural amino acids.
  • antibodies are glycoproteins and the domains above may be
  • the binding protein specific to human LAG-3 has a non- immunoglobulin scaffold having loops connecting elements of secondary structure which can be engineered to include CDR regions.
  • Non immunoglobulin scaffolds include CTLA-4 (Evibodies; Journal Immunological Methods 248(1-2): 31-45, 2001), lipocalin, Protein A derived molecules such as Z-domain of Protein A (Affibodies, Protein Eng Des Sel 17: 455- 462, 2004 and EP1641818), A-domain (Avimer/Maxibody), heat shock proteins such as GroEI and GroES, transferrin (trans-body), ankyrin repeat protein (DARPin), peptide aptamer, C- type lectin domain (Tetranectin), human ⁇ -crystallin and human ubiquitin (affilins), PDZ domains, scorpion toxin kunitz-type domains of human protease inhibitors, and
  • fibronectin/adnectin These scaffolds are subjected to protein engineering to arrange the CDRs in a functional manner (for a summary of alternative antibody formats see Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No 9, 1126-1136).
  • the binding protein specific to human LAG-3 has the CDR sequences listed above for any particular embodiment of the antibody specific for human LAG-3 present in the binding protein specific for human PD-1 and human LAG-3.
  • the binding protein specific for human LAG-3 contains one or more CDR variants
  • the binding protein specific human LAG-3 containing one or more CDR variants retains biological activity, defined as >50% inhibition of LAG3-MHCII interaction in a competition flow cytometry assay.
  • the binding protein specific to human LAG-3 is an antibody comprising two identical heavy chains comprising the sequence defined as SEQ ID NO.l or a variant of SEQ ID NO. 1 that differs in having up to 10 amino acid additions, deletions or substitutions, and/or an antibody comprising two identical light chains comprising the sequence defined as SEQ. ID NO.2 or a variant of SEQ ID NO. 2 that differs in having up to 10 amino acid additions, deletions or substitutions as described above in relation to the corresponding embodiments of the antibody specific for human LAG-3 described above.
  • the binding protein specific to human LAG-3 is an antibody having two identical heavy chains comprising the sequence defined as SEQ ID NO.l or a sequence that has at least 90% sequence identity to the sequence of SEQ ID NO. 1 and/or two identical light chains comprising the sequence defined as SEQ ID NO.2 or a sequence that has at least 90% sequence identity to the sequence of SEQ ID NO. 2 as described above in relation to the corresponding embodiments of the antibody specific for human LAG-3 present in the binding protein specific for human PD-1 and human LAG-3 described above.
  • the binding protein specific to human LAG-3 is additionally capable of neutralising a human checkpoint inhibitor other than LAG-3.
  • a human checkpoint inhibitor other than LAG-3 As defined above,
  • neutralisation of a human checkpoint inhibitor refers to blockade of the biological activity of the said human checkpoint inhibitor. Because the biological activity of a human checkpoint inhibitor is to inhibit signalling cascades that modulate the activation, proliferation or cytokine production of an immune cell following stimulation, neutralisation of the human checkpoint inhibitor increases or restores the biological activity of the immune cell following stimulation. Methods of measuring activation, proliferation or cytokine production of immune cells enabling the assessment of neutralisation are outlined above (in relation to neutralisation of a human checkpoint inhibitor other than PD-1).
  • the human checkpoint inhibitor other than LAG-3 is selected from the group consisting of CTLA-4, TIM-3, CD160 and TIGIT.
  • the binding protein comprises an antibody specific for binding human LAG-3 (which may be that described in any of the foregoing embodiments) attached by a linker to one or more domains specific to a human checkpoint inhibitor other than LAG-3.
  • a domain specific for binding a further human checkpoint inhibitor is attached by a linker to the C terminus of the heavy chain of an antibody specific for binding human LAG-3.
  • the domains specific for binding a further human checkpoint inhibitor may be single variable domains of an antibody that retains their tertiary structure independent of the rest of the binding protein and which are capable of binding the further checkpoint inhibitor independently of a different variable region or domain (i.e. the domain specific for binding a further checkpoint inhibitor may be a domain antibody or dAb).
  • the term epitope binding domain specific to human PD1 refers to a folded protein structure which retains its tertiary structure independent of the rest of the binding protein.
  • the epitope binding domain specific to human PD1 has CDRs permitting the binding protein to bind human PD-1.
  • the epitope binding domain specific to human PD1 has CDRs permitting the binding protein to exhibit an IC50 of less than or equal to 5 nM in the PD- 1/PDL-l competition assay.
  • the epitope binding domain specific to human PD1 is a single variable domain of an antibody (in other words the protein scaffold is an immunoglobulin scaffold). This single variable domain may be capable of binding human PD1 independently of a different variable region or domain.
  • the linker may be a bond (such as a peptide bond) or a peptide linker.
  • the linker is a peptide linker from 1 to 100 amino acids in length, more particularly, from 1 to 50 or from 1 to 10 amino acids in length. Suitable linkers are described in published PCT patent application WO2010/136483.
  • the linker has the sequence set out as SEQ. ID NO. 30 (STGLDSPT).
  • STGLDSPT SEQ. ID NO. 30
  • the linker is a peptide bond or a peptide linker
  • the one or more domains specific for binding a further checkpoint inhibitor are expressed as a genetic fusion with one of the chains of the antibody specific to LAG-3.
  • the invention provides a binding protein having the general formula (III):
  • H(LAG-3) is an antibody heavy chain of the IgG class comprising CDRHl, CDRH2 and CDRH3, wherein said CDRHl is selected from: CDRHl present in SEQ ID NO:l, and CDRHl that differs from the CDRHl present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids; wherein CDRH2 is selected from: CDRH2 present in SEQ ID NO:l and CDRH2 that differs from the CDRH2 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRH3 is selected from: CDRH3 as present in SEQ ID NO: 1 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids;
  • L(LAG-3) is an antibody light chain of the IgG class comprising CDRL1 and CDRL2, wherein said CDRL1 is selected from: CDRL1 present in SEQ ID NO:2 and CDRL1 that differs from the CDRL1 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRL2 is selected from: CDRL2 present in SEQ ID NO:2 and CDRL2 that differs from the CDRL2 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids; n is an integer selected from 2, 4 and 11;
  • A is a bond or a peptide linker
  • CPI is a domain that is capable of binding a human checkpoint inhibitor other than LAG-3.
  • the binding protein of formula (III) is a binding protein specific to human LAG-3 and another checkpoint inhibitor.
  • the binding protein of formula (III) exhibits >50% inhibition of LAG3- MHCII interaction in competition flow cytometry assay, and exhibits neutralisation of the human checkpoint inhibitor other than LAG-3.
  • H(LAG-3) and L( LAG-3) are as defined above in any of the embodiments relating to a binding protein of formula (I).
  • the binding protein of formula (III) containing one or more CDR variants in H(LAG-3) and L(LAG-3) retains biological activity, defined as exhibiting >50% inhibition of LAG3-MHCII interaction in competition flow cytometry assay.
  • CPI is a single variable domain that may be a complete antibody variable domain such as VH, VHH and VL or a modified domain which has been truncated or contains N or C-terminal extensions, or in which one or more loops have been replaced by sequences which are not characteristic of the domain in question.
  • Other modifications are also included, for example, the addition of other post-translational modifications such as phosphorylation,
  • CPI is single variable domain that retains its tertiary structure independent of the rest of the binding protein of formula (III) and which is capable of binding the further checkpoint inhibitor independently of a different variable region or domain.
  • CPI may comprise a non-immunoglobulin scaffold having loops connecting elements of secondary structure which can be engineered to include CDR regions.
  • Non immunoglobulin scaffolds include CTLA-4 (Evibodies; Journal Immunological Methods 248(1-2): 31-45, 2001), lipocalin, Protein A derived molecules such as Z-domain of Protein A (Affibodies, Protein Eng Des Sel 17: 455-462, 2004 and EP1641818), A-domain (Avimer/Maxibody), heat shock proteins such as GroEI and GroES, transferrin (trans-body), ankyrin repeat protein (DARPin), peptide aptamer, C-type lectin domain (Tetranectin), human ⁇ -crystallin and human ubiquitin (affilins), PDZ domains, scorpion toxin kunitz-type domains of human protease inhibitors, and fibronectin/adnectin.
  • CTLA-4 Evibodies; Journal Immunological Methods
  • A is as defined above in any of the embodiments relating to a binding protein of formula (I).
  • A is a peptide bond or a peptide linker from 1 to 100 amino acids in length.
  • A is a peptide linker from 1 to 100 amino acids in length, more particularly, from 1 to 50 or from 1 to 10 amino acids in length.
  • Suitable linkers are described in published PCT patent application WO2010/136483.
  • the linker has the sequence set out as SEQ. ID NO. 30 (STGLDSPT).
  • STGLDSPT SEQ. ID NO. 30
  • PD-1 and/or LAG-3 blockade reduces immunosuppression and reverses T-ce 11 exhaustion and is believed to be useful in the treatment of infectious diseases and cancer.
  • the invention provides a method of treating infectious diseases which comprises administering to a human in need thereof a therapeutically effective amount of the binding protein of the invention.
  • infectious diseases that may be treated include bacterial infections (such as tuberculosis and infection with Listeria sp., Streptococcus sp. and Salmonella sp.), parasitic infections (such as malaria and
  • viral infections including respiratory infections with, for example influenza virus, respiratory syncytial virus or parainfluenza virus, and chronic viral infections including infection with hepatitis B virus, hepatitis C virus, cytomegalovirus, herpes simplex virus, human papillomavirus, ebola virus, epstein barr virus and human immunodeficiency virus) and sepsis.
  • latently infected CD4 T cells express LAG-3 and PD-1 and inhibiting the interaction between PD-1 and PD-L1/2 has been shown to enhance the production of HIV by CD4+ cells. This may make the viral reservoir (cells infected with HIV) "visible" to the immune system.
  • LAG-3 and PD-1 blockade has been shown increase the HIV specific CD8+ T cell response. This may result in immune clearance of the now visible viral reservoir. This approach to the depletion of the viral reservoir is commonly referred to as "kick and kill” (kicking the viral reservoir into expressing viral antigens followed by killing - immune clearance - of the infected cells).
  • the invention provides a method of curing HIV which comprises administering to a human in need thereof a therapeutically effective amount of the binding protein of the invention.
  • a treatment period is anticipated to be between 3-12 months.
  • curing HIV refers to e.g. inducing and maintaining sustained viral control (undetectable levels of plasma viremia as measured by an assay capable of detecting a single copy of HIV type-1 RNA in 1 ml plasma) of human immunodeficiency virus for a minimum of two years in the absence of any therapy. Any assay having suitable sensitivity may be used. In one embodiment, the Single Copy Assay described in Palmer et al. (J. Clin. Microbiol., 2003, 41(10): 4531-4536) is used. In other words, following the treatment period, no HIV is detectable in the plasma for a period of at least two years, during which period there is no other anti-HIV therapy administered.
  • the invention provides a method of treating HIV which comprises administering to a human in need thereof a therapeutically effective amount of the binding protein of the invention.
  • HIV treatment with the binding protein of the invention may result in a reduction in the incidence of several non-AIDS morbidities and mortalities.
  • HIV treatment with the binding protein of the invention may result in reduction in inflammation, reduction of the reservoir, or an increase in HIV specific T cell function during the course of treatment.
  • treatment with the binding protein may be ongoing, for the purposes of assessing reduction in inflammation, reduction of the reservoir or an increase in specific T cell function, a treatment period must be specified.
  • HIV treatment refers to treatment resulting in a reduction in inflammation over the treatment period.
  • the treatment period could be a period between 3-12 months.
  • a reduction in inflammation can be determined by reduction in levels of activated monocytes as measured by levels of soluble CD163 or soluble CD14, by a reduction in expression of key inflammatory markers of cardiovascular disease (CVD) risk, or by a reduction in vascular inflammation.
  • Markers of CVD risk include high-sensitivity C- reactive protein (hsCRP), interleukin-6 (IL-6), and D-dimer.
  • vascular inflammation levels can be measured by arterial fluorodeoxyglucose (FDG) uptake.
  • FDG arterial fluorodeoxyglucose
  • monocytes shed the scavenger receptor CD163 into the plasma as well as CD14 a receptor involved in sensing bacterial products.
  • the levels of soluble CD163 or soluble CD14 can be measured by ELISA in the plasma.
  • a reduction in the level of soluble CD163 or soluble CD14 refers to the situation where the mean level of soluble CD163 or soluble CD14 determined from at least three plasma samples is lower at the end of the treatment period than at the beginning.
  • a reduction in the level of soluble CD163 or soluble CD14 refers to the situation where the mean level of soluble CD163 or soluble CD14 determined from at least three plasma samples at the end of the treatment period is less than or equal to the mean level of soluble CD163 or soluble CD14 at the beginning of the treatment period minus one standard deviation (the larger of the pre-treatment/post-treatment standard deviations being used).
  • a reduction in the level of soluble CD163 or soluble CD14 refers to the situation where the mean level of soluble CD163 or soluble CD14 determined from at least three plasma samples at the end of the treatment period is less than or equal to the mean level of soluble CD163 or soluble CD14 at the beginning of the treatment period minus two standard deviations (the larger of the pre-treatment/post-treatment standard deviations being used).
  • a reduction in the level of soluble CD163 or soluble CD14 refers to the situation where the mean level of soluble CD163 or soluble CD14 determined from at least three plasma samples at the end of the treatment period is less than or equal to the mean level of soluble CD163 or soluble CD14 at the beginning of the treatment period minus three standard deviations (the larger of the pre-treatment/post- treatment standard deviations being used).
  • a reduction in the level of soluble CD163 or soluble CD14 refers to the situation where the mean level of soluble CD163 or soluble CD14 determined from at least three plasma samples is 2 fold lower at the end of the treatment period than at the beginning.
  • a reduction in the level of soluble CD163 or soluble CD14 refers to the situation where the mean level of soluble CD163 or soluble CD14 determined from at least three plasma samples is 5 fold lower at the end of the treatment period than at the beginning.
  • HIV treatment refers to a reduction in expression of high-sensitivity C- reactive protein (hsCRP), interleukin-6 (IL-6), or D-dimer.
  • hsCRP high-sensitivity C- reactive protein
  • IL-6 interleukin-6
  • D-dimer D-dimer
  • markers of CVD risk may be measured in any biological sample.
  • levels of these markers are measured in plasma by any suitable method.
  • a reduction in the level of a marker of CVD risk refers to the situation where the mean level of the marker determined from at least three samples at the end of the treatment period is less than or equal to the mean level of the marker at the beginning.
  • a reduction in the level of a marker of CVD risk refers to the situation where the mean level of the marker determined from at least three samples at the end of the treatment period is less than or equal to the mean level of the marker at the beginning of the treatment period minus one standard deviation (the larger of the pre-treatment/post-treatment standard deviations being used).
  • a reduction in the level of a marker of CVD risk refers to the situation where the mean level of the marker determined from at least three samples at the end of the treatment period is less than or equal to the mean level of the marker at the beginning of the treatment period minus two standard deviations (the larger of the pre- treatment/post-treatment standard deviations being used).
  • a reduction in the level of a marker of CVD risk refers to the situation where the mean level of the marker determined from at least three samples at the end of the treatment period is less than or equal to the mean level of the marker at the beginning of the treatment period minus three standard deviations (the larger of the pre-treatment/post-treatment standard deviations being used).
  • a reduction in the level of a marker of CVD risk refers to the situation where the mean level of the marker determined from at least three samples at the end of the treatment period is 2 fold lower at the end of the treatment period than at the beginning.
  • a reduction in the level of a marker of CVD risk refers to the situation where the mean level of the marker determined from at least three samples at the end of the treatment period is 5 fold lower at the end of the treatment period than at the beginning.
  • HIV treatment refers to a reduction in vascular inflammation as measured by a reduction in arterial fluorodeoxyglucose (FDG) uptake.
  • FDG uptake may be assessed by FDG-PET/CT.
  • a reduction in FDG uptake refers to the situation where the mean FDG uptake determined from at least three experiments at the end of the treatment period is less than or equal to the mean FDG- uptake determined from at least three experiments at the beginning.
  • a reduction FDG-uptake refers to the situation where the mean FDG uptake determined from at least three experiments at the end of the treatment period is less than or equal to the mean FDG-uptake determined from at least three experiments at the beginning of the treatment period minus one standard deviation (the larger of the pre-treatment/post- treatment standard deviations being used).
  • a reduction FDG-uptake refers to the situation where the mean FDG uptake determined from at least three experiments at the end of the treatment period is less than or equal to the mean FDG- uptake determined from at least three experiments at the beginning of the treatment period minus two standard deviations (the larger of the pre-treatment/post-treatment standard deviations being used.
  • a reduction FDG-uptake refers to the situation where the mean FDG uptake determined from at least three experiments at the end of the treatment period is less than or equal to the mean FDG-uptake determined from at least three experiments at the beginning of the treatment period minus three standard deviations (the larger of the pre-treatment/post-treatment standard deviations being used.
  • a reduction FDG-uptake refers to the situation where the mean FDG uptake determined from at least three experiments is 2 fold lower at the end of the treatment period than at the beginning.
  • a reduction FDG-uptake refers to the situation where the mean FDG uptake determined from at least three experiments is 5 fold lower at the end of the treatment period than at the beginning.
  • HIV treatment refers to treatment resulting in a reduction of the reservoir over the treatment period.
  • CD4+ T cells which accounts for most of the HIV reservoir
  • a suitable treatment period in this embodiment would be no less than 6 months, for example between 6-24 months.
  • the size of the HIV reservoir may be determined by quantifying cell associated HIV DNA and/or RNA at the beginning and end of the treatment period.
  • peripheral blood mononuclear cells, CD4+ T cells, or lymphoid tissues e.g. a lymph node mononuclear cell suspension derived from an inguinal lymph node biopsy
  • CD4+ T cells or subsets of CD4+ T cells are used. Methods of obtaining these cell populations are well known.
  • nucleic acid may be extracted by conventional techniques. Any suitable technique may be used for quantification of HIV RNA.
  • the Amplicor Monitor assay (run according to the manufacturer's specifications) may be used (NOTE- where the "copies per ml" value exceeds 10 6 , quantification should be repeated with appropriate dilutions of fresh RNA extract; where the copies/ml value is lower than 50, quantification may be alternatively be performed using the Single Copy Assay described supra).
  • a mathematical conversion can be used to convert copies/ml to copies/mg where the weight of the tissue (mg), quantity of total RNA (ng) extracted in a given volume, and RNA copies/ng are measured.
  • quantitative PCR techniques may be used to quantify integrated HIV DNA (e.g.
  • PCR a quantitative version of inverse PCR, or real-time PCR using a 7500 Real-Time PCR system, Applied Biosystems.
  • the PCR product may be quantified using a phosphorimager and suitable software (e.g.
  • An alternative method for measuring the HIV reservoir within CD4+ T cells is the quantitative viral outgrowth assay (Q.VOA) described for the first time in Chun et al. (Nature, 1997, 387: 183-8), and well known variants thereof.
  • Q.VOA quantitative viral outgrowth assay
  • This assay determines the levels of replication competent HIV virus measured as infectious HIV units per million CD4+ T cells. This method requires approximately 50 million resting CD4 T cells and thus is generally performed leukapheresis blood samples.
  • Replicate CD4 T cells are plated in a limiting dilution manner. Cells are maximally activated to induce HIV expression with PHA, allogeneic irradiated PBMCs from a seronegative donor, and IL2 for 24 hours.
  • Cultures are washed and co-cultured with target cells that are susceptible to HIV infection and replication. After 2 weeks of culture, supernatants are harvested and assayed for virus protein production. The number of positive wells at each cell dilution is determined and the number of resting CD4 + T cells in infected units per million is estimated using a maximum likelihood method.
  • a reduction in the reservoir refers to the situation where the mean levels of replication competent virus measured by QVOA or HIV genetic material measured by cell associated HIV DNA and/or RNA is lower at the end of the treatment period than at the beginning.
  • the mean level of replication competent virus or HIV genetic material from at least three samples/experiments at the end of the treatment period is equal to or less than the mean level of replication competent virus or HIV genetic material from at least three samples/experiments at the beginning of the treatment period less one standard deviation (the larger of the pre- and post- treatment standard deviations being used).
  • the mean level of replication competent virus or HIV genetic material from at least three samples/experiments at the end of the treatment period is equal to or less than the mean level of replication competent virus or HIV genetic material from at least three samples/experiments at the beginning of the treatment period less two standard deviations (the larger of the pre- and post- treatment standard deviations being used).
  • the mean level of replication competent virus or HIV genetic material from at least three samples/experiments at the end of the treatment period is equal to or less than the mean level of replication competent virus or HIV genetic material from at least three samples/experiments at the beginning of the treatment period less three standard deviations (the larger of the pre- and post- treatment standard deviations being used).
  • the mean level of replication competent virus from at least three experiments at the end of the treatment period is 10%, 20% or 50% lower than the mean level of replication competent virus from at least three samples at the beginning of the treatment period.
  • the mean level of HIV genetic material from at least three samples at the end of the treatment period is 0.5logio copies per 10 6 cells less than the mean level of HIV genetic material from at least three samples at the beginning of the treatment period.
  • HIV treatment refers to treatment that results in an increase in HIV specific T cell function following antigen stimulation.
  • the increase in HIV-specific T-cell function may be measured by cytokine production, increased cytotoxic potential or T-cell proliferation.
  • the treatment period could be a period between 3-12 months.
  • Cytokine production and increased cytotoxic potential may be measured by ELISA, ELISPOT or by intracellular staining of cells and acquisition on a flow cytometer. These are standard techniques and some suitable protocols are outlines in Chapter 24, HIV Protocols, Second Ed., vol. 485, 2009. ELISAs may be performed on blood samples to measure the levels of cytokines or the cytotoxic products of immune cells. ELISPOT assays can be performed on peripheral blood mononuclear cells or CD4+ or CD8+ T cell populations whilst cytokine flow cytometry is of course capable of providing separate information on CD4+ and CD8+ cells when mixed cell populations (e.g. peripheral blood mononuclear cells) are used.
  • mixed cell populations e.g. peripheral blood mononuclear cells
  • HIV peptides may be used for antigen stimulation, including gag, pol, env, nef or pooled peptides.
  • One or more cytokines e.g. IFN- ⁇ , IL-2, TNF
  • cytotoxic products e.g. CD107a, perforin or granzyme
  • the skilled person would be aware which cytokines/cytotoxic products should be measured for different cells types.
  • suitable cytokines include IFN- ⁇ , IL-2, TNFa.
  • suitable cytokines include IFN- ⁇ , IL-2, TNFa and suitable cytotoxic products include CD107a.
  • an increase in the production of a cytokine or cytotoxic product refers to the situation where the mean level of the measured cytokine or cytotoxic product determined from at least three samples is higher at the end of the treatment period than at the beginning. In one embodiment, an increase in cytokine production or an increase in the release of cytotoxic product refers to the situation where the mean level of the measured cytokine/cytotoxic product determined from at least three samples at the end of the treatment period is equal to or higher to the mean level of the measured
  • an increase in cytokine production or an increase in the release of cytotoxic product refers to the situation where the mean level of the measured cytokine/cytotoxic product determined from at least three samples at the end of the treatment period is equal to or higher to the mean level of the measured cytokine/cytotoxic product at the beginning of the treatment period determined from at least three samples plus two standard deviations (using the larger of the pre- and post- treatment standard deviations).
  • an increase in cytokine production or an increase in the release of cytotoxic product refers to the situation where the mean level of the measured cytokine/cytotoxic product determined from at least three samples at the end of the treatment period is equal to or higher to the mean level of the measured cytokine/cytotoxic product at the beginning of the treatment period determined from at least three samples plus three standard deviations (using the larger of the pre- and post- treatment standard deviations).
  • an increase in cytokine production or an increase in the release of cytotoxic product refers to the situation where the mean level of the measured cytokine/cytotoxic product determined from at least three samples at the end of the treatment period is 5%, 15%, 50% or 100% higher than the mean level of the measured cytokine/cytotoxic product at the beginning of the treatment period determined from at least three samples.
  • T cell proliferation following antigen stimulation may be measured by a technique that uses carboxy fluorescein diacetate succinimidyl ester (CSFE), a cell permeable dye which allows monitoring of cell division by flow cytometry essentially as described in Chapter 19, Methods in Cell Biology, Vol. 75, 2004.
  • CSFE carboxy fluorescein diacetate succinimidyl ester
  • Peripheral blood mononuclear cells may be used in this method and various HIV peptides may be used for antigen stimulation, including gag, pol, env, nef or pooled peptides.
  • an increase in proliferation refers to the situation where the mean percentage of antigen specific T cells (CD4+ and/or CD8+) that had divided (calculated by determining the percentage that had diluted CSFE after subtracting background division, namely the percentage of T cells that divided in the negative control) determined from at least three samples is higher at the end of the treatment period than at the beginning.
  • an increase in proliferation refers to the situation where the mean percentage of antigen specific T cells that had divided determined from at least three samples at the end of the treatment period is equal to or higher to the mean percentage of antigen specific T cells that had divided determined from at least three samples at the beginning of the treatment period plus one standard deviation.
  • an increase in proliferation refers to the situation where the mean percentage of antigen specific T cells that had divided determined from at least three samples at the end of the treatment period is equal to or higher to the mean percentage of antigen specific T cells that had divided determined from at least three samples at the beginning of the treatment period plus two standard deviations.
  • an increase in proliferation refers to the situation where the mean percentage of antigen specific T cells that had divided determined from at least three samples at the end of the treatment period is equal to or higher to the mean percentage of antigen specific T cells that had divided determined from at least three samples at the beginning of the treatment period plus three standard deviations.
  • an increase in proliferation refers to the situation where the mean percentage of antigen specific T cells that had divided determined from at least three samples at the end of the treatment period is 1.2, 1.5, 2, 5, or 20 fold greater than the mean percentage of antigen specific T cells that had divided determined from at least three samples at the beginning of the treatment period.
  • the invention provides a method of treating cancer which comprises administering to a human in need thereof a therapeutically effective amount of the binding protein of the invention.
  • cancer refers to cells that have undergone a malignant transformation that makes them pathological to the host organism.
  • the term cancer refers to both primary cancers, which can be readily distinguished from non-cancerous cells by well-established techniques, particularly histological examination and metastasized cancer cells.
  • treating means: (1) to ameliorate or prevent the condition or one or more of the biological manifestations of the condition, (2) to alleviate one or more of the symptoms, effects or side effects associated with the condition or treatment thereof, or (3) to slow the progression of the condition or one or more of the biological manifestations of the condition.
  • the cancer is a solid tumour selected from brain cancer (gliomas), glioblastomas, astrocytomas, glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast cancer, inflammatory breast cancer, Wilm's tumor, Ewing's sarcoma, Rhabdomyosarcoma, ependymoma, medulloblastoma, colon cancer, colorectal cancer, head and neck cancer (including squamous cell carcinoma of head and neck), kidney cancer, lung cancer (including lung squamous cell carcinoma, lung adenocarcinoma, lung small cell carcinoma and non-small cell lung carcinoma), liver cancer (including hepatocellular carcinoma), melanoma, ovarian cancer, pancreatic cancer
  • kidney clear cell cancer including kidney papillary cancer, renal cell carcinoma
  • mesothelioma mesothelioma, esophageal cancer, salivary gland cancer, gastric cancer, nasopharangeal cancer, buccal cancer, cancer of the mouth, stomach cancer, GIST (gastrointestinal stromal tumor) and testicular cancer.
  • the cancer is a haematological cancer (liquid tumour), including both lymphoid and myeloid malignancies.
  • Myeloid malignancies include, but are not limited to, acute myeloid (or myelocytic or myelogenous or myeloblastic) leukemia
  • myeloid malignancies also include myeloproliferative disorders (MPD) which include, but are not limited to, chronic myeloid (or promyelocytic or promyelogenous or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia and megakaryocytic (or megakaryoblastic) leukemia.
  • AML acute myeloid leukemia
  • Myeloid malignancies also include myeloproliferative disorders (MPD) which include, but are not limited to, chronic myeloid (orlocytic or promyelogenous or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia and megakaryocytic (or mega
  • myelogenous (or myeloid) leukemia CML
  • chronic myelomonocytic leukemia CMML
  • essential thrombocythemia or thrombocytosis
  • PCV polcythemia vera
  • Myeloid malignancies also include myelodysplasia (or myelodysplastic syndrome or MDS), which may be referred to as refractory anemia (RA), refractory anemia with excess blasts (RAEB), and refractory anemia with excess blasts in transformation (RAEBT); as well as myelofibrosis (MFS) with or without agnogenic myeloid metaplasia.
  • myelodysplasia or myelodysplastic syndrome or MDS
  • RA refractory anemia
  • RAEB refractory anemia with excess blasts
  • RAEBT refractory anemia with excess blasts in transformation
  • MFS myelofibrosis
  • Hematopoietic cancers also include lymphoid malignancies, which may affect the lymph nodes, spleens, bone marrow, peripheral blood, and/or extranodal sites.
  • Lymphoid cancers include B-cell malignancies, which include, but are not limited to, B-cell non-Hodgkin's lymphomas (B-NHLs).
  • B-NHLs may be indolent (or low-grade), intermediate-grade (or aggressive) or high-grade (very aggressive).
  • Indolent B cell lymphomas include follicular lymphoma (FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma (MZL) including nodal MZL, extranodal MZL, splenic MZL and splenic MZL with villous lymphocytes; lymphoplasmacytic lymphoma (LPL); and mucosa-associated-lymphoid tissue (MALT or extranodal marginal zone) lymphoma.
  • FL follicular lymphoma
  • SLL small lymphocytic lymphoma
  • MZL marginal zone lymphoma
  • LPL lymphoplasmacytic lymphoma
  • MALT mucosa-associated-lymphoid tissue
  • Intermediate-grade B-NHLs include mantle cell lymphoma (MCL) with or without leukemic involvement, diffuse large cell lymphoma (DLBCL), follicular large cell (or grade 3 or grade 3B) lymphoma, and primary mediastinal lymphoma (PML).
  • MCL mantle cell lymphoma
  • DLBCL diffuse large cell lymphoma
  • follicular large cell or grade 3 or grade 3B lymphoma
  • PML primary mediastinal lymphoma
  • High-grade B-NHLs include Burkitt's lymphoma (BL), Burkitt-like lymphoma, small non-cleaved cell lymphoma (SNCCL) and lymphoblastic lymphoma.
  • B-NHLs include immunoblastic lymphoma (or immunocytoma), primary effusion lymphoma, HIV associated (or AIDS related) lymphomas, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma.
  • B-cell malignancies also include, but are not limited to, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), Waldenstrom's macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphocyte (LGL) leukemia, acute lymphoid (or lymphocytic or lymphoblastic) leukemia, and Castleman's disease.
  • CLL chronic lymphocytic leukemia
  • PLL prolymphocytic leukemia
  • WM Waldenstrom's macroglobulinemia
  • HCL hairy cell leukemia
  • LGL large granular lymphocyte
  • LAman's disease Castleman's disease.
  • NHL may also include T-cell non-Hodgkin's lymphoma s(T-NHLs), which include, but are not limited to T-cell non-Hodgkin's lymphoma not otherwise specified (NOS), peripheral T-cell lymphoma (PTCL), anaplastic large cell lymphoma (ALCL), angioimmunoblastic lymphoid disorder (AILD), nasal natural killer (NK) cell / T-cell lymphoma, gamma/delta lymphoma, cutaneous T cell lymphoma, mycosis fungoides, Sezary syndrome, lymphoblastic T-cell leukaemia, acute lymphoblastic T-cell leukaemia and lymphoblastic T-cell lymphoma.
  • T-NHLs T-cell non-Hodgkin's lymphoma s
  • T-NHLs T-cell non-Hodgkin's lymphoma not otherwise specified
  • PTCL peripheral T-cell lymphoma
  • ALCL
  • Hematopoietic cancers also include Hodgkin's lymphoma (or disease) including classical Hodgkin's lymphoma, nodular sclerosing Hodgkin's lymphoma, mixed cellularity Hodgkin's lymphoma, lymphocyte predominant (LP) Hodgkin's lymphoma, nodular LP Hodgkin's lymphoma, and lymphocyte depleted Hodgkin's lymphoma.
  • Hematopoietic cancers also include plasma cell diseases or cancers such as multiple myeloma (MM) including smoldering MM, monoclonal gammopathy of undetermined (or unknown or unclear) significance (MGUS), plasmacytoma (bone, extramedullary), lymphoplasmacytic lymphoma (LPL), Waldenstrom's Macroglobulinemia, plasma cell leukemia, primary amyloidosis (AL), and multiple myeloma magakaryoblastic leukaemia.
  • MM multiple myeloma
  • MGUS monoclonal gammopathy of undetermined (or unknown or unclear) significance
  • MGUS monoclonal gammopathy of undetermined (or unknown or unclear) significance
  • plasmacytoma bone, extramedullary
  • LPL lymphoplasmacytic lymphoma
  • Waldenstrom's Macroglobulinemia plasma cell leukemia
  • A primary amyloidosis
  • haematopoietic cancers include plasmacytoma, immunoblastic large cell leukaemia, mantle cell leukaemia, acute megakaryocytic leukaemia, promyelocytic leykamia, erythroleukaemia and follicular lymphoma.
  • the cancer is a solid tumour.
  • the solid tumour may be selected from glioma, head and neck cancer (including squamous cell carcinoma of head and neck), stomach cancer, gastric cancer, melanoma, renal cell carcinoma (RCC), esophageal cancer, lung squamous cell carcinoma, lung adenocarcinoma, lung small cell carcinoma, non-small cell lung carcinoma, hepatocellular carcinoma, kidney clear cell cancer, kidney papillary cancer, prostate cancer, esophageal cancer, colorectal cancer, breast cancer, bladder cancer, cervical cancer, cancer of the uterus, ovarian cancer and pancreatic cancer.
  • the human has a liquid tumour such as diffuse large B cell lymphoma (DLBCL), multiple myeloma, chronic lyphomblastic leukemia (CLL), follicular lymphoma, acute myeloid leukemia and chronic myelogenous leukemia.
  • DLBCL diffuse large B cell lymphoma
  • CLL chronic lyphomblastic leukemia
  • follicular lymphoma acute myeloid leukemia and chronic myelogenous leukemia.
  • the binding protein When used for the treatment of infectious diseases other than HIV, the binding protein may be co-administered together with one or more epigenetic modifying agents, immune checkpoint agonists or antagonists or immune modulators.
  • Epigenetic modifying agents include, but are not limited to, histone deacetylase inhibitors (HDACi), bromodomain inhibitors (BETi), protein kinase C (PKC) agonists, PTEFb activators, histone methyl transferase inhibitors (HMTi) and cytokines (e.g. IL21).
  • HDACi histone deacetylase inhibitors
  • BETi bromodomain inhibitors
  • PLC protein kinase C
  • PTEFb activators histone methyl transferase inhibitors
  • cytokines e.g. IL21
  • Immune checkpoint agonists or antagonists include antibodies directed to CTLA-4, TIM -3, CD160, TIGIT, OX40 and ICOS.
  • the binding protein of the invention may be employed alone or in combination with other therapeutic agents. Therefore, in other embodiments, the methods of treating or curing HIV infection in a subject may in addition to administration of the binding protein further comprise administration of one or more additional pharmaceutical agents that may be useful in the treatment or cure of HIV. Examples of such agents include:
  • Nucleotide reverse transcriptase inhibitors such as zidovudine, didanosine, , tenofovir, lamivudine, zalcitabine, abacavir, stavudineadefovir, adefovir dipivoxil, fozivudine, todoxil, emtricitabine, alovudine, amdoxovir, elvucitabine, and similar agents;
  • Non-nucleotide reverse transcriptase inhibitors include an agent having anti- oxidation activity such as immunocal, oltipraz, etc.
  • an agent having anti- oxidation activity such as immunocal, oltipraz, etc.
  • nevirapine delavirdine, efavirenz, loviride
  • immunocal immunocal
  • oltipraz immunocal
  • capravirine capravirine
  • lersivirine GSK2248761
  • TMC- 278, TMC-125 etravirine, and similar agents
  • Protease inhibitors such as saquinavir, ritonavir, indinavir, nelfinavir, amprenavir, fosamprenavir, brecanavir, lopinavir, darunavir, atazanavir, tipranavir, palinavir, lasinavir, and similar agents;
  • Integrase inhibitors such as raltegravir, elvitegravir, dolutegravir, cabotegravir, bictegravir and similar agents;
  • Maturation inhibitors such as PA-344 and PA-457, and similar agents;
  • Inhibitors of HIV Entry include enfuvirtide; T-20; T-1249, PRO-542, PRO-140, TNX-355, BMS-378806, Fostemsavir (BMS-663068), temsavir (BMS-626529), 5-Helix, vicriviroc (Sch-C), Sch-D, TAK779, maraviroc (UK 427,857), TAK449, as well as those disclosed in WO 02/74769, PCT/US03/39644, PCT/US03/39975, PCT/US03/39619, PCT/US03/39618, PCT/US03/39740, and PCT/US03/39732, and similar agents.
  • Highly active antiretroviral therapy generally includes a combination of drugs from several different classes for example a reverse transcriptase inhibitor, an integrase inhibitor and a protease inhibitor.
  • the binding protein would be administered with one or more antiretroviral agents, for example one or more agents selected from the group consisting of: a reverse transcriptase inhibitor, an integrase inhibitor and a protease inhibitor.
  • the binding protein would be administered with a reverse transcriptase inhibitor an integrase inhibitor and a protease inhibitor. Any reverse transcriptase inhibitor, integrase inhibitor and protease inhibitor listed in Table 3 can be used in these
  • the binding protein of present invention may be used in combination with one or more agents useful as pharmacological enhancers as well as with or without additional compounds for the treatment or cure of HIV.
  • agents useful as pharmacological enhancers include, but are not limited to, ritonavir, GS-9350, and SPI-452.
  • Ritonavir is 10-hydroxy-2-methyl-5-(l-methyethyl)-l-l[2-(l-methylethyl)-4-thiazolyl]-3,6- dioxo-8,ll-bis(phenylmethyl)-2,4,7,12-tetraazatridecan-13-oic acid, 5-thiazolylmethyl ester, [5S-(5S*,8R*,10R*,11R*)] and is available from Abbott Laboratories of Abbott park, Illinois, as NORVIR.
  • Ritonavir is an HIV protease inhibitor indicated with other antiretroviral agents for the treatment of HIV infection.
  • Ritonavir also inhibits P450 mediated drug metabolism as well as the P-gycoprotein (Pgp) cell transport system, thereby resulting in increased concentrations of active compound within the organism.
  • Pgp P-gycoprotein
  • GS-9350 is a compound being developed by Gilead Sciences of Foster City California as a pharmacological enhancer.
  • SPI-452 is a compound being developed by Sequoia Pharmaceuticals of Gaithersburg, Maryland, as a pharmacological enhancer.
  • Latency reversing agents include, but are not limited to, histone deacetylase inhibitors (HDACi), bromodomain inhibitors (BETi), protein kinase C (PKC) agonists, PTEFb activators, histone methyl transferase inhibitors (HMTi) and cytokines (e.g. IL21).
  • HDACi histone deacetylase inhibitors
  • BETi bromodomain inhibitors
  • PLC protein kinase C
  • PTEFb activators histone methyl transferase inhibitors
  • cytokines e.g. IL21
  • Immune checkpoint agonists or antagonists include antibodies directed to CTLA-4, TIM-3, CD160, TIGIT, OX40 and ICOS.
  • Immune modulators include indolamine 2,3 dioxygenase-1 (IDO-1) inhibitors.
  • binding protein of the invention and any other pharmaceutically active agent(s) may be administered together or separately and, when administered separately, administration may occur simultaneously or sequentially, in any order.
  • the amounts of the binding protein of the present invention and the other pharmaceutically active agent(s) and the relative timings of administration will be selected in order to achieve the desired combined therapeutic effect.
  • the administration of binding proteins in combination with other treatment agents may be by administration
  • a unitary pharmaceutical composition including both pharmaceutically active agents concomitantly in: (1) a unitary pharmaceutical composition including both pharmaceutically active agents; or (2) separate pharmaceutical compositions each including one of the pharmaceutically active agents.
  • the combination may be administered separately in a sequential manner wherein one treatment agent is administered first and the other second or vice versa. Such sequential administration may be close in time or remote in time.
  • the binding protein When the binding protein is intended for use in the treatment cancer, the binding protein may be used in combination with at least one neoplastic agent.
  • the binding protein or binding protein/neoplastic agent combination When used for the treatment of cancer, the binding protein or binding protein/neoplastic agent combination may be co-administered together with one or more epigenetic modifying agents, immune checkpoint agonists or antagonists or immune modulators.
  • Epigenetic modifying agents include, but are not limited to, histone deacetylase inhibitors (HDACi), bromodomain inhibitors (BETi), protein kinase C (PKC) agonists, PTEFb activators, histone methyl transferase inhibitors (HMTi) and cytokines (e.g. IL21).
  • Immune checkpoint agonists or antagonists include antibodies directed to CTLA-4, TIM -3, CD160, TIGIT, OX40 and ICOS.
  • Immune modulators include indolamine 2,3 dioxygenase-1 (IDO-1) inhibitors.
  • Binding proteins according to the invention may be used to ameliorate immunosuppression in a tumour microenvironment.
  • the binding protein may be
  • an engineered cytotoxic cell e.g., a naturally or non-naturally occurring T cell, natural killer (NK) cell or cytotoxic T cell or NK cell line expressing an antigen receptor (CAR) or a T cell receptor (TCR).
  • ACT adoptive cell therapy
  • an engineered cytotoxic cell e.g., a naturally or non-naturally occurring T cell, natural killer (NK) cell or cytotoxic T cell or NK cell line expressing an antigen receptor (CAR) or a T cell receptor (TCR).
  • ACT adoptive cell therapy
  • an engineered cytotoxic cell e.g., a naturally or non-naturally occurring T cell, natural killer (NK) cell or cytotoxic T cell or NK cell line expressing an antigen receptor (CAR) or a T cell receptor (TCR).
  • CAR antigen receptor
  • TCR T cell receptor
  • the CAR or TCR is specific for a tumour-specific antigen (TSA) or a tumour-associated antigen (TAA) that is present on a cancer cell.
  • Tumour antigens are well known in the art.
  • TSAs or TAAs include the following: differentiation antigens such as MART-l/MelanA (MART-1), gplOO (Pmet 17), tyrosinase, TRP-1, TRP-2 and tumour- specific multilineage antigens such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, pl5;
  • overexpressed embryonic antigens such as CEA
  • overexpressed oncogenes and mutated tumour-suppressor genes such as p53, Ras, HER-2/neu
  • unique tumour antigens resulting from chromosomal translocations such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR
  • viral antigens such as the Epstein Barr virus antigens EBVA and the human
  • papillomavirus (HPV) antigens E6 and E7 Other large, protein-based antigens include TSP- 180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, pl85erbB2, pl80erbB-3, c-met, nm-23Hl, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p 15, p 16, 43-9F, 5T4, 791Tgp72, alpha-fetoprotein, beta-HCG, BCA225, BTAA, CA 125, CA 15-3 ⁇ CA 27.29 ⁇ BCAA, CA 195, CA 242, CA-50, CAM43, CD68 ⁇ P1, CO-029, FGF-5, G250,
  • the cytotoxic cell is a human cell and the CAR or TCR are human antigens.
  • the active agents may be administered simultaneously, separately or sequentially in one or more pharmaceutical compositions.
  • binding protein of the invention may conveniently be presented for use in the form of a pharmaceutical formulation and thus pharmaceutical formulations comprising the binding protein together with a pharmaceutically acceptable excipient comprise a further aspect of the invention.
  • compositions can be administered to patients by any convenient route.
  • Particular pharmaceutical compositions are those adapted for intravenous or sub-cutaneous injection.
  • Pharmaceutical compositions adapted for intravenous or sub-cutaneous administration include aqueous and non-aqueous sterile injection solutions (which may contain anti-oxidants, buffers, bacteriostats and solutes which render the composition isotonic with the blood of the intended recipient) and aqueous and non-aqueous sterile suspensions (which may include suspending agents and thickening agents).
  • compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • sterile liquid carrier for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • the pharmaceutical composition is administered fortnightly or monthly via intravenous injection. In another embodiment, the pharmaceutical composition is administered fortnightly or monthly via sub-cutaneous injection.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a population of engineered human cytotoxic cells (e.g., a naturally or non-naturally occurring T cell, natural killer (NK) cell or cytotoxic T cell or NK cell line) capable of co-expressing a binding protein described herein and a human CAR or human TCR.
  • the invention provides a method of treating cancer in a human patient, the method comprising administering to the patient an effective amount of this pharmaceutical composition.
  • the invention also provides isolated nucleic acids encoding the binding protein.
  • this will be polynucleotide sequences encoding the amino acid sequences represented by H-A- VH(PD-I) and L.
  • this will be polynucleotide sequences encoding the amino acid sequences represented by H(CPI )-A-VH(PD-1) and L(CPI ).
  • the sequences will vary (e.g.
  • polynucleotide sequences could be cloned into a vector.
  • the polynucleotides could be cloned into an expression vector.
  • An expression vector may be produced by placing the polynucleotide coding sequences in operative association with conventional regulatory control sequences capable of controlling the replication and expression in, and/or secretion from, a host cell. Regulatory sequences include promoter sequences, e.g., CMV promoter, and signal sequences which can be derived from other known antibodies.
  • each polynucleotide is cloned into a separate expression vector.
  • the expression vectors are identical except insofar as the coding sequences and selectable markers are concerned. The use of different selectable markers ensures, as far as possible, that each polypeptide chain is functionally expressed.
  • all coding sequences may reside on a single vector, for example in separate expression cassettes in the same vector.
  • a selected host cell is co-transfected by conventional techniques with all expression vectors required to create the transfected host cell of the invention.
  • the invention thus provides a host cell capable of expressing the binding protein of the invention.
  • the invention provides a transfected host cell comprising an expression vector comprising one or more polynucleotides encoding the binding proteins described herein.
  • Suitable host cells or cell lines for the expression of the binding proteins of the invention include mammalian cells such as NSO, Sp2/0, CHO (e.g. DG44), COS, HEK, a fibroblast cell (e.g., 3T3), and myeloma cells, for example it may be expressed in a CHO or a myeloma cell.
  • mammalian cells such as NSO, Sp2/0, CHO (e.g. DG44), COS, HEK, a fibroblast cell (e.g., 3T3), and myeloma cells, for example it may be expressed in a CHO or a myeloma cell.
  • Human cells may be used, thus enabling the molecule to be modified with human glycosylation patterns.
  • other eukaryotic cell lines may be employed.
  • the selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening and product production and purification are known in the art.
  • Bacterial cells may prove useful as host cells suitable for the expression of the binding proteins of the present invention (see, e.g., Pluckthun, A., Immunol. Rev., 130:151-188 (1992)).
  • any binding protein produced in a bacterial cell would have to be screened for retention of biological activity.
  • the molecule expressed by the bacterial cell was produced in a properly folded form, that bacterial cell would be a desirable host, or in alternative embodiments the molecule may express in the bacterial host and then be subsequently re-folded.
  • various strains of E. coli used for expression are well-known as host cells in the field of
  • strains of yeast cells known to those skilled in the art are also available as host cells, as well as insect cells, e.g. Drosophila and Lepidoptera and viral expression systems. See, e.g. Miller et al., Genetic Engineering, 8:277-298, Plenum Press (1986) and references cited therein.
  • the invention further provides a method for the production of any of the binding proteins described herein which method comprises a step of culturing the host cell described herein and recovering the binding protein produced.
  • the culture method of the present invention is a serum-free culture method, usually by culturing cells serum-free in suspension.
  • the antigen binding constructs of the invention may be purified from the cell culture contents according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Such techniques are well known in the art.
  • the invention provides a method of making an engineered cytotoxic cell capable of co- expressing a binding protein described herein and a CAR or TCR, the method comprising introducing into a cytotoxic cell (e.g., a naturally or non-naturally occurring T cell, natural killer (NK) cell or cytotoxic T cell or NK cell line) all expression vectors required to express the binding protein and CAR or TCR.
  • a cytotoxic cell e.g., a naturally or non-naturally occurring T cell, natural killer (NK) cell or cytotoxic T cell or NK cell line
  • the one or more expression vectors are viral vectors selected from the group consisting of an adeno-associated viral vector, an adenoviral vector, a lentiviral vector, and a retroviral vector.
  • Embodiment 1 A binding protein specific to human PD-1 that comprises one or more of CDRHl, CDRH2 and CDRH3, wherein CDRHl is selected from the group consisting of: CDRHl as present in SEQ. ID NO:3 and CDRHl that differs from the CDRHl present in SEQ.
  • CDRH2 is selected from the group consisting of: CDRH2 as present in SEQ ID NO:3 and CDRH2 that differs from the CDRH2 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids
  • CDRH3 is selected from the group consisting of: CDRH3 as present in SEQ ID NO: 3 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids.
  • Embodiment 2 A binding protein according to embodiment 1, which is single variable domain.
  • Embodiment 3 A binding protein according to embodiment 2, wherein CDRHl is THYMX 4 , wherein X 4 is V or A; wherein CDRH2 is FIGPAGDX 5 TYYADSVX 6 G wherein X 5 is T, F or S and X 6 is K or E; and wherein CDRH3 is YTX TSXgXgDXioYDV, wherein X 7 is A or E, X 8 is G, S or D, X 9 is V, F or Y, and Xio is T or S.
  • Embodiment 4 A binding protein according to embodiment 3, wherein CDRHl, CDRH2 and CDRH3 are as present in SEQ ID NO. 3.
  • Embodiment 5 A binding protein according to embodiment 4, wherein CDRHl has the sequence defined as SEQ ID NO: 10, CDRH2 has the sequence defined as SEQ ID NO: 11 and CDRH3 has the sequence defined as SEQ ID NO: 12.
  • Embodiment 6 A binding protein according to any one of embodiments 2 to 5, which comprise the sequence defined as SEQ ID NO.3 or a variant of SEQ ID NO. 3 that differs in having up to 10 amino acid additions, deletions or substitutions.
  • Embodiment 7 A binding protein according to embodiment 6, wherein the up to 10 amino acid additions, deletions or substitutions are not within the CDR regions.
  • Embodiment 8 A binding protein according to any one of embodiments 2 to 5, which comprises the sequence defined as SEQ ID NO.3 or a sequence that has 90% sequence identity to the sequence of SEQ ID NO. 3.
  • Embodiment 9 A binding protein according to embodiment 8, wherein variation occurs outside of the CDR regions.
  • Embodiment 10 A binding protein according to any one of embodiments 2 to 9, which comprises the sequence defined as SEQ. ID NO.3.
  • Embodiment 11 A binding protein according to any preceding embodiment which exhibits an IC50 of less than or equal to 5 nM in the PD-l/PDL-1 competition assay
  • Embodiment 12 A binding protein according to any preceding embodiment, which comprises a domain specific for binding human PD-1 attached by a linker to one or more domains specific for a human checkpoint inhibitor other than PD-1.
  • Embodiment 13 A binding protein according to embodiment 12, wherein the binding protein comprises a domain specific for binding human PD-1 attached by a linker to the C terminus of the heavy chain of an antibody specific for the human checkpoint inhibitor other than PD-1.
  • Embodiment 14 A binding protein according to embodiment 13, wherein there are two binding domains specific for PD-1, one attached to the C-terminus of each of the two heavy chains of the antibody specific for a human checkpoint inhibitor other than PD-1.
  • Embodiment 15 A binding protein according to any one of embodiments 12 to 14, which binding protein is capable of neutralising said human checkpoint inhibitor other than PD-1.
  • Embodiment 16 A binding protein having the general formula (II):
  • H(CPI) is an antibody heavy chain of the IgG class and L(CPI) is an antibody light chain of the IgG class such that H(CPI) and L(CPI) together form an antibody specific for a human checkpoint inhibitor other than PD-1;
  • n is an integer selected from 2, 4 and 11;
  • A is a bond or a peptide linker
  • VH(PD-I) is an antibody heavy chain variable domain having CDRHl, CDRH2 and CDRH3, wherein said CDRHl is selected from: CDRHl present in SEQ ID NO:3, and CDRHl that differs from the CDRHl present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids; wherein CDRH2 is selected from: CDRH2 present in SEQ ID NO:3 and CDRH2 that differs from the CDRH2 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRH3 is selected from: CDRH3 as present in SEQ ID NO : 3 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:3 by the addition or deletion or substitution of 1, 2 or 3 amino acids.
  • Embodiment 17 A binding protein according to embodiment 16, wherein CDRHl of VH(PD- 1) is THYMX 4 , wherein X 4 is V or A; wherein CDRH2 of V H (PD-1) is FIGPAGDX 5 TYYADSVX 6 G wherein X5 is T, F or S and e is K or E; and wherein CDRH3 of VH(PD-I) is
  • YTX7TSX8X9DX10YDV wherein X 7 is A or E, X 8 is G, S or D, X 9 is V, F or Y, and ⁇ ⁇ is T or S.
  • Embodiment 18 A binding protein according to embodiment 16 or embodiment 17, wherein the V H (PD-1) has CDRHl, CDRH2 and CDRH3 as present in SEQ ID NO. 3.
  • Embodiment 19 A binding protein according to embodiment 18, wherein CDRHl has the sequence defined as SEQ ID NO : 10, CDRH2 has the sequence defined as SEQ ID N O : 11 and CDRH3 has the sequence defined as SEQ ID N O : 12.
  • Embodiment 20 A binding protein according to any one of embodiments 16 to 19, wherein VH(PD-I) comprises the sequence defined as SEQ ID NO.3 or a variant of SEQ ID NO. 3 that differs in having up to 10 amino acid additions, deletions or substitutions.
  • Embodiment 21 A binding protein according to embodiment 20, wherein the up to 10 amino acid additions, deletions or substitutions are not within the CDR regions.
  • Embodiment 22 A binding protein according to any one of embodiments 16 to 21, wherein the VH(PD-I) comprises the sequence defined as SEQ ID NO.3 or a sequence that has 90% sequence identity to the sequence of SEQ ID N O. 3.
  • Embodiment 23 A binding protein according to embodiment 22, wherein variation occurs outside of the CDR regions.
  • VH(PD-I) comprises the sequence defined as SEQ ID NO.3.
  • Embodiment 25 A binding protein according to any one of embodiments 16 to 24, wherein the linker or A is a peptide linker
  • Embodiment 26 A binding protein according to embodiment 25, wherein the linker or A has the sequence of SEQ. ID NO. 30.
  • Embodiment 27 A binding protein according to any one of embodiments 16 to 26, which has an IC50 of less than or equal to 5 nM in the PD-l/PDL-1 competition assay and exhibits neutralisation of the human checkpoint inhibitor other than PD-1.
  • Embodiment 28 An isolated nucleic acid encoding the binding protein as defined in any one of embodiments 1 to 15.
  • Embodiment 29 An isolated nucleic acid encoding H(CPI)-A- VH(PD-I), wherein H(CPI), A and VH(PD-I) are as defined in any one of embodiments 16 to 27.
  • Embodiment 30 An isolated nucleic acid encoding L(CPI), wherein L(CPI) as defined in any one of embodiments 16 to 27.
  • Embodiment 31 A vector comprising a nucleic acid as defined in embodiment 28, 29 or 30.
  • Embodiment 32 A vector according to embodiment 31, which is an expression vector.
  • Embodiment 33 A host cell comprising a vector according to embodiment 32.
  • Embodiment 34 A method of producing a binding protein as defined in any one of embodiments 1 to 26, comprising culturing a host cell according to embodiment 33 under conditions suitable for protein expression, and isolating the binding protein, wherein the host cell either contains the nucleic acid defined in embodiment 28 or contains the nucleic acids defined in embodiment 29 and embodiment 30 in one or more expression vectors.
  • Embodiment 35 A pharmaceutical composition comprising a binding protein according to any one of embodiments 1 to 26 and a pharmaceutically acceptable excipient.
  • Embodiment 36 A method of treating cancer which comprises administering to a human in need thereof a therapeutically effective amount of a binding protein as defined in any one of embodiments 1 to 26.
  • Embodiment 37 A method of treating an infectious disease which comprises administering to a human in need thereof a therapeutically effective amount of a binding protein as defined in any one of embodiments 1 to 26.
  • Embodiment 38 A method of treating an infectious disease according to embodiment 37, wherein the infectious disease is a bacterial infection, a parasitic infection, a viral infection or sepsis.
  • Embodiment 39 A method of treating HIV which comprises administering to a human in need thereof a therapeutically effective amount of a binding protein as defined in any one of embodiments 1 to 26.
  • Embodiment 40 A method of treating HIV according to embodiment 39 which further comprises administering one or more a nti retroviral agents.
  • Embodiment 41 A method of curing HIV which comprises administering to a human in need thereof a therapeutically effective amount of a binding protein as defined in any one of embodiments 1 to 26.
  • Embodiment 42 A method of curing HIV according to embodiment 41 which further comprises administering one or more a nti retroviral agents.
  • Embodiment 43 A binding protein as defined in any one of embodiments 1 to 26 for use in medicine.
  • Embodiment 44 A binding protein as defined in any one of embodiments 1 to 26 for use in the treatment of cancer.
  • Embodiment 45 A binding protein as defined in any one of embodiments 1 to 26 for use in the treatment of infectious diseases.
  • Embodiment 46 A binding protein for use according to embodiment 45, wherein the infectious disease is a bacterial infection, a parasitic infection, a viral infection or sepsis.
  • Embodiment 47 A binding protein as defined in any one of embodiments 1 to 26 for use in the treatment of HIV.
  • Embodiment 48. A binding protein as defined in any one of embodiments 1 to 26 for use in curing HIV.
  • Embodiment 49 A binding protein as defined in any one of embodiments 1 to 26 and one or more anti-retroviral agents for separate simultaneous or sequential use in treating HIV.
  • Embodiment 50 A binding protein as defined in any one of embodiments 1 to 26 and one or more anti-retroviral agents for separate simultaneous or sequential use in curing HIV.
  • Embodiment 51 Use of a binding protein as defined in any one of embodiments 1 to 26 in the manufacture of a medicament for the treatment of cancer.
  • Embodiment 52 Use of a binding protein as defined in any one of embodiments 1 to 26 in the manufacture of a medicament for the treatment of infectious diseases.
  • Embodiment 53 Use of a binding protein according to embodiment 52, wherein the infectious disease is a bacterial infection, a parasitic infection, a viral infection or sepsis.
  • Embodiment 54 Use of a binding protein as defined in any one of embodiments 1 to 26 in the manufacture of a medicament for the treatment of HIV.
  • Embodiment 55 Use of a binding protein as defined in any one of embodiments 1 to 26 in the manufacture of a medicament for curing HIV.
  • Embodiment 56 Use of a binding protein as defined in any one of embodiments 1 to 26 and one or more anti-retroviral agents in the manufacture of medicaments for separate simultaneous or sequential use in treating HIV.
  • Embodiment 57 Use of a binding protein as defined in any one of embodiments 1 to 26 and one or more anti-retroviral agents for use in the manufacture of medicaments for separate simultaneous or sequential use in curing HIV.
  • Embodiment 1 A binding protein specific to human LAG-3, which comprises: one or more of CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: CDRH1 as present in SEQ. ID NO:l and CDRH1 that differs from the CDRH1 present in SEQ ID N0:1 by the addition or deletion or substitution of 1, 2 or 3 amino acids, wherein CDRH2 is selected from the group consisting of: CDRH2 as present in SEQ ID NO:l and CDRH2 that differs from the CDRH2 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids, and wherein CDRH3 is selected from the group consisting of: CDRH3 as present in SEQ ID NO: 1 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids; and one or more of CDRL1, CDRL2 and CDRL3, wherein CDRL1 is
  • Embodiment 2 A binding protein according to embodiment 1, which comprises:
  • CDRH2 is selected from the group consisting of: CDRH2 as present in SEQ ID NO:l and CDRH2 that differs from the CDRH2 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids
  • CDRH3 is selected from the group consisting of: CDRH3 as present in SEQ ID NO: 1 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids
  • the antibody specific for human LAG-3 comprises CDRL1 and CDRL2, wherein CDRL1 is selected from the group consisting of: CDRL1 as present in SEQ ID NO:2
  • Embodiment 3 A binding protein according to embodiment 1 or paragraph 2, which is an antibody.
  • Embodiment 4. A binding protein according to embodiment 3, wherein the antibody specific to LAG-3 is of the IgA or IgG class.
  • Embodiment 5 A binding protein according to embodiment 4, wherein the antibody specific to LAG-3 is of the IgG class.
  • Embodiment 6 A binding protein according to any preceding embodiment, which exhibits >50% inhibition of LAG3-MHCII interaction in competition flow cytometry assay.
  • Embodiment 7 A binding protein according to any one of embodiments 3 to 6, wherein one or more domains specific for a human checkpoint inhibitor other than LAG-3 are attached by a linker to the C terminus of the heavy chain of the antibody specific to human LAG-3.
  • Embodiment 8 A binding protein according to embodiment 7, wherein there are two domains specific for a human checkpoint inhibitor other than LAG-3, one attached to the C- terminus of each of the two heavy chains of the antibody specific to LAG-3.
  • Embodiment 9 A binding protein according to embodiment 7 or embodiment 8, which binding protein is capable of neutralising said human checkpoint inhibitor other than LAG-3.
  • Embodiment 10 A binding protein having the general formula (III): L (LAG-3)
  • H(LAG-3) is an antibody heavy chain of the IgG class comprising CDRHl, CDRH2 and CDRH3, wherein said CDRHl is selected from: CDRHl present in SEQ. ID N0:1, and CDRHl that differs from the CDRHl present in SEQ ID N0:1 by the addition or deletion or substitution of 1, 2 or 3 amino acids; wherein CDRH2 is selected from: CDRH2 present in SEQ ID N0:1 and CDRH2 that differs from the CDRH2 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRH3 is selected from: CDRH3 as present in SEQ. ID NO: 1 and CDRH3 that differs from the CDRH3 present in SEQ ID NO:l by the addition or deletion or substitution of 1, 2 or 3 amino acids;
  • L(LAG-3) is an antibody light chain of the IgG class comprising CDRLl and CDRL2, wherein said CDRLl is selected from: CDRLl present in SEQ ID NO:2 and CDRLl that differs from the CDRLl present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids; and wherein CDRL2 is selected from: CDRL2 present in SEQ ID NO:2 and CDRL2 that differs from the CDRL2 present in SEQ ID NO:2 by the addition or deletion or substitution of 1, 2 or 3 amino acids; n is an integer selected from 2, 4 and 11;
  • A is a bond or a peptide linker
  • CPI is a domain that is capable of binding a human checkpoint inhibitor other than LAG-3.
  • Embodiment 11 A binding protein according to any preceding embodiment, which comprises CDRH1, CDRH2 and CDRH3 as present in SEQ ID NO. 1.
  • Embodiment 12 A binding protein according to any preceding embodiment, which comprises CDRLl and CDRL2 as present in SEQ ID NO. 2.
  • Embodiment 13 A binding protein according to paragraph 12, which comprises CDRLl, CDRL2 and CDRL3 as present in SEQ ID NO. 2.
  • Embodiment 14 A binding protein according to paragraph 11, wherein CDRH1 has the sequence defined as SEQ ID NO: 4, CDRH2 has the sequence defined as SEQ ID NO: 5 and CDRH3 has the sequence defined as SEQ ID NO: 6.
  • Embodiment 15 A binding protein according to embodiment 13, wherein CDRLl has the sequence defined as SEQ ID NO: 7, CDRL2 has the sequence defined as SEQ ID NO: 8 and CDRL3 has the sequence defined as SEQ ID NO: 9.
  • Embodiment 16 A binding protein according to any one of embodiments 3 to 15, wherein the heavy chains of the antibody specific for human LAG-3 or H comprises the sequence defined as SEQ ID NO.l or a variant of SEQ ID NO. 1 that differs in having up to 10 amino acid additions, deletions or substitutions.
  • Embodiment 17 A binding protein according to embodiment 16, wherein the up to 10 amino acid additions, deletions or substitutions are not within the CDR regions.
  • Embodiment 18 A binding protein according to any one of embodiments 3 to 17, wherein the light chains of the antibody specific for human LAG-3 or L comprises the sequence defined as SEO ID NO. 2 or a variant of SEO ID NO. 2 that differs in having up to 10 amino acid additions, deletions or substitutions.
  • Embodiment 19 A binding protein according to embodiment 18, wherein the up to 10 amino acid additions, deletions or substitutions are not within the CDR regions.
  • Embodiment 20 A binding protein according to any one of embodiments 3 to 15, wherein the heavy chains of the antibody specific for human LAG-3 or H comprises the sequence defined as SEO ID NO.l or a sequence that has 90% sequence identity to the sequence of SE ID NO. 1.
  • Embodiment 21 A binding protein according to embodiment 20, wherein variation occurs outside of the CDR regions.
  • Embodiment 22 A binding protein according to any one of embodiments 3 to 15 or 20 to 21, wherein the light chains of the antibody specific for human LAG-3 or L comprises the sequence defined as SEO ID NO.2 or a sequence that has 90% sequence identity to the sequence of SEO ID NO. 2.
  • Embodiment 23 A binding protein according to claim 22, wherein variation occurs outside of the CDR regions.
  • Embodiment 24 A binding protein according to any one of paragraphs 3 to 23, wherein the heavy chains of the antibody specific for human LAG-3 or H comprises the sequence defined as SEO ID NO.l.
  • Embodiment 25 A binding protein according to any one of embodiments 3 to 24, wherein the light chains of the antibody specific for human LAG-3 or L comprises the sequence defined as SEO ID NO.2.
  • Embodiment 26 A binding protein according to any one of embodiments 7 to 25, wherein the linker or A is a peptide linker
  • Embodiment 27 A binding protein according to embodiment 26, wherein the linker or A has the sequence of SEO ID NO. 30.
  • Embodiment 28. A binding protein according to any one of embodiments 10 to 27 which exhibits >50% inhibition of LAG3-MHCII interaction in competition flow cytometry assay, and exhibits neutralisation of the human checkpoint inhibitor other than LAG-3.
  • Embodiment 29 Isolated nucleic acid encoding the binding protein defined in embodiment 1 or embodiment 2.
  • Embodiment 30 An isolated nucleic acid encoding the heavy chain of the binding protein defined in any one of embodiments 3 to 9.
  • Embodiment 31 An isolated nucleic acid encoding the light chain of the binding protein defined in any one of embodiments 3 to 9.
  • Embodiment 32 An isolated nucleic acid encoding H-A- CPI, wherein H, A and VH(PD-I) are as defined in any one of embodiments 10 to 28.
  • Embodiment 33 An isolated nucleic acid encoding L, wherein L is as defined in any one of embodiments 10 to 28.
  • Embodiment 34 A vector comprising a nucleic acid as defined in any one of embodiments 29 to 33.
  • Embodiment 35 A vector according to embodiment 34, which is an expression vector.
  • Embodiment 36 A host cell comprising a vector according to embodiment 35.
  • Embodiment 37 A method of producing a binding protein as defined in embodiment 1 or embodiment 2, comprising culturing a host cell according to embodiment 36 under conditions suitable for protein expression, and isolating the binding protein, wherein the host cell contains the nucleic acid defined in embodiment 29 in one or more expression vectors.
  • Embodiment 38 A method of producing a binding protein as defined in any one of embodiments 3 to 9, comprising culturing a host cell according to embodiment 36 under conditions suitable for protein expression, and isolating the binding protein, wherein the host cell contains the nucleic acids defined in embodiment 30 and embodiment 31 in one or more expression vectors.
  • Embodiment 39 A method of producing a binding protein as defined in any one of embodiments 10 to 28, comprising culturing a host cell according to paragraph 36 under conditions suitable for protein expression, and isolating the binding protein, wherein the host cell contains the nucleic acids defined in paragraph 32 and paragraph 33 in one or more expression vectors.
  • Embodiment 40 A pharmaceutical composition comprising a binding protein according to any one of embodiments 1 to 28 and a pharmaceutically acceptable excipient.
  • Embodiment 41 A method of treating cancer which comprises administering to a human in need thereof a therapeutically effective amount of a binding protein as defined in any one of embodiments 1 to 28.
  • Embodiment 42 A method of treating an infectious disease which comprises administering to a human in need thereof a therapeutically effective amount of a binding protein as defined in any one of embodiments 1 to 28.
  • Embodiment 43 A method of treating an infectious disease according to embodiment 42, wherein the infectious disease is a bacterial infection, a parasitic infection, a viral infection or sepsis.
  • Embodiment 44 A method of treating HIV which comprises administering to a human in need thereof a therapeutically effective amount of a binding protein as defined in any one of embodiments 1 to 28.
  • Embodiment 45 A method of treating HIV according to embodiment 44 which further comprises administering one or more antiretroviral agents.
  • Embodiment 46 A method of curing HIV which comprises administering to a human in need thereof a therapeutically effective amount of a binding protein as defined in any one of embodiments 1 to 28.
  • Embodiment 47 A method of curing HIV according to embodiment 46 which further comprises administering one or more antiretroviral agents.
  • Embodiment 48 A binding protein as defined in any one of embodiments 1 to 28 for use in medicine.
  • Embodiment 49 A binding protein as defined in any one of embodiments 1 to 28 for use in the treatment of cancer.
  • Embodiment 50 A binding protein as defined in any one of embodiments 1 to 28 for use in the treatment of infectious diseases.
  • Embodiment 51 A binding protein for use according to embodiment 50, wherein the infectious disease is a bacterial infection, a parasitic infection, a viral infection or sepsis.
  • Embodiment 52 A binding protein as defined in any one of embodiments 1 to 28 for use in the treatment of HIV.
  • Embodiment 53 A binding protein as defined in any one of embodiments 1 to 28 for use in curing HIV.
  • Embodiment 54 A binding protein as defined in any one of embodiments 1 to 28 and one or more anti-retroviral agents for separate simultaneous or sequential use in treating HIV.
  • Embodiment 55 A binding protein as defined in any one of embodiments 1 to 28 and one or more anti-retroviral agents for separate simultaneous or sequential use in curing HIV.
  • Embodiment 56 Use of a binding protein as defined in any one of embodiments 1 to 28 in the manufacture of a medicament for the treatment of cancer.
  • Embodiment 57 Use of a binding protein as defined in any one of embodiments 1 to 28 in the manufacture of a medicament for the treatment of infectious diseases.
  • Embodiment 58 Use of a binding protein according to embodiment 57, wherein the infectious disease is a bacterial infection, a parasitic infection, a viral infection or sepsis.
  • Embodiment 59 Use of a binding protein as defined in any one of embodiments 1 to 28 in the manufacture of a medicament for the treatment of HIV.
  • Embodiment 60 Use of a binding protein as defined in any one of embodiments 1 to 28 in the manufacture of a medicament for curing HIV.
  • Embodiment 61 Use of a binding protein as defined in any one of embodiments 1 to 28 and one or more anti-retroviral agents in the manufacture of medicaments for separate simultaneous or sequential use in treating HIV.
  • Embodiment 62 Use of a binding protein as defined in any one of embodiments 1 to 28 and one or more anti-retroviral agents for use in the manufacture of medicaments for separate simultaneous or sequential use in curing HIV. Examples
  • Example 1 Identification of naive variable domains that bind PD-1
  • Epitope binding domains also known as domain antibodies or dAbs
  • dAbs domain antibodies
  • phage displaying dAbs that bind to hPD-1 Three rounds of selection were used to identify phage displaying dAbs that bind to hPD-1.
  • the phage library was pre-incubated in empty immunotubes in round 1, and in round 2, the phage library was pre- incubated in immunotubes coated with glycated Human Serum Albumin (Sigma A8301), in round 3, no pre-incubation was carried out. I n all 3 rounds, the library was then added to immunotubes coated with hPD-1 antigen and incubated to allow binding of phage to the antigen. Following binding, unbound (and weakly bound) phage were washed off by several rounds of washing.
  • the retained phage are then eluted with trypsin (which cleaves the c- myc tag) thereby eluting phage.
  • the eluted phage were then used to infect E.coli and infected cells were plated on selective media, with cultures from the resulting colonies used to amplify the eluted phage ready for the next round of selection.
  • hPD-1 50 ⁇ hPD-1 (1 ⁇ g/ml) in PBS were added to the wells of a 96 well MaxisorpTM immunoplates (Nunc, Denmark) and the plates were incubated overnight at 4°C (2 mg/ml albumin glycated HSA (Sigma A83Q.1) was used as a control). Wells were washed with PBS and then blocked with 2% Marvel in PBS. A 1:1 mixture of supernatant containing a monoclonal phage-dAb population and 2% Marvel in PBS was added to each well. Bound phage-dAb particles are detected with an anti-M 13 (phage coat protein) specific monoclonal antibody-HRP conjugate.
  • M 13 phage coat protein
  • a colorimetric substrate (SureBlue 1 - active component TM B Microwell Peroxidase solution) was added and optical density (OD) was measured at 450 nm.
  • the dAb-phage clones from round 3 of the passive selection were considered as positive binders in the ELISA assay if the ratio of OD450 (hPD-1 antigen)/ OD450 (HSA) was greater than 3.
  • the supernatant from the transfected cells was screened for the ability to inhibit the interaction between hPDl and hPDLl using the hPD-l/hPDL-1 ligand binding inhibition assay (method 3) at a single concentration and for binding to hPD-1 by SPR (Surface Plasmon resonance assay) to allow ranking of the clones by dissociation constant.
  • test samples were prepared by mixing 30 ⁇ of antibody (diluted as described above) and 30 ⁇ of 4 ⁇ g/ml Human PDL-1 Fc.flag-ST. Negative controls were prepared by replacing the test antibody and Human PDL-1 Fc.flag-ST with buffer. Positive controls were prepared by replacing just the test antibody with buffer.
  • the background signal was subtracted by deducting the average of the negative control for each plate. % inhibition of the positive control was then determined using the formula: 100- ((data value/positive control) x 100).
  • test samples were prepared by mixing 30 ⁇ of antibody (diluted as described above) and 30 ⁇ of 2 ⁇ g/ml Cynomolgus PDL-1 Fc.flag-ST. Negative controls were prepared by replacing the test antibody and Cynomolgus PDL-1 Fc.flag-ST with buffer. Positive controls were prepared by replacing just the test antibody with buffer.
  • the background signal was subtracted by deducting the average of the negative control for each plate. % inhibition of the positive control was then determined using the formula: 100- ((data value/positive control) x 100).
  • mAbdAbs were assessed for binding kinetics and affinity for binding to PD-1.
  • 2050 resonance units (RU) of biotinylated human PD-1, 835 RU biotinylated rhesus PD1 were loaded onto streptavidin coated Biacore chips.
  • Test mAbdAbs were passed over the chips at a single concentration in HBS-EP buffer and binding curves were recorded (where a series of concentrations were used). This was run in duplicate at 25°C within the same Biacore run. The curves were double-referenced using a buffer injection curve and then fitted to the 1:1 binding model inherent to the Biacore Evaluation software.
  • Method 5 Assay to determine concentration of mAb-dAbs in supernatants and to determine the concentration of mAbs following small scale purification
  • Samples were quantified using the Octet Red 384 instrument and software version 8.0.2.5, using the basic wizard template 'Quantitation with regeneration. Firstly, Protein A sensor tips were soaked in PBSF for 10 minutes prior to use. Then, tenfold dilutions (15 ⁇ of each sample and 135 ⁇ PBSF) were incubated with the sensor tips. A standard curve: was generated using a mAb-dAb standard with serial twofold dilutions in PBSF from 100 ⁇ g/ml down to 0.78 ug/ml. Sensors were regenerated with 10 mM glycine pH 1.5 between each sample. Data was then analysed using Data Analysis software version 8.0.
  • the concentration of purified mAbdAbs in buffer was determined spectrophotometrically by measurement of the absorbance of UV light at 280 nm using a Nanodrop 1000 instrument (Thermo Scientific)
  • the 51A09 clone which exhibited inhibition in the hPDl-hPDL-1 ligand binding inhibition assay and a slow dissociation rate by SPR was subjected to affinity maturation to increase the potency of the dAb.
  • the dAb sequence of clone 51A09 was subjected to diversification using the MutazymeTM DNA polymerase (Stratagene catalogue number 200550) at a mutation rate of 3.5 amino acid changes per dAb (including both framework and CDR regions).
  • the library of diversified dAb genes were then cloned into pre-cut phage vector DNA (pDOM4) before transformation into TGI electrocompetent cells (C2987).
  • the diversified clones were selected as outlined in method 7.
  • the phage library based on the diversified 51A09 clone and sufficient streptavidin coated (or neutravidin coated) magnetic beads were each blocked separately to reduce non-specific binding of phage during subsequent selection steps. Before each round of selection, the phage library was pre-incubated twice with unloaded beads as an additional step to reduce the levels of non-specific phage. In round 1, the the diversified phage library based on 51A09 was incubated with 50 nM biotinylated hPD-l-His for 1 hour at room temperature. Following incubation, the antigen -phage complexes are captured on the beads through interaction of biotin with streptavidin (or neutravidin).
  • the beads are pulled out of solution through use of a magnetic rack and washed repeatedly to remove non-specific phage and weak binders. The remaining phage are eluted with trypsin and recovered. Following infection of E.coli, the selected phage were subjected to further rounds of selection under the following conditions :
  • Round 2 5 nM hPDl for 15 minutes (+100 nM Glycated HSA) at room temperature
  • Round 3 0.5nM hPDl for 30 minutes at room temperature
  • Phage DNA was isolated from the Round 4 selection output.
  • the pool of dAb coding genes from the pDOM4 phage displace vector was amplified with primers (forward primer 5'- cccggaaagggatccacaggactggactccccgacagaggtgcagctgttggagtct-3' (SEQ. ID NO: 46); reverse primer 3'-ggggatctagaattcatcagctcgagacggtgaccagggtt-5' (SEQ. ID NO: 47)) to generate fragments suitable for In-Fusion cloning into the mammalian expression vector pTT5 containing anti-RSV mAb heavy chain (IgGl Fc disabled).
  • a BamHl site was introduced as a silent mutation at the C-terminus of the anti-RSV mAb heavy chain sequence to generate an open reading frame with the anti-RSV mAb heavy chain fused in frame to the PD-1 dAb.
  • the pTT5 anti-RSV vector was linearised with BamHl and EcoRl and an In Fusion reaction was performed with the purified PCR products from the selected clones using the In-Fusion HD Cloning Kit (Clontech) according to the manufacturer's instructions.
  • the In Fusion reaction was transformed into NEB 5-alpha competent E.coli (High Efficiency; Catalogue Number C2987) cells.
  • DNA isolated from the transformed individual clones was sequenced allowing identification of unique clones and providing material for small scale transfection and expression in HEK293 cells.
  • the supernatant from the transfected cells was screened using the hPDl-hPDL-1 ligand binding inhibition assay (method 3).
  • a group of clones were then selected for repeat transfection and purification based on their potency and sequence and were screened for potency in the Jurkat PD1+ assay (method 8).
  • PDL1+ CHO cells containing an NFAT drive reporter gene were mixed with PD1+ Jurkat cells in the presence an absence of test mAbdAbs.
  • the mAbdAbs were tested for their ability to block expression from the reporter gene utilising the following method:A functional PD-1 assay using the Promega Jurkat NFAT-luc2/PD-l effector T cells stimulated by CHO PD-L1+ cells was performed essentially following the protocol published by Promega Corporation (CS187109).
  • Antibodies were generally prepared as nine point dose curves starting at 200 ⁇ g/ml with 3-fold dilution steps. Plates were incubated for 6 hours at 37°C, 5% C02 then at ambient temperature for 5min. To each well was then added 80 ⁇ of BioGloTM Reagent (prepared as described in the Bio-Glo Luciferase Assay System, Promega catalogue number G7940). Following incubation for 5-10 minutes, luminescence was measured in a plate reader. Raw data values were divided by those of no antibody control wells to give a 'fold over no treatment' value also referred to as 'fold induction'. The EC50 of the antibody response was determined using nonlinear regression curve fitting software with the minimum asymptote constrained to a value of 1 (fitting method: least squares (ordinary) fit).
  • Anti-LAG3/PD-1 antibodies were assessed for binding to cell surface expressed LAG3 and PD-1 using LAG3 expressing Jurkat cells and PD-1 expressing Jurkat cells purchased from Promega (CS194801and CS187102 respectively). Both Promega cell lines were individually incubated at 4°C for 1 hour with antibody at concentrations from ⁇ to O.OOlnM made up in 1% FBS in PBS, a 'no antibody' control was included. Wild-type Jurkat cells were incubated at 4°C for 1 hour alongside with ⁇ antibody as a negative control. After incubation the cells were washed three times in 1% FBS in PBS.
  • Polyclonal PE-linked anti- human IgGl Fc F(ab')2 (Invitrogen, # H10104) was added to treated cells at a 1/250 dilution in 1%FBS in PBS. Cells were incubated at 4°C for 30 minutes in the dark. Cells were washed once with 1% FBS in PBS and again with PBS. NIR dead cell dye (Invitrogen # L10119) diluted 1 in 1000 in PBS was added to the cells to select for live cells only. Cells were incubated at 4°C for 30 minutes in the dark. Cells were washed once with PBS and resuspended in ⁇ PBS for measurement using the MACSQuant flow cytometer (Miltenyi).
  • the MFI data generated from the Jurkat WT cells treated with ⁇ top concentration were all very close to 1 after dividing by the MFI from 'PE-linked anti-human IgGl Fc F(ab')2 Ab only' proving that the antibodies did not bind to the wild type cells.
  • Table 5 shows that dAbs having one amino acid substitution in CDRH1 (at one position within CDRH1), 2 amino acid substitutions in CDRH2 (at two positions within CDRH2) and 2 amino acid substitutions in CDRH3 (at any of four positions within CDRH3) exhibit an IC50 ⁇ 5 nM in the hPDl-hPDL-1 ligand binding inhibition assay and rhesus cross reactivity.
  • FIGPAG DX 5 TYYADSVX 6 G (SEQ I D NO: 58), wherein X 5 is T, F or S and X 6 is K or E;
  • 51A09-188 is an example of a dAb identified after affinity maturation which binds human and rhesus monkey PD1.
  • an alanine residue was added to the C-terminus of the domain antibody to make the C-terminal amino acid sequence LVTVSSA (SEQ ID NO: 81) by In Fusion cloning, as described in Example 2, with the reverse primer being mutated.
  • the mutated version was cloned into the pTT5 anti-RSV heavy chain vector by In Fusion homologous recombination as described in
  • Example 2 This clone is referred to as 51A09-188001.
  • Mutations from other affinity improved clones in the 51A09 lineage were introduced into the 51A09-188001 sequence in the hope of achieving further improvements in affinity and potency in biological assays. Mutations were introduced by site directed mutagenesis using primers containing the desired mutations as follows:
  • Mutant 22D034-51A09-188.3001 T28A and V105F
  • the mutated versions were cloned into the pTT5 anti-RSV heavy chain vector by In Fusion homologous recombination as described for Example 2.
  • the mAbdAb proteins were purified and analysed in the PD1+ Jurkat functional assay.
  • naive antibody selection Three rounds of naive antibody selection were performed using a yeast based platform to generate anti-hLAG3 binding molecules.
  • lOOnM human LAG3-His hLAG3-His
  • Antibody heavy chains from the isolated binding populations were then rearrayed within diverse light chain libraries and rounds of selection performed to optimise the heavy and light pairing of selected antibodies.
  • Rearrayed heavy and light chain pairings were then subjected to selection to enrich a binding population.
  • Magnetic bead selection using 50nM hLAG3-His was performed for one round of enrichment, followed by FACS selection using 50nM rhesus LAG3-His. This yielded pure binding populations to both human and rhesus LAG3-His.
  • the binding populations were then subjected to negative FACS selection with a non-specificity reagent mixed with an irrelevant antigen possessing a 6-His tag, and the resultant outputs were then subjected to affinity pressuring by binding to 0.5nM hLAG3-His.
  • Final selections outputs were generated by gating and sorting cells in FACS selection, and isolating individual clones by plating on selective media. Isolated clones were sequenced and unique mAbs were expressed and tested for binding to soluble antigen and to cells expressing hLAG3. Over 350 clones were tested the following methods and 82 clones meeting the following criteria were identified:
  • LAG-3 antibodies were assessed for binding kinetics and affinity for binding to LAG-3.
  • Method 11 Competition flow cytometry assay 1. Transfer ⁇ of Daudi cell suspension (Biocat, catalogue number 117942) at 1x106 cells/ml in assay buffer (prepared by mixing Dulbecco's PBS 10:1 with foetal bovine serum) into each well of a 96-well V-bottomed plate.
  • test antibodies All antibodies at a concentration range of from 60pM to ⁇ . Incubate plates on ice for 60 minutes.
  • test antibodies at concentrations of between 1-100 nM. Incubate plates on ice for 30 minutes.
  • Dilute detection antibody PE linked anti-human Fc, Invitrogen, Catalogue number H10104 for test antibodies
  • PE 2R - phycoerthyrin.
  • Open Access Analytical Size Exclusion Chromatography was carried out on the Agilent 1100 (including degasser, Quat pump, autosampler ALS, column compartment and DAD detector) using the TSK G300SWXL column at ambient temperature.
  • the column was equilibrated with buffer (200 mM sodium phosphate monobasic, 250 mM NaCI, pH 6 .0) followed by loading c.20 ⁇ g test antibody.
  • the sample was run in standard SEC method utilising equilibration buffer at a flow rate of 0.5 ml/min.
  • the eluted protein was detected by monitoring absorption at 214 nm. Percentage of monomers and aggregation was determined using peak integration in Chemstation.
  • a functional LAG-3 assay using the Promega LAG-3+ Jurkat/NFAT-luc effector T cells stimulated by Raji cells and SEE was performed essentially following the protocol published by Promega Corporation (CS194801). Briefly, into each well of a 96 well plate was added: 25 ⁇ anti-LAG-3 antibody ten-point dose curve starting at 25 ⁇ g/ml (or control antibody dose curve)
  • the plates were incubated for six hour at 37°C in a C0 2 incubator. They were then allowed to reach room temperature and 75 ⁇ BioGloTM Reagent (prepared as described in the Bio-Glo Luciferase Assay System, Promega catalogue number G7940) was added to each well.
  • the EC50 of the antibody response was determined using nonlinear regression curve fitting software (fitting method: least squares (ordinary) fit).
  • Clones meeting these criteria were subjected to epitope binning.
  • the method was run on an Octet Red 384 using software version 8.0.2.5 at 25 °C.
  • Each clone (diluted to 15 ⁇ g/ml) was, in turn, loaded onto ProA sensors (180 s, soaked in PBSF for at least 10 minutes before use).
  • the sensors were then blocked using EpoFix IgGl W T(50 ⁇ g/ml, 300 s), then washed in PBSF and dipped into each clone (15 ⁇ g/ml, 60 s). This was a self binding check to ensure that no binding occurred in the absence of LAG3.
  • Clones 57B02, 57E02, 57C06 and 57H08 competed for binding to LAG3 and were assigned to the same epitope bin. They have the following properties:
  • Table 8 shows that clones show variation in their CDRL1 and CDRL3 sequences. mAbs having three amino acid substitutions in CDRL1 (at 3 positions within CDRL1 compared to the CDRL1 sequence of 57E02) meet the criteria set out in Example 4. The table also shows that a large degree of variation is permitted at CDRL3, suggesting that the precise sequence of this CDR may not be essential.
  • VH and VL sequences for clone 57E02 was codon optimised using an automated
  • amino acid sequence of the light chain is given by SEQ ID NO: 50
  • the DNA sequence of the light chain is given by SEQ ID NO: 51.
  • amino acid sequence of the heavy chain is given by SEQ I D NO: 52
  • DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK and the DNA sequence of the heavy chain is given by SEQ. ID NO : 53.
  • the heavy and light chain DNA sequences were ordered as gBlocks from IDT and cloned into pTT5 expression vector containing the IgGl LAGA disabled heavy and light chain constant regions digested with Agel-HF and Apal-HF using the In-Fusion HD Cloning Kit (Clontech) according to the manufacturer's instructions.
  • Example 6 Cloning and expression of PD-l/LAG-3 mAbdAb mAb-dAbs were initially generated by amplifying the mAb and the dAb sequences and then assembling the PCR products using SOE PCR followed by cloning into the pTT5 vector utilising Agel and EcoRI restriction endonuclease recognition sites to enable In Fusion cloning which was carried out according to manufacuturer's instructions.
  • the VH region in the mAb-dAb containing the 51A09-188001 dAb was replaced with a gBlock containing the 57E02 VH region using the same cloning strategy as described above for cloning the 57E02 mAb (see Example 7).
  • the light chain amino acid and DNA sequences of the mAbdAb is identical to the light chain amino acid and DNA sequences of the mAb 57E02 (generated in Example 6).
  • the heavy chain amino acid sequence of the 57E02x51A09-188001 mAbdAb is given by SEQ ID NO: 54:
  • DKSRWQQGNVFSCSVM HEALHN HYTQKSLSLSPGKGSTGLDSPTEVQLLESGGGLVQPGGSLRLSCAA
  • the heavy chain nucleotide sequence of the 57E02x51A09-188001 mAbdAb is given by SEQ. I D NO: 55:
  • LAG3/PD1 mAbdAbs were identified and profiled in the suite of in-vitro assays described. Three molecules with desired functional activity and distinct CDR binding sites for either LAG3 or PD1 were evaluated in-vivo. 57E02-51A09-188001 was determined to have the best combination of in-vitro and in-vivo characteristics including: affinity, ability to enhance T cell activation and function, in-vivo target engagement, pharmocokinetic and pharmocodynamic profile.
  • the mixed lymphocyte reaction (MLR) assay is an in-vitro correlate of T cell function. It evaluates the ability of T cells to make cytokines in response to an allogenic stimulation. In this assay, the stimulation is due to the major histocompatibility (MHC) antigenic differences of dendritic cells from three distinct donors.
  • MHC major histocompatibility
  • PBMCs Peripheral blood mononuclear cells
  • RPMI 1640 medium containing 10% FBS.
  • CD4+ T cells from rested PBMCs were isolated (using Stemcell Technologies CD4 negative selection/ enrichment kit - catalogue number 19052).
  • CD4 T cells were resuspended at 1 x 10 6 /mL j n RPMI + 10% FBS.
  • CD4 T cells were plated in triplicate with 1X10 5 cells in ⁇ / ⁇ in tissue culture treated U-bottom plates.
  • the final concentrations of each antibody tested was 200, 50, 12.5, 3.125, 0.781 and 0.195nM. Plates were placed in a 37 ° C humidified incubator with 5% C0 2 for 5 days. On Day 6, the supernatant was collected and tested for IFNy production using a Meso Scale Diagnostics kit: V-PLEX Proinflammatory Panel 1 (human), catalogue number K151A0H-4.
  • the MLR assay was used to compare the functional activity of the bispecific LAG3/PD1 mAbdAb (57E02-51A09-188001) to the combination of its component LAG 3 mAb (57E02) and PD1 dAb (51A09-188001) functional arms.
  • Antibodies evaluated in the assay 1) the LAG3/PD1 bispecific 57E02-51A09-188; 2) a LAG 3 monoclonal antibody 57E02; 3) a control anti- respiratory syncytial virus (aRSV) mAb linked to the PD1 dAb present in the bispecific aRSV- 51A09-188001; 4) a negative control antibody (VHDUM) that contains the RSV antibody linked to an irrelevant dAb (that has no known binding activity); 5) the combination of the LAG3mAb (57E02) and the PD1 mAbdAb-(aRSV-51A09-188001).
  • This assay was also used to evaluate the CTLA4/PD1 mAbdAb by comparing the levels of IFNy produced by the bispecific to the same control and PD1 mAbdAbs listed above as well as to a CTLA4 mAb.
  • LAG3/PD1 bispecific mAbdAb facilitates significantly greater cytokine production from T cells than either LAG3, PD1 or the combination of the two antibodies.
  • T cells proliferate or expand both in-vitro and in-vivo when they recognize their cognate antigen displayed on antigen presenting cells.
  • Blocking the inhibitory signals from LAG3 a PD1 improves the antiviral response by facilitating the expansion/proliferation of virus specific CD8 T cells.
  • the following assay protocol was used to characterize the ability of LAG3-PD1 mAb-dAbs to enhance the proliferation of HIV Gag Specific CD8 T cells.
  • PBMCs Peripheral blood mononuclear cells from HIV-infected stably antiretroviral treated donors were thawed in AIM-V Serum Free Media (Thermofisher) with Efavirenz (EFV) and DNASe (Stem cell technologies) added.
  • AIM-V Serum Free Media Thermofisher
  • Efavirenz Efavirenz
  • DNASe Stem cell technologies
  • PBMCs were washed, counted and resuspend cells at 5 million/ml in DPBS.
  • Carboxyfluorescein succinimidyl ester obtained from Life Technologies Cat # C34554 is a cell permeable fluorescent cell staining dye.
  • CFSE was resuspended in dimethyl sulfoxide (DMSO) at a ImM stock concentration.
  • DMSO dimethyl sulfoxide
  • CFSE labelling was performed adding 0.25 ⁇ of diluted CFSE per 1ml of PBMCs (0.25 ⁇ solution) for 5mins in the 37C incubator with gentle mixing during the incubation.
  • the labelling reaction was stopped with 25ml of cold FBS and the labelled cells washed and counted.
  • CFSE labelled PBMCs were resuspended at lOmillion/ml and 50 ⁇ of cells to triplicate well of the 96 well U Bottom plate containing the appropriately diluted antibody from step #1.
  • HIV Gag overlapping peptides were obtained from JPT Inovative Peptide Solutions (PepMix Gag Ultra #PM-HIV-GAG) and resuspended in DMSO at a 0.5 ⁇ / ⁇ stock concentration.
  • a Gag Peptide stimulation mix was made adding Gag peptides to AIMV+EFV media ⁇ g/ml, 2X concentration).
  • Gag peptide mix was added to each well of the plate containing 50 ⁇ of labelled PBMCs and 50 ⁇ of diluted antibody.
  • Each plate has triplicate control wells that were either unstimulated (negative control), Gag stimulated in the absence of binding proteins, or Phytohemagglutinin- M (Sigma-Aldrich ⁇ g/ml stimulated cells (positive control).
  • Proliferating CD8 T Cells are defined as: CFSE dim, Live (Amine -ve), CD8+ T cells i.e.( CD3+, CD8+, CD4).
  • results shown are the average from two independent runs of the assay, each run performed in triplicate, from an HIV infected stably treated donor.
  • Method 17 Intracellular Cytokine Assay: This assay was used to evaluate the ability of the LAG3/PD1 antagonist mAbdAb (57E02-51A09-188001) to increase the function of T cells.
  • T cells are activated by MHC mediated recognition of antigens displayed on antigen presenting cells. Binding of the T cell Receptor (TCR) to the MHC-peptide complex results in the activation of a signaling pathway that induces the production of cytokines and/or increased cytotoxic activity.
  • Bacterial toxins such as staphylococcal enterotoxin can be used to crosslink the TCR to MHC molecules in-vitro leading to T cell activation and cytokine production. Cytokine production and cytotoxic activity are hallmarks of functional CD4 and CD8 T cells respectively. Intracellular cytokine staining is a powerful method to measure the ability of T cells to produce multiple cytokines and increase their cytotoxic potential, it is commonly used in clinical trials to monitor the quality and quantity of the T cell response.
  • PBMC peripheral blood mononuclear cells
  • Rested PBMCs were resuspended at lOmillion/mL in R10+EFV containingGolgiStop protein transport inhibitor containing monensin (BD Biosciences Cat. No. 554724) at 0.7 ⁇ / ⁇ -. 50 ⁇ of the cell culture mixture was added to the non-tissue culture- treated 96 well U bottom plates containing the corresponding 4x antibodies.
  • CD107a also known as Lysosome-Associated Membrane Protein 1 (LAMP-1), (Clone:
  • H4A3, BioLegend Cat. No. 328620 was included in the culture medium as a measure of cytotoxic potential/degranulation.
  • SEB/A staphylococcal enterotoxin A & B
  • List Biologicals Bacterial antigens such as SEB/A crosslink the TCR& MHC molecules in-vitro leading to T cell activation and cytokine production.
  • PD1 (Clone: EH12) from Biolegend
  • CD45RO (Clone: UCHL1) from Beckman Coulter and the Live/Dead Amine Aqua Dye from Life Technologies.
  • TNFa Clone: Mabll
  • IFNy Clone: 4S.B3
  • IL2 Clone: MQ1-17H12
  • Cells were acquired on a BD LSRII- Fortessa using the high throughput sampler.
  • Flow cytometric gating was used to identify single cell populations based on area and height, lymphocytes were gated based forward and side scatter properties. The fraction of viable/live cells was then determined by the exclusion of an amine dye. T cells were gated based on CD3, CD4 and CD8 expression. Na ' ive cells express high levels of CD27 and lack CD45RO expression. Na ' ive cells (CD45RO- &CD27+) were excluded from further analysis focusing on memory and effector populations.
  • CD4 helper T cells The primary function of CD4 helper T cells is to produce cytokines such as IL2 that can augment the function of antibody producing B cells and cytotoxic CD8 T cells.
  • This assay was used to evaluate the ability of the LAG3/PD1 antagonist mAbdAb (57E02- 51A09-188001) to increase HIV RNA production.
  • Persistent latently infected CD4 T cells are a major barrier to an HIV cure. These latently infected cells express no/low levels of HIV RNA and protein rendering them invisible from the immune system. TCR stimulation can lead to both cellular activation and viral reactivation. Inducing viral reactivation and the expression of viral RNA/ protein from CD4 T cells allows the immune system to recognize and specifically target these infected cells for clearance. Latency reversal or HIV reactivation can be assessed in-vitro by increased levels of HIV cell associated RNA and proteins produced by CD4 T cells.
  • PBMCs Peripheral blood mononuclear cells
  • CD4+ T cells from rested PBMCs were isolated (using Stemcell Technologies CD4 negative selection/ enrichment kit - catalogue number 19052).
  • CD4 T cells were resuspended at 1 x 10 6 /mL j n RPMI + 10% FBS.
  • CD4 T cells Twenty replicates of 1X10 5 CD4 T cells were place in tissue culture treated U-bottom plates at ⁇ / ⁇ . Control or LAG3/PD1 mAbdAbs were used at a concentration of 125nM. Each antibody was added per well to CD4 T cells and the mixture was incubated for 15 mins at room temperature. During the incubation, monocyte derived dendritic cells (MDDC) expressing PDL-1 and MHC-II from three healthy donors were thawed and resuspended at 0.2 x 10 6 /mL in RPMI + 10% FBS. MDDCs were added to appropriate wells containing CD4 T cells and binding proteins. Wells containing only CD4 T cells were used as negative controls.
  • MDDC monocyte derived dendritic cells
  • a master mix using Fast Virus 1-step Master Mix reagent was used. A total of 5ul of isolated sample was used for the input. The final concentration of primers in the master mix were 900nM and the probe was 250nM. Samples were run on the OuantStudio 3 under the following PCR cycle conditions: 50 degrees at 10 minutes for reverse transcription, 95 degrees for 20 seconds to denature for 1 cycle, then 50 cycles of a 95 degree 3 second denature followed by a 60 degree 30 second anneal/extend step. The concentration of Gag was calculated by taking the number of copies per reaction and adjusting it for the volume of RNA eluted and normalized to the number of cells that were extracted.
  • Persistent latently infected CD4+ T cells remain a major barrier for an HIV Cure.
  • the first step in eliminating this viral reservoir is to induce HIV RNA and protein expression such that the antiviral response can recognize and eliminate these cells.
  • CTLA4/PD-1 mAbdAb clone CTLA-4 x 51A09-188001 was cloned, expressed and purified.
  • the amino acid sequence of the light chain is given by SEO ID NO: 79
  • the MLR assay (method 15) was used to assess the functional activity of CTLA4/PD1 mAbdAb.
  • Antibodies to CTLA4, PD1 (aRSV-51A09-188), the combination of the two antibodies, the CTLA4/PD1 mAbdAb or a control antibody were cocultured with monocyte derived dendritic cells and CD4 T cells as previously described. The levels of I FNy produced after 6 days of culture was determined. Co-culture with the bispecific CTLA4/PD1 mAbdAb enhances cytokine production compared to CTLA4 or PD1 blockade alone or the control antibody (1.42-12.28 fold).
  • SEQ ID NO: 2 Light chain variable domain of clone 37Y056-57E02-1)
  • SEQ ID NO: 7 (CDRL1 LAG 3, Kabat definition)
  • SEQ ID NO: 8 (CDRL2 LAG 3, Kabat definition)
  • SEQ ID NO: 10 (CDRHl (Kabat definition) of clones 22D034-51A09-188, 22D034-51A09-183, 22D034-51A09-140, 22D034-51A09-135, 22D034-51A09-126, 22D034-51A09-110, 22D034- 51A09-109, 22D034-51A09-73, 22D034-51A09-55, 22D034-51A09-54, 22D034-51A09-30, 22D034-51A09-16, 22D034-51A09-13, 22D034-51A09-10 and 22D034-51A09-4)
  • THYMV SEQ ID NO: 11 CDRH2 (Kabat definition) of clones 22D034-51A09-188, 22D034-51A09-140, 22D034-51A09-109, 22D034-51A09-73, 22D034-51A09-30 and 22D034-51A09-13)
  • SEQ ID NO: 12 (CDRH3 (Kabat definition) of clones 22D034-51A09-188, 22D034-51A09-183, 22D034-51A09-110, 22D034-51A09-109, 22D034-51A09-73, 22D034-51A09-30, 22D034- 51A09-13 and 22D034-51A09-4)
  • WMG WINPNSGGTN SEQ ID NO: 23 (CDRH3 LAG -3, Contact definition)
  • SEQ ID NO: 24 (CDRL1 LAG-3, Contact definition)
  • SEQ ID NO: 38 (CDRH3 (Kabat definition) of clones 22D034-51A09-126 and 22D034-51A09- 55)
  • SEQ ID NO: 46 forward primer oligonucleotide
  • SEQ ID NO: 47 reverse primer oligonucleotide
  • SEQ ID NO: 48 forward primer oligonucleotide
  • SEQ. I D NO: 50 (Amino acid sequence of light chain of LAG -3 mAb 57E02 and PD-l/LAG-3 mAbdAb 57E02x51A09-188001)
  • SEQ I D NO: 51 DNA sequence of light chain of LAG -3 mAb 57E02 and PD-l/LAG-3 mAbdAb 57E02x51A09-188001)
  • SEQ I D NO: 52 (Amino acid sequence of heavy chain of LAG-3 mAb 57E02)
  • DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ I D NO: 53 (DNA sequence of heavy chain of LAG -3 mAb 57E02)

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Abstract

La présente invention concerne une protéine de liaison spécifique pour au moins l'un des récepteurs de points de contrôle immunitaires PD-1 et LAG-3. Dans un mode de réalisation, l'invention concerne une protéine de liaison spécifique à la fois pour PD-1 et LAG -3. L'invention concerne également des procédés de préparation des protéines de liaison, des compositions pharmaceutiques contenant les protéines de liaison et des utilisations médicales des protéines de liaison.
PCT/EP2017/077891 2016-11-02 2017-10-31 Protéines de liaison WO2018083087A2 (fr)

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