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1. (WO2018083087) BINDING PROTEINS
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BINDING PROTEINS

Field of the Invention

The present invention relates to a binding protein specific for at least one of the immune checkpoint receptors PD-1 and LAG-3. In one embodiment, 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.

Background of the Invention

T-cell co-stimulatory and co-inhibitory molecules (collectively named co-signalling 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). Following recognition of cognate peptide-MHC complexes on antigen-presenting cells by the T-cell receptor, 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). The ultimate immune response is regulated by a balance between co-stimulatory and co-inhibitory signals ("immune checkpoints") (Pardoll 2012, Nature 12: 252-264). Programmed death-1 (PD-1) and Lymphocyte Activation Gene 3 (LAG-3) function as immune checkpoints as outlined below.

PD-1

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). 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 (PD-L1 and PD-L2) 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).

It is well characterized that in the context of infection, antigen specific CD8+ T cells initially acquire effector function but gradually become less functional during chronic infections, allowing infected cells to survive and evade immune surveillance (Wherry et al., Immunity, 27(4): 670-684, 2007). This 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).

Bacterial Infections

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).

Parasitic Infections

The role of PD-1 in exhaustion during malaria infection is reviewed by Wykes and Colleagues (Front Microbiol, 2014, 27(5): 249) and potential treatment by blockade of PD-1 in combination with other immune checkpoint inhibitors has been proposed (see section entitled PD-l/LAG-3).

Viral Infections

Erikson and colleagues have shown that PD-1 mediated CD8+ T cell impairment occurred in mice following infection with human metapneumovirus or influenza virus and PD1 and PDL1 have been shown to upregulated in the lungs of patients with 2009 H1N1 influenza virus, respiratory syncytial virus or parainfluenza virus suggesting that PD-1 blockade may represent a therapeutic target in the treatment of viral respiratory infections (Erikson et al., J Clin Invest 2012 122(8) 2967-82).

PD-1 blockade is also thought to have utility in the treatment of other chronic viral infections. In one clinical trial, PD-1 blockade with an anti-PDl fully human monoclonal antibody (BMS-936558) lead to persistent suppression of HCV replication in some patients with chronic infection. Ye and colleagues (Cell Death Dis, 2015, 6: el694) 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. Similarly, 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. 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. Even though existing HIV treatment can suppress viral replication to low levels, HIV is not completely eliminated and low level viral expression persists in tissues with poor drug penetrance and/or from the activation of latently infected cells. It is believed that the continuous stimulation of the immune system by viral antigens results in persistent inflammation in HIV infected individuals despite anti-retroviral therapy (ART). There is an increased incidence of several non-AIDS morbidities and mortalities including cardiovascular disease, neurocognitive impairment and frailty in patients considered well controlled by ART (Deeks, Annu Rev Med., 2011, 62: 141-55). This increased incidence occurs in the context of elevated systemic inflammation, related to the persistant immunologic damage caused by HIV and other opportunistic infections (Hunt et al., J Infect Dis., 2014, 210(8): 1228-1238; Tenorio et al., J Infect Dis., 2014, 210(8): 1248-1259; Kuller et al., PLOS Med, 2008, 5(10): e203). 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. De Fonseca et al., showed that 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. Additional work by Bristol Myers Squibb found that PDL-1 blockade in ART suppressed SIV infected macaques resulted in reduced viremia in approximately half of the animals upon treatment interruption (Whitney et al., 6th International Workshop on HIV persistence during therapy, Miami, FL, 2009).

Sepsis

Treatment of cells from septic patients with anti-PD-1 or anti-PD-Ll antibody decreased apoptosis and increased IFN-γ and IL-2 production (Chang et al., Crit Care, 2014, 18(1): R3). Also, fewer PD1 knockout mice compared to wild type mice died in an experimentally induced sepsis model (Huang et al., Proc. Natl. Acad. Sci., 2009, 106(15): 6303-8). Another group found that, PD-Ll expression in the liver was increased in this animal model. In the same study, it was found that administration of anti-PD-Ll decreased levels of certain proinflammatory cytokines (Zhu et al., Mediators Inflamm, 2013, 361501).

Cancer

In addition, as mentioned above, 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).

Known PD-1 binding proteins

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.

US20110008369; US20130017199; US20130022595; US20110171220; US20110171215; and

US20110271358 and in WO2006121168; WO20091154335; WO2012145493;

WO2013014668; WO2009101611; EP2262837; and EP2504028. Combinations of CTLA-4 and PD-1 antibodies are described in US Patent No. 9,084,776.

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. The amino acid sequence for nivolumab and methods of using and making it are disclosed in US Patent No. US 8,008,449. A phase I double blind placebo controlled ascending single dose study assessing the safety and immune response of this antibody in fully suppressed HIV infected patients under combination antiretroviral therapy is underway. 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

Endpoints, presentation at the International AIDS Symposium in Paris, 2017).

The anti-PD-Ll antibody BMS-936559 also targets the PD1-PDL1 axis. In 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).

The ability of 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

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. Several cell types

express LAG-3. For example, 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. For example, 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.

Immunol. 7(2):428-439).

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. For example, 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-γ 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). Furthermore, CD4+CD25+ regulatory T cells (Treg) 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).

Like PD-1, 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

Whilst the T cell response in LAG-3-deficient mice is similar in size and function to that in wild type animals, it has recently been found that in mice infected with LCMV, CD8(+) T cells exhibit a slightly reduced rate of cell division in comparison with LAG-3-deficient cells, showing that LAG-3 directly modulates the size of the T cell response (Cook et al., 2016, J Immunol., 197(1): 119-127). Erikson et al., 2016, J Immunol., 197(1): 233-43 showed that blockade of LAG-3 in PD-1 deficient mice infected with human metapneumovirus resulted in increased CD8 effector function (compared to mice not treated with an anti-LAG-3 mAb).

Specifically in relation to HIV, Fromentin and colleagues identified that in HIV individuals controlled on anti-retroviral therapy, expression of LAG-3 on CD4+ T cells was associated with integrated HIV genomes), a proportion of which can be induced to express HIV genes (Fromentin et al., PLoS Pathog., 2016, 12(7): el005761). Tian and colleagues (J. Immunol. 194:3873-3882, 2015) showed that treatment of peripheral blood mononuclear cells derived from HIV infected individuals naive to anti-retroviral therapy with a LAG-3-Fc chimera increased proliferation and cytokine produced by CD4+ and CD8+ cells in response to B-Gag stimulation.

Antibodies to LAG-3, and methods of using in treatment of disease are described in

US20110150892, US 20150259420, and US Patent No. 6,143,273. Several anti-LAG-3 monoclonal antibodies including BMS-986016 (BMS) and LAG525 (Novartis) have also progressed into clinical trials for the treatment of cancer.

Parasitic infections

Doe et al. (2016, Microbiol. Immunol., 60(2): 121-131) showed that CD4+ T cells from mice infected by Plasmodium expressed LAG-3. In vivo blockade of PD-L1 and LAG-3 restored CD4(+) T cell function, amplified the number of follicular helper T cells, germinal-center B cells and plasmablasts, enhanced protective antibodies and rapidly cleared blood-stage malaria in mice (Butler et al., 2011, Nat Immunol., 13(2): 188-95).

Sepsis

Boomer et al., 2012, Crit Care, 16(3): R112 showed that the levels of LAG 3 (and Tim 3) on CD4+ cells from patients admitted to hospital with severe sepsis increased over a period of 7 days.

Cancer

Grosso and colleagues (J Clin I nvest., 2007, 117:3383-3392) showed that antigen-specific CD8+ T cells within antigen-expressing organs or tumors exhibited increased levels of LAG-3 protein. LAG-3 blockade or genetic ablation resulted in increased accumulation and effector function of antigen-specific CD8+ T cells within organs and tumors that express their cognate antigen and combining LAG-3 blockade with specific antitumor vaccination resulted in a significant increase in activated CD8+ T cells in the tumor and disruption of the tumor parenchyma.

Known LAG-3 binding proteins

Antibodies to LAG-3, and methods of using in treatment of disease are described in

US20110150892, US 20150259420, and US Patent No. 6,143,273. Several anti-LAG-3 monoclonal antibodies including BMS-986016 (BMS) LAG525 (Novartis) have also progressed into clinical trials for the treatment of cancer.

PD-l/LAG-3

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)). I n addition, the degree of CD8+ T cell exhaustion, e.g., as shown by the percentages of dual I FNY/TN FCX producers, correlates with the number of inhibitory receptors expressed (Blackburn et al. (2009) Nat. I mmunol. 10(1): 29-37). At the subcellular level, 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. For example, 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. 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). In the context of a murine model of Plasmodium infection that induces T cell exhaustion, antagonizing LAG-3 and PD-1 restored CD4+, CD8+ and Tfh function and resulted in the clearance of

Plasmodium infection in blood (Butler et al., Nat Immunol. 13(2): 188-95, 2011).

Several clinical trials are underway for the treatment of solid tumours, melanoma, glioblastoma, colorectal cancer, virus associated tumors and hematological tumours using an anti-LAG-3 monoclonal antibody (LAG525, RGN3767, IMP321, and BMS 986016 in combination with an anti-PD-1 antibody (PDR001, REGN2810, pembrolizumab and nivolumab). Further, Macrogenics are conducting phase I trials with MGD013, a tetravalent, bsispecific (PD-1 x LAG3), Fc bearing DART protein with a human lgG4 backbone for the treatment of solid tumours and hematologic neoplasms. (Motte-Mohs, MGD013, a

Bispecific PD-1 x LAG-3 Dual Affinity Re-Targeting (DART) Protein with T-cell

Immunomodulatory Activity for Cancer Treatment, Poster 3217, presented at the 2016 American Association for Cancer Research Annual Meeting, April 16-20, 2016, New Orleans, Louisiana). TESARO are also reportedly in preclinical development with a bispecific anti-LAG-3/PD-1 antibody for cancer.

Other Immune Checkpoints

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).

Several other inhibitory receptors are now targets to improve T cell responses. In animal models, it has been shown that combining blockade of Tim-3 with blockade of the PD-1 pathway can further improve T cell rescue (Sakuishi Ket al., J Exp Med., 2010, 207:2187-2194; Jin et al. Proc Natl Acad Sci U S A., 2010, 107:14733-14738). Similarly, Jun et al. (Jun et al., Generation of antagonistic anti-TIM-3 and anti-LAG-3 monoclonal antibodies for potential novel immunotherapy combinations, Poster LB266, presented at the 2016

American Association for Cancer Research Annual Meeting, April 16-20, 2016, New Orleans, Louisiana) showed that anti-Tim in combination with anti-PD-1 increases IL-2 secretion in the mixed lymphocyte reaction and decreases the effective EC50 of each single agent.

Summary of the Invention

In a first aspect, 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. 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:l by the addition or deletion or substitution of 1, 2 or 3 amino acids;

wherein 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 amino acids, wherein CDRL2 is selected from the group consisting of: CDRL2 as 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 the group consisting of: 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;

wherein the one or more epitope binding domains specific to human PD-1 comprise 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 I D 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 I D 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 I D 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; and

wherein the linker is selected from the group consisting of a bond or a peptide linker.

I n one embodiment, 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.

I n a more particular aspect, the invention provides a binding protein specific to human LAG-3 and human PD-1 having the general formula (I):

L (LAG-3)

VH(PD-1) - A - H '(LAG-3)

( 4 )n

VH(PD-1) - A -H (LAG-3)

L '(LAG-3)

(I)

wherein:

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 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; 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.

In one embodiment, 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.

In a second aspect, 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. In one embodiment, the binding protein exhibits an IC50 of less than or equal to 5 nM in the PD-l/PDL-1 competition assay.

In a more particular aspect, the binding protein specific to human PD-lis additionally capable of neutralising a human checkpoint inhibitor other than PD-1. In the context of this invention, 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.

In a third aspect, 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. 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: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 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, wherein CDRL2 is selected from the group consisting of: CDRL2 as 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 the group consisting of: 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.

In one embodiment, the binding protein specific to human LAG-3 exhibits >50% inhibition of LAG3-MHCII interaction in a competition flow cytometry assay.

In a more particular aspect, the binding protein specific to human LAG-3 is additionally capable of neutralising a human checkpoint inhibitor other than LAG-3. As explained above, 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.

In further aspects, 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. For example, the invention provides isolated nucleic acids encoding H-A-VH(PD-I), H and L, wherein H, A, VH(PD-I) and L are as defined above. The invention also provides a method of producing a binding protein of the invention comprising culturing a host cell containing appropriate vector(s) under conditions suitable for protein expression, and isolating the binding protein.

In one aspect, 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. Specifically, the invention provides methods of treating cancer and methods of treating and curing HIV.

Brief Description of the Figures

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. Note that 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 Data0,2 transformed scale to make variances more homogeneous.

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.

Detailed Description of the Invention

Binding Proteins

Several different binding proteins are provided. These can be distinguished by the targets to which they bind. In separate aspects, 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).

In a first aspect, 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:l by the addition or deletion or substitution of 1, 2 or 3 amino acids;

wherein 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 amino acids, wherein CDRL2 is selected from the group consisting of: CDRL2 as 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 the group consisting of: 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;

wherein the one or more epitope binding domains specific to human PD1 comprise 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; and

wherein the linker is selected from the group consisting of a bond or a peptide linker. This defines the binding protein specific to human PD-1 and human LAG-3.

In one embodiment, the binding protein (i.e. the binding protein specific to human PD-1 and human LAG-3) 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.

In the context of the present invention, 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).

Alternatively, 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).

In one embodiment, the heavy chains comprise the CHI, CH2, and VH domains and the light chains comprise the CL and VL domains. In a more particular embodiment, 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. For the avoidance of doubt, antibodies are glycoproteins and the domains above may be glycosylated.

In the context of the invention, 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. In one embodiment, the epitope binding domain specific to human PD1 has CDRs permitting the binding protein to bind human PD-1. In a more particular embodiment, 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. In order to bind to PD-1, it is apparent that the CDR regions of the epitope binding domain must be held in an appropriate conformation by a protein scaffold. In one embodiment, 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.

In one embodiment, 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. In a more particular embodiment, 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. Other modifications may also be 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. In one embodiment, the single variable domain may be a VH domain.

In other embodiments, 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 γ-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).

In one embodiment, 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.

In another embodiment, 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.

In yet another embodiment, 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.

In another embodiment, 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.

In another embodiment, 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.

In yet another embodiment, 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) 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.

In yet another embodiment, 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) 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.

In the foregoing embodiments, 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:


As described above, each CDR may be modified by one, two or three amino acid substitutions, deletions or additions to form CDR variants. I n certain embodiment where 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. 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.

Typically, the modification is a substitution. In one embodiment, 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. In this regard, the skilled person would appreciate that 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.

Table 2:


In one embodiment, 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, X2 is W, F or Y, and X3 is A or N.

In another embodiment, 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, wherein CDRH1 has the sequence THYMX4 (SEQ ID NO: 57), wherein X4 is V or A, wherein CDRH2 has the sequence FIGPAGDX5TYYADSVX6G (SEQ ID NO: 58) wherein

X5 is T, F or S and X6 is K or E, and wherein CDRH3 is YTX7TSX8X9DXi0YDV (SEQ I D NO : 59), wherein X7 is A or E, Xs is G, S or D, Xg is V, F or Y, and Xio is T or S.

In one embodiment, 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.

In one embodiment, the antibody specific for LAG (that forms part of the binding protein specific to human PD-1 and human LAG-3) 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.

In one embodiment, the antibody specific for LAG (that forms part of the binding protein specific to human PD-1 and human 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 SEQID NO: 7, CDRL2 has the sequence defined as SEQ ID NO: 8 and CDRL3 has the sequence defined as SEQ ID NO:9.

In one embodiment, 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.

In one embodiment, 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. In a more particular embodiment, 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.

In another embodiment, 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. In a more particular embodiment, 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.

In another embodiment, 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 variant of SEQ ID NO. 3 that differs in having up to 10 amino acid additions, deletions or substitutions. In a more particular embodiment, 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.

It will be appreciated by one of skill in the art that 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. Typically, the modification is a substitution. In one embodiment, the sequences are modified by the substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In a more particular embodiment, the modification is a conservative substitution (a substitution of one amino acid residue for another residue in the same group of Table 2).

In one embodiment, 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.

In another embodiment, 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.

In another embodiment, 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. Alternatively, 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.

In a particular embodiment, the heavy chain and light chain of the antibody specific for human LAG-3 (that form part of the binding protein specific to human PD-1 and human LAG-3) and the one or more epitope binding domains specific to human PD1 (that forms part of the binding protein specific to human PD-1 and human LAG-3) 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 the heavy chain), SEQ ID NO. 2 (for the light chain) and SEQ ID NO.3 (for the epitope binding domain).

In one embodiment, 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.

In another embodiment, 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.

In yet another embodiment, 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.

In one embodiment, the one or more epitope binding domains specific to human PD-1 (that form part of the binding protein specific to human PD-1 and human LAG-3) has a reduced ability to bind to pre-existing antibodies (ADAs) as compared to the equivalent unmodified molecule. By 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.

In one embodiment, 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. In one embodiment, X is absent. In another embodiment, 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. In a further embodiment, 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.

In one embodiment, 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.

I n one embodiment, 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. In a more particular embodiment, there are two epitope binding domains, one attached to the C-terminus of each of the two heavy chains of the antibody specific to LAG-3.

The linker may be a bond (such as a peptide bond) or a peptide linker. I n one embodiment, 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). For the avoidance of doubt, where the linker is a peptide bond or a peptide linker, the epitope binding domain(s) specific to PD1 are expressed as a genetic fusion with one of the chains of the antibody specific to LAG-3.

I n another aspect, the invention provides a binding protein having the general formula (I):

L (LAG-3)

VH(PD-1) - A - H '(LAG-3)

( 4 )n

VH(PD-1) - A -H (LAG-3)

L '(LAG-3)

(I)

wherein:

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 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; 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 (I) is a binding protein specific to human PD-1 and human LAG -3.

In one embodiment, 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.

In one embodiment, H comprises the VH and CHI domains and L comprises the CL and VL domains. In a more particular embodiment, H comprises the CHI, CH2, and VH domains and L comprises the CL and VL domains. Even more particularly, 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. For the avoidance of doubt, antibodies are glycoproteins and the domains above may be

glycosylated.

In one embodiment H is a heavy chain of the IgGl, lgG2, lgG3, lgG4 or lgG4PE subclasses. When H is of the IgGl and lgG4 subclasses, n is 2. When H is of the lgG2 subclass, n is 4. When H is of the lgG3 subclass, n is 11. In a more particular embodiment, 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. In one embodiment, 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.

As described above, each CDR may be modified by one, two or three amino acid

substitutions, deletions or additions to form CDR variants. In certain embodiments, 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.

Typically, the modification is a substitution. In one embodiment, a CDR is modified by the substitution of 1, 2 or 3 amino acids. More particularly, the modification is a conservative substitution (a substitution of one amino acid residue for another residue in the same group of Table 2).

In one embodiment of the binding protein of formula (I), 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:

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.

In one embodiment of the binding protein of formula (I), 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.

In yet another embodiment of the binding protein of formula (I), 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.

In another embodiment of the binding protein of formula (I), 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.

In another embodiment of the binding protein of formula (I), 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.

In one embodiment of the binding protein of formula (I), L(LAG-3) comprises CDRL1 having the sequence RASQX1ISSX2LX3 (SEQ ID NO: 56), wherein Xi is G or S, X2 is W, F or Y, and X3 is A or N.

In one embodiment, VH(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.

In yet another embodiment, 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.

In a further embodiment, VH(PD-1) comprises CDRH1, CDRH2 and CDRH3, wherein CDRH1 has the sequence THYMX4 (SEQ I D NO: 57), wherein X4 is V or A, wherein CDRH2 has the sequence FIGPAGDX5TYYADSVX6G (SEQ I D NO: 58) wherein X5 is T, F or S and X6 is K or E, and wherein CDRH3 is YTX7TSX8X9DXi0YDV (SEQ I D NO: 59), wherein X7 is A or E, X8 is G, S or D, Xg is V, F or Y, and Xio is T or S.

In one embodiment, H(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.

In another embodiment), L(LAG-3) 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 SEQ ID NO: 7 and CDRL2 has the sequence defined as SEQ ID NO: 8.

In another embodiment), 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.

In one embodiment, VH(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.

In one embodiment, 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. In a more particular embodiment, 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.

In another embodiment, 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. In a more particular embodiment, 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.

In another embodiment, 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. In a more particular embodiment, 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.

It will be appreciated by one of skill in the art that 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.

Typically, the modification is a substitution. In one embodiment, the sequences are modified by the substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids. In a more particular embodiment, the modification is a conservative substitution (a substitution of one amino acid residue for another residue in the same group of Table 2).

In one embodiment, 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.

In another embodiment, 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.

In another embodiment, 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. Alternatively, 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.

In a particular embodiment, 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 VH), SEQ ID NO. 2 (for VL) and SEQ ID NO.3 (for VH(PD-l)).

In one embodiment, VH has the sequence defined as SEQ ID NO.l.

In another embodiment, VL has the sequence defined as SEQ ID NO.2.

In yet another embodiment, VH(PD-I) has the sequence defined as SEQ ID NO.3.

I n one embodiment, A is a peptide bond or a peptide linker from 1 to 100 amino acids in length. I n one embodiment, 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). For the avoidance of doubt, where A is a peptide bond or a peptide linker, VH(PD-1)-A- H(LAG-3) may be expressed as a genetic fusion.

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. It is believed that the effects on T cell activation and proliferation result from the blockade of PD-1 and LAG-3 signalling. As a result, it is reasonable to believe that this mAbdAb has potential to treat other medical indications (disclosed in the medical uses section) where there is a rationale for therapy with PD-l/LAG-3 blockade.

It is also noted that 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. Similarly, 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. However, of those tested, 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).

Based on all of the above data, it is reasonable to expect that the beneficial properties observed with 57E02x51A09-188001 mAbdAb would also be observed with related binding proteins having variant CDR and framework regions as disclosed in the above embodiments.

In a second aspect, 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. In one embodiment, the binding protein specific to human PD-1 exhibits an IC50 of less than or equal to 5 nM in the PD-l/PDL-1 competition assay.

In order to bind to PD-1, it is apparent that the CDR regions of the binding protein specific to human PD-1 must be held in an appropriate conformation by a protein scaffold. In one embodiment, 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. In one embodiment, 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. 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.

In other embodiments, 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 γ-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).

In particular embodiments, 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. In embodiments in which the binding protein specific to human PD-1 contains one or more CDR variants, the skilled person will appreciate that, in a particular embodiment, 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.

In particular embodiments, the binding protein specific to human PD-1 comprises the sequence defined as 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.

In one embodiment, 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. 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.

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. Levels of 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.

For cytokines whose production is normally increased upon stimulation, 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. In one embodiment, 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). In a more particular embodiment, 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). Even more particularly, 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). In one embodiment 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.

For cytokines whose production is normally decreased upon stimulation, 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. In one embodiment, 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). In a more particular embodiment, 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). Even more particularly, 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). In one embodiment 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.

Proliferation of immune cells is normally inhibited by human checkpoint inhibitors.

Accordingly, 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. In one embodiment, 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. In one embodiment, 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). In a more particular

embodiment, 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). Even more particularly, 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). In one embodiment, 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.

Accordingly, 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.

For transcription factors whose production, activation or phosphorylation status is normally increased upon stimulation, 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). In a more particular 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 two standard deviations (using the larger of the standard deviations taken in the presence or absence of binding protein). Even more particularly, 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). In one embodiment 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.

For transcription factors whose production, activation or phosphorylation status is normally decreased upon stimulation, 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). In a more particular 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 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.

In embodiments in which 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.

Several well characterized 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.

In one embodiment, 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

In one embodiment, 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). In one

embodiment, 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). In a more particular embodiment, 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). Even more particularly, 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). In one embodiment, 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.

In one embodiment, the human checkpoint inhibitor other than PD-1 is selected from the group consisting of CTLA-4, TIM-3, CD160 and TIGIT. In a more particular embodiment, 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. In a more particular embodiment, 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). In a more particular embodiment, 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. In one embodiment, 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. In a more particular embodiment, the linker has the sequence set out as SEQ. ID NO. 30 (STGLDSPT). For the avoidance of doubt, where the linker is a peptide bond or a peptide linker, the one or more domains specific for binding human PD-1 are expressed as a genetic fusion with one or more domains specific for a human checkpoint inhibitor other than PD-1.

In a third aspect, the invention provides a binding protein having the general formula (II):

L (CPI)

VH(PD-1) - A - H (CPI)

{ )η

VH(PD-1) - A -H (CPI)


(N)

wherein:

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.

In one embodiment, 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.

In one embodiment, H(CPI) comprises the VH and CHI domains and L(CPI) comprises the CL and VL domains. In a more particular embodiment, H(CPI) comprises the CHI, CH2, and VH domains and L(CPI) comprises the CL and VL domains. Even more particularly, 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. For the avoidance of doubt, antibodies are glycoproteins and the domains above may be

glycosylated.

In one embodiment H(CPI) is a heavy chain of the IgGl, lgG2, lgG3, lgG4 or lgG4PE subclasses. When H(CPI) is of the IgGl and lgG4 subclasses, n is 2. When H(CPI) is of the lgG2 subclass, n is 4. When H(CPI) is of the lgG3 subclass, n is 11. In a more particular embodiment, 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). In one embodiment, 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). For example, in one embodiment, A is a peptide bond or a peptide linker from 1 to 100 amino acids in length. In one embodiment, 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. In a more particular embodiment, the linker has the sequence set out as SEQ. ID NO. 30 (STG LDSPT). For the avoidance of doubt, where A is a peptide bond or peptide linker, VH(PD-1)-A- H(CPI ) may be expressed as a genetic fusion.

The PD-1 dAb 51A09-188001 is capable of functioning to block PD-1 signalling in the context of a mAbdAb as shown by its function in the anti-RSV-PDl mAbdAb (RSV-51A09-188001) the LAG-3/PD-1 mAbdAb (57E02x51A09-188001) and in a mAbdAb with an anti CTLA-4 mAb (CTLA4-51A09-188001, it was determined to have an IC50 of 0.24 (n=l) and 0.29 (n=2) in the human PD1-PDL1 inhibition assay. This CTLA4/PD1 bispecific also facilitates cytokine production in the mixed lymphocyte reaction assay. Based on this it is reasonable to expect that it will be able to form functioning mAbdAbs of formula (II) with mAbs to other checkpoint inhibitors, including but not limited to CTLA4, OX40, TIM-3, CD160 and TIGIT. mAbs to these antibodies are already known in the art.

As explained above, 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.

In a fourth aspect, 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. 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: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 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, wherein CDRL2 is selected from the group consisting of: CDRL2 as 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 the group consisting of: 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.

In one embodiment, the binding protein specific to human LAG-3 exhibits >50% inhibition of LAG3-MHCII interaction in a competition flow cytometry assay.

In one embodiment, the binding protein specific to human LAG-3 is an antibody. In the context of this embodiment, 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). Alternatively, 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).

In one embodiment of the binding protein specific to human LAG-3, the heavy chains comprise the CHI, CH2, and VH domains and the light chains comprise the CL and VL domains. In a more particular embodiment, 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. For the avoidance of doubt, antibodies are glycoproteins and the domains above may be

glycosylated.

In other embodiments, 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 γ-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).

In one embodiment, 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. In embodiments in which the binding protein specific for human LAG-3 contains one or more CDR variants, the skilled person will appreciate that, in a particular embodiment, 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.

In particular embodiments, 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.

In particular embodiments, 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.

In one embodiment, the binding protein specific to human LAG-3 is additionally capable of neutralising 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).

In one embodiment, the human checkpoint inhibitor other than LAG-3 is selected from the group consisting of CTLA-4, TIM-3, CD160 and TIGIT. In a more particular embodiment, 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. In a more particular embodiment, 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. In a more particular embodiment, there are two binding 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 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).

In the context of the invention, 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. In one embodiment, the epitope binding domain specific to human PD1 has CDRs permitting the binding protein to bind human PD-1. In a more particular embodiment, 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. In order to bind to PD-1, it is apparent that the CDR regions of the epitope binding domain must be held in an appropriate conformation by a protein scaffold. In one embodiment, 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. In one embodiment, 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. In a more particular embodiment, the linker has the sequence set out as SEQ. ID NO. 30 (STGLDSPT). For the avoidance of doubt, where 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.

In a fifth aspect, the invention provides a binding protein having the general formula (III):

L (LAG-3)

1/1

CPI - A - H (LAG-3)

( )n

CPI - A -H (LAG-3)

1/1

t i

L ( LAG-3)

(IN)

wherein:

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; and

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.

In one embodiment, 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). In one embodiment, 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,

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.

In one embodiment, 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.

In other embodiments, 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 γ-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).

A is as defined above in any of the embodiments relating to a binding protein of formula (I). For example, in one embodiment, A is a peptide bond or a peptide linker from 1 to 100 amino acids in length. In one embodiment, 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. In a more particular embodiment, the linker has the sequence set out as SEQ. ID NO. 30 (STGLDSPT). For the avoidance of doubt, CPI-A- H(LAG-3) may be expressed as a genetic fusion.

Medical uses

As discussed in the section entitled Background of the Invention, 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.

Accordingly, in one embodiment, 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

leishmaniasis), 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.

Specifically in relation to HIV, 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. In parallel, 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). Therefore the inventors hypothesise that blockade of both receptors may provide a synergistic effect in treating or curing HIV infection.

In one embodiment, 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.

In the context of this invention, 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.

In another embodiment, 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.

On a population level, HIV treatment with the binding protein of the invention may result in a reduction in the incidence of several non-AIDS morbidities and mortalities. At the level of the individual patient, 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. Whilst it will be apparent to the skilled reader that 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.

Accordingly, in one embodiment, 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.

During an inflammatory response 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. In one embodiment, 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). In a more particular embodiment, 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). Even more particularly, 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). In one embodiment, 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. In one embodiment, 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.

In one embodiment, HIV treatment refers to a reduction in expression of high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), or D-dimer. These markers of CVD risk may be measured in any biological sample. In one embodiment, levels of these markers are measured in plasma by any suitable method. In one embodiment, 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. In one embodiment, 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). In a more particular embodiment, 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). Even more particularly, 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). In one embodiment, 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. In one embodiment, 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.

In one embodiment, HIV treatment refers to a reduction in vascular inflammation as measured by a reduction in arterial fluorodeoxyglucose (FDG) uptake. In one embodiment, FDG uptake may be assessed by FDG-PET/CT. In one embodiment, 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. In one embodiment, 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). In a more particular embodiment, 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. Even more particularly, 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. In one embodiment, 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. In one embodiment, 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.

In another embodiment, HIV treatment refers to treatment resulting in a reduction of the reservoir over the treatment period. Given CD4+ T cells (which accounts for most of the HIV reservoir) have a 6 month half life, 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. In one embodiment, 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) are used as the source of cells. Most particularly, CD4+ T cells or subsets of CD4+ T cells are used. Methods of obtaining these cell populations are well known. Following isolation of the cells, nucleic acid may be extracted by conventional techniques. Any suitable technique may be used for quantification of HIV RNA. In one embodiment, the Amplicor Monitor assay (run according to the manufacturer's specifications) may be used (NOTE- where the "copies per ml" value exceeds 106, 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. In one embodiment, quantitative PCR techniques may be used to quantify integrated HIV DNA (e.g. a quantitative version of inverse PCR, or real-time PCR using a 7500 Real-Time PCR system, Applied Biosystems). Alternatively, where PCR incorporates a 32P labeled nucleotide, the PCR product may be quantified using a phosphorimager and suitable software (e.g.

I MAG EQUANT, Molecular Dynamics). 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. 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. In one embodiment, 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). In a more particular embodiment, 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). Even more particularly, 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). In one embodiment, 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. In one embodiment, the mean level of HIV genetic material from at least three samples at the end of the treatment period is 0.5logio copies per 106 cells less than the mean level of HIV genetic material from at least three samples at the beginning of the treatment period.

In a further embodiment, 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. In this embodiment, 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. Various HIV peptides may be used for antigen stimulation, including gag, pol, env, nef or pooled peptides. One or more cytokines (e.g. IFN-γ, IL-2, TNF) or cytotoxic products (e.g. CD107a, perforin or granzyme) produced following antigen stimulation may be monitored. The skilled person would be aware which cytokines/cytotoxic products should be measured for different cells types. For CD4 cells, suitable cytokines include IFN-γ, IL-2, TNFa. For CD8 cells, suitable cytokines include IFN-γ, IL-2, TNFa and suitable cytotoxic products include CD107a. In one embodiment, 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

cytokine/cytotoxic product at the beginning of the treatment period determined from at least three samples plus one standard deviation (using the larger of the pre- and post-

treatment standard deviations). In a more particular 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 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). Even more particularly, 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). 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 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. 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. In one embodiment, 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. In one embodiment, 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. In a more particular embodiment, 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. Even more particularly, 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. In one embodiment, 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.

In a further embodiment, 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.

As used herein, the term "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. In the context of cancer treatment, the term "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.

In one embodiment, 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

(including squamous pancreatic cancer), prostate cancer, sarcoma, osteosarcoma, giant cell tumor of bone, thyroid cancer, neuroblastoma, bladder cancer, urothelial cancer, lung cancer, vulval cancer, cervical cancer, cancer of the uterus, endometrial cancer, renal cancer (including kidney clear cell cancer, kidney papillary cancer, renal cell carcinoma),

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.

In another embodiment, 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

(undifferentiated or differentiated), acute promyeloid (or promyelocytic or promyelogenous or promyeloblastic) leukemia, acute myelomonocytic (or myelomonoblastic) leukemia, acute monocytic (or monoblastic) leukemia, erythroleukemia and megakaryocytic (or megakaryoblastic) leukemia. These leukemias may be referred together as acute myeloid (or myelocytic or myelogenous) leukemia (AML). Myeloid malignancies also include myeloproliferative disorders (MPD) which include, but are not limited to, chronic

myelogenous (or myeloid) leukemia (CML), chronic myelomonocytic leukemia (CMML), essential thrombocythemia (or thrombocytosis), and polcythemia vera (PCV). 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.

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. 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). High-grade B-NHLs include Burkitt's lymphoma (BL), Burkitt-like lymphoma, small non-cleaved cell lymphoma (SNCCL) and lymphoblastic lymphoma. Other 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. 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.

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. Hematopoietic cancers may also include other cancers of additional hematopoietic cells, including polymorphonuclear leukocytes (or neutrophils e.g. chronic neutrophilic leukaemia), basophils, eosinophils, dendritic cells, platelets, erythrocytes and natural killer cells. For example, haematopoietic cancers include plasmacytoma, immunoblastic large cell leukaemia, mantle cell leukaemia, acute megakaryocytic leukaemia, promyelocytic leykamia, erythroleukaemia and follicular lymphoma.

In a more particular embodiment, 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. In another aspect 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.

Combinations

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). 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.

When the binding protein is intended for use in the treatment or cure of HIV, 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 (including an agent having anti- oxidation activity such as immunocal, oltipraz, etc.) such as nevirapine, delavirdine, efavirenz, loviride, immunocal, oltipraz, 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: These agents inhibit viral attachment, co-receptor binding or membrane fusion: examples 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.

Table 3 lists several FDA approved antiretroviral therapies. Highly active antiretroviral therapy (HAART) generally includes a combination of drugs from several different classes for example a reverse transcriptase inhibitor, an integrase inhibitor and a protease inhibitor.

Table 3:

saquinavir (no Roche

1997 Fortovase

longer marketed) Pharmaceuticals

1999 Agenerase amprenavir, APV GlaxoSmithKline lopinavir+ ritonavir, Abbott

2000 Kaletra

LPV/RTV Laboratories atazanavir sulfate, Bristol-Myers

2003 Reyataz

ATV Squibb

fosamprenavir

2003 Lexiva GlaxoSmithKline calcium, FOS-APV

Boehringer

2005 Aptivus tripranavir, TPV

Ingelheim

Tibotec

2006 Prezista Darunavir

Therapeutics

Fusion Inhibitors

Roche

2003 Fuzeon Enfuvirtide, T-20 Pharmaceuticals &

Trimeris

Entry Inhibitors

2007 Selzentry Maraviroc Pfizer

Integrase Inhibitors

2007 Isentress Raltegravir Merck

2013 Tivicay Dolutegravir ViiV Healthcare

— — Cabotegravir

Thus, in one embodiment, 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. In another embodiment, 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

embodiments.

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. Examples of such pharmacological enhancers (or pharmakinetic boosters) 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.

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.

When used for the treatment or cure of HIV, the binding protein or binding

protein/antiretroviral combination may be co-administered together with one or more latency reversing agents, immune checkpoint agonists or antagonists or immune modulators. 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). 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.

When used for the treatment or cure of HIV, the 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

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. Alternatively, 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.

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.

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. In one embodiment, the binding protein may be

administered in conjunction with a treatment comprising adoptive cell therapy (ACT) of 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). In another embodiment, the invention provides an engineered cytotoxic cell capable of co-expressing a binding protein and a CAR or TCR.

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. Non-limiting examples of 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; and 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,

Ga733\EpCAM, HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB/70K, NY-CO-1, RCAS1, SDCCAG16, TA-90\Mac-2 binding protein\cyclophilin C-associated protein, TAAL6, TAG72, TLP, and TPS. In one embodiment, the cytotoxic cell is a human cell and the CAR or TCR are human antigens.

Where combination therapy is envisaged, the active agents may be administered simultaneously, separately or sequentially in one or more pharmaceutical compositions.

Pharmaceutical compositions

The 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.

Pharmaceutical 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). The

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. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.

In one embodiment, 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 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. In one aspect, 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.

Manufacture

The invention also provides isolated nucleic acids encoding the binding protein. For the binding protein of formula (I), this will be polynucleotide sequences encoding the amino

acid sequences represented by H-A- VH(PD-I) and L. For the binding protein of formula (II), this will be polynucleotide sequences encoding the amino acid sequences represented by H(CPI )-A-VH(PD-1) and L(CPI ). For other binding proteins, the sequences will vary (e.g.

according to the disposition of one or more epitope binding domains), but will readily be identifiable by one of ordinary skill in the art. It will be understood that such

polynucleotide sequences could be cloned into a vector. In one embodiment, 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. In some embodiment, each polynucleotide is cloned into a separate expression vector. In certain embodiments, 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. Alternatively, 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. In one embodiment, 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. Human cells may be used, thus enabling the molecule to be modified with human glycosylation patterns. Alternatively, 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)). However, due to the tendency of proteins expressed in bacterial cells to be in an unfolded or improperly folded form or in a non-glycosylated form, any binding protein produced in a bacterial cell would have to be screened for retention of biological activity. If 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. For example, various strains of E. coli used for expression are well-known as host cells in the field of

biotechnology. Various strains of B. subtilis, Streptomyces, other bacilli and the like may also be employed in this method.

Where desired, 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. Typically, the culture method of the present invention is a serum-free culture method, usually by culturing cells serum-free in suspension. Likewise, once produced, 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. In one embodiment, 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.

Other embodiments

Specific embodiments relating to binding proteins specific to human PD-1 are set out in the following numbered embodiments:

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. 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.

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 THYMX4, wherein X4 is V or A; wherein CDRH2 is FIGPAGDX5TYYADSVX6G wherein X5 is T, F or S and X6 is K or E; and wherein CDRH3 is YTX TSXgXgDXioYDV, wherein X7 is A or E, X8 is G, S or D, X9 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):

L (CPI)

t

VH( PD-1) - A - H ( CPI )

( ) n

VH( PD-1) - A -H (CPI )

CO CO

L '(CPI )

(ID

Wherein 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 THYMX4, wherein X4 is V or A; wherein CDRH2 of VH(PD-1) is FIGPAGDX5TYYADSVX6G wherein X5 is T, F or S and e is K or E; and wherein CDRH3 of VH(PD-I) is

YTX7TSX8X9DX10YDV, wherein X7 is A or E, X8 is G, S or D, X9 is V, F or Y, and Χω is T or S.

Embodiment 18. A binding protein according to embodiment 16 or embodiment 17, wherein the VH(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.

Paragraph 24. A binding protein according to any one of embodiments 16 to 23, wherein 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.

Specific embodiments relating to binding proteins specific to human LAG-3 are set out in the following numbered embodiments:

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 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, wherein CDRL2 is selected from the group consisting of: CDRL2 as 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 the group consisting of: 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.

Embodiment 2. A binding protein according to embodiment 1, which comprises:

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:l by the addition or deletion or substitution of 1, 2 or 3 amino acids; and

wherein 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 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 the group consisting of: CDRL2 as 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.

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)

00

CPI - A - hi (LAG-3)

( ¾ )n

CPI - A -H (LAG-3)

00

οό

L '(LAG-3)

(III)

wherein:

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; and

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) specific for PD-1 were identified by selecting clones from a naive phage library displaying individual VH variable domains (dAbs) fused via a c-myc tag to phage coat protein I I I against human PD-1 (hPD-1) extracellular domain as antigen adsorbed onto an immunotube according to method 1.

Method 1: Passive phage selection

Three rounds of selection were used to identify phage displaying dAbs that bind to hPD-1. To deplete phage displaying dAbs that display non-specific binding, 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.

After these 3 rounds of selection, individual phage infected E.coli colonies were picked and grown overnight and the resulting phage containing supernatant was screened by ELISA according to method 2.

Method 2: ELISA screening

50 μΙ hPD-1 (1 μg/ml) in PBS were added to the wells of a 96 well Maxisorp™ 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. 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.

Positive clones were sequenced. Unique clones were re-formatted as an anti-RSV (IgGl Fc disabled heavy chain) mAb-dAb fusion by splicing by overlap extension (SOE) PCR with the flanking fragments (5' fragment - promoter-signal-anti-RSV VH-CHl-3- 3' flanking fragment-stop codon-polyA). The product of SOE PCR was used for linear DNA transfection of mammalian HEK293 cells. 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.

Over 2000 clones were screened and 18 anti-RSV-PDl-mAb-dAb clones that demonstrated a combination of inhibition in the hPD-l/hPDL-1 ligand binding inhibition assay and slow dissociation rate by SPR were cloned into the pTT5 mammalian expression vector containing anti-RSV mAb heavy chain (IgGl Fc disabled). Anti-RSV-PDl-mAb-dAb molecules were expressed and the mAb-dAb was purified. hPDl-hPDL-1 ligand binding inhibition and steady state kinetics SPR was performed with purified mAbdAbs.

Method 3: PD-l/PDL-1 ligand binding inhibition assays

Human PD-1/ PDL-1 ligand binding inhibition assay

Serial dilutions (1:3) of each test mAb-dAb or the positive control GRITS50150 (all at maximal concentrations of 34 μg/ml) were included in the assay. Test samples were prepared by mixing 30 μΙ of antibody (diluted as described above) and 30 μΙ of 4 μg/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.

96 well plates were coated with 1 μΙ/well of 20 μg/ml Human PDl.his in PBS using the Mosquito liquid handler. The plates were sealed and incubated overnight at 4°C. The plates were then washed using an automated plate washer followed by the addition of 200 μΙ blocking buffer and incubation at room temperature for 1 hour with gentle agitation. The plates were then washed again using an automated plate washer, blotted dry and 25μΙ test sample added immediately. The plates were incubated with gentle agitation for 2 hours,

before washing with an automated plate washer. 150 μΙ/well of 2X read buffer T with surfactant reagent was added to each well and the plate immediately read on a MSD Sector Imager 6000.

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). Graph Pad Prism was used to plot data as the mean of two replicate wells with error bars showing the SEM, using an X=log[x] transformation and a non-linear regression "log(inhibitor) vs. Response - Variable slope (four parameters" fit to determine the IC50 (concentration resulting in a 50% inhibition of the difference between top and bottom of the dose response curve). Note that the MW of mAb-dAbs was assumed to be 170,000 kDa. The MW of GRITS50150 was assumed to be 150,000 kDa.

Rhesus PD-1/ Cynomolgus PDL-1 ligand binding inhibition assay

Serial dilutions (1:3) of each test mAb-dAb or the positive control GRITS50150 (all at maximal concentrations of 34 μg/ml) were included in the assay. Test samples were prepared by mixing 30 μΙ of antibody (diluted as described above) and 30 μΙ of 2 μg/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.

96 well plates were coated with 1 μΙ/well of 20 μg/ml Human PDl.his in PBS using the Mosquito liquid handler. The plates were sealed and incubated overnight at 4°C. The plates were then washed using an automated plate washer followed by the addition of 200 μΙ blocking buffer and incubation at room temperature for 1 hour with gentle agitation. The plates were then washed again using an automated plate washer, blotted dry and 25μΙ test sample added immediately. The plates were incubated with gentle agitation for 2 hours, before washing with an automated plate washer. 150 μΙ/well of 2X read buffer T with surfactant reagent was added to each well and the plate immediately read on a MSD Sector Imager 6000.

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). Graph Pad Prism was used to plot data as the mean of two replicate wells with error bars showing the SEM, using an X=log[x] transformation and a non-linear regression "log(inhibitor) vs. Response - Variable slope (four parameters" fit to determine the IC50 (concentration resulting in a 50% inhibition of the difference between top and bottom of the dose response curve). Note that the MW of mAb-dAbs was assumed to be 170,000 kDa. The MW of GRITS50150 was assumed to be 150,000 kDa.

Method 4: Surface Plasmon Resonance (SPR)

Using a BIACORE™ 3000 or 4000, 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 (15μΙ of each sample and 135μΙ 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 μg/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.

Method 6: Assay to determine concentration of purified mAbs and mAb-dAbs

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.

Example 2: Affinity Maturation

The dAb sequence of clone 51A09 was subjected to diversification using the Mutazyme™ 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.

Method 7: Soluble phage selection

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

Round 4: 60°C for 2 hours followed by 0.5 nM hPDl for 15 minutes at room temperature (the phage library was incubated at 60°C prior to blocking and antigen binding steps to select for clones with good biophysical properties)

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). To facilitate cloning, 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).

Method 8: PD1+ Jurkat assay

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). Briefly, into each well of a 96 well plate was transferred ΙΟΟμΙ of CHO PDL1+ cells at 4x105 cell/ml (40000 cells/well) in F-12 nutrient mix (HAM) containing 10% FBS, 0.25mg/ml G418 and 0.2mg/ml hygromycin B. Cultures were incubated at 37°C, 5% C02 for 16h. 95μΙ of culture medium was removed from each well to which was then added 40μΙ of test antibody or control in RPM I media containing HEPES supplemented with 2% FBS and 40μΙ of Jurkat NFAT-Iuc2/PD1 cells at 1.25x106 cells/ml (50000 cells/well) in RPM I media containing HEPES supplemented with 2% FBS. Antibodies were generally prepared as nine point dose curves starting at 200μg/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 80μΙ of BioGlo™ 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).

Method 9: PD1+/LAG3+ Jurkat Cell Binding Assay

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 ΙΟΟηΜ 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 ΙΟΟηΜ 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 ΙΟΟμΙ PBS for measurement using the MACSQuant flow cytometer (Miltenyi). Cells were run through the flow cytometer selecting an uptake volume of 50μΙ, fast mode and high flow rate. The instrument settings were adjusted for appropriate voltages for PE fluoresence, NIR fluorescence in addition to forward and side scatter. A live gate was set on the NIR fluorescence scatter plot (non-fluorescing cells) and 10,000 events collected from within this gate. The data were exported from the MACSQuant and analysed using FlowJo version 10 software. Cells were selected on forward scatter area (FSC) and side scatter area (SSC), FSC versus NIR fluorescence for live cell selection and on FSC height vs FSC area to gate single cell population. The median PE fluorescence intensity (MFI) of these live single cells were determined in FlowJo and exported to Windows Excel. In Excel the MFI values for each antibody treatment were divided by the MFI value of cells treated with the PE-linked anti-human IgGl Fc F(ab')2 only ('no antibody' control). This MFI data were copied into

GraphPad prism V 5.0.4 where the pM concentrations were transformed to log values and the data plotted as a sigmoidal dose response (variable slope) curve. The EC50 values generated from the curves represented half the maximal effective concentration (a concentration that induces a response halfway between the baseline and maximum after a specified exposure).

The MFI data generated from the Jurkat WT cells treated with ΙΟΟηΜ 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.

Results

Certain clones exhibiting an IC50 <5 nM in the hPDl-hPDL-1 ligand binding inhibition assay and exhibiting rhesus cross reactivity on Biacore are listed below (Table 4):

Table 4


CDR sequences for these clones are given in Table 5.

Table 5


THYMV (SEQ. FIGPAGDFTYYADSVKG YTATSGVDTYDV

22D034-51A09-4 ID NO: 10) (SEQ ID NO: 32) (SEQ ID NO: 12)

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.

Further comparison of these sequences permits consensus CDR sequences to be identified as follows:

CDRH1 of VH(PD-I): THYMX4 (SEQ ID NO: 57), wherein X4 is V or A;

CDRH2 of VH(PD-I) : FIGPAG DX5TYYADSVX6G (SEQ I D NO: 58), wherein X5 is T, F or S and X6 is K or E;

CDRH3 of VH(PD-I) : YTX7TSX8X9DXI0YDV (SEQ ID NO: 59), wherein X7 is A or E, X8 is G, S or D, Xg is V, F or Y, and Xio is T or S.

Example 3: Further mutations

51A09-188 is an example of a dAb identified after affinity maturation which binds human and rhesus monkey PD1. To reduce the impact of any pre-existing anti-drug antibodies, 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.

Purified mAbdAb was tested in the PD1+ Jurkat functional assay to determine whether the C-terminal modification has any impact on dAb function.

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 22D034-51A09-188.1001: T28A

Mutant 22D034-51A09-188.2001: V105F

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.

Results

Table 6


Example 4: Identification of monoclonal antibodies that bind LAG -3

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) was incubated with naive yeast antibody libraries and the outputs selected by magnetic bead separation for two initial rounds of enrichment. This was followed by a round of FACS selection, whereby populations of yeast cells capable of binding to lOOnM hLAG3-His were isolated by gating and cell sorting. This yielded pure hLAG3-His binding populations. 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:

• KD by SPR (Surface Plasmon Resonance; method 9) <lnM for human and

cynomolgus LAG-3

• Inhibition of LAG3-MHCII interaction in competition flow cytometry assay (method 10) >50%

• Binding to LAG3 positive cells in cell binding flow cytometry assay (method 11) >2 fold over background

• Binding to LAG3 negative cells in cell binding flow cytometry assay (method 11) <2 fold over background

• 65% monomer having a retention time of < 16.5 or >80% monomer and a retention time of >16.5 by aSEC analysis (method 12)

• EC50 <5 μg/ml in functional LAG-3 assay (method 13)

• No CDR cysteines identified on (DNA) sequencing

Method 10: Surface Plasmon Resonance

Using a BIACORE™ 4000, antibodies were assessed for binding kinetics and affinity for binding to LAG-3. LAG-3 antibodies were captured on spots 1 and 5 of each flow cell of a protein A coated CM5 series S sensor chip. Human or cynomolgus LAG-3 was flowed over the antibodies at 50 nM, 12.5 nM, 3.125 nM and 0.78 nM (blank = 0 nM) at 37°C. Data was analysed using Biacore 4000 Data Analysis software version 1.0, applying a 1:1 binding model. For some curves, kinetics could not be determined but a steady state model could be applied to determine the affinities only. For some antibodies, a longer dissociation time is required to accurately determine the kd (off rate), as a 10 % drop in response was not observed in the 10 minute dissociation time. For some clones (typically those with affinities of around 100 pM or less), a lower concentration range would be required to accurately determine the kinetics and affinities as a good fit could not be obtained at the higher concentrations used in this experiment.

Method 11: Competition flow cytometry assay

1. Transfer ΙΟΟμΙ 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.

2. Centrifuge at 4°C for 3min at 250-350g then remove supernatant.

3. Resuspend cells in 25μΙ of assay buffer containing 16 nM soluble biotinylated human LAG- 3. Fc.

4. Add 25μΙ of either assay buffer alone, containing control antibody 11E3

(Caltag/Abioscience, catalogue number AG-20B-0011-C100), control antibody 17B4 (GSK), or control antibody 6D511 (US Biological), or containing test antibodies (all antibodies at a concentration range of from 60pM to ΙΟΟΟηΜ). Incubate plates on ice for 60 minutes.

5. Add 150μΙ assay buffer to wells. Centrifuge at 4°C for 3min at 250-350g then remove supernatant.

6. Wash cells twice with 200μΙ assay buffer.

7. Resuspend cells in 50μΙ Streptavidin PE (Biolegend, catalogue number 45204) diluted 1: 200 in assay buffer, or 50μΙ assay buffer containing 0.5μΙ I3-RD1 PE linked anti-MHCII (Beckman Coulter Inc., catalogue number 6604366)

8. Incubate on ice in the dark for 30 minutes.

9. Add 150μΙ of PBS to assay wells and centrifuge the plate at 4°C for 3min at 250-350g then remove supernatant. Wash cells twice with Dulbecco's PBS (200μΙ/ννθΙΙ).

10. Resuspend cell pellets in 50-100μΙ Dulbecco's PBS.

11. Add equivalent volume (50-100μΙ) of 2μΜ Sytox Blue (Molecular Probes Catalogue Number S11348) to each assay well then acquire data using the FACS Canto II (BD

Biosciences) and FACSDiva 8.01 software.

12. Determine the appropriate flow cytometry parameters using an unstained sample well then apply settings to all data samples. Identify live and dead cells by Sytox blue dead cell exclusion staining.

Method 12: Cell binding flow cytometry assay

1. Transfer ΙΟΟμΙ of EL4 cell suspension overexpressing human LAG3 (LAG3 positive cells) or not expressing LAG3 (LAG3 negative cells) 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.

2. Centrifuge at 4°C for 3min at 250-350g, and remove supernatant.

3. Resuspend cells in 50μΙ assay buffer with or without control antibody 11E3

(Catag/Abioscience, catalogue number AG-20B-0011-C100), control antibody 17B4 (Enzo Life Sciences, catalogue number ALX-804-806PF-C100) or test antibodies at concentrations of between 1-100 nM. Incubate plates on ice for 30 minutes.

4. Add 150μΙ of assay buffer, centrifuge the plate at 4°C for 3min at 250-350g, and remove supernatant.

5. Wash cells twice with 200μΙ assay buffer.

6. Dilute detection antibody (PE linked anti-human Fc, Invitrogen, Catalogue number H10104 for test antibodies) 1: 500 in assay buffer (for control antibodies, the detection antibody is PE linked goat anti mouse Ig diluted 1: 50 in assay buffer). PE= 2R -phycoerthyrin.

7. Resuspend cells in 50μΙ of detection antibody or assay buffer alone

8. Incubate on ice in the dark for 30 minutes.

9. Add 150μΙ of PBS to assay wells and centrifuge the plate at 4°C for 3min at 250-350g then remove supernatant. Wash cells twice with Dulbecco's PBS (200μΙ/ννθΙΙ).

10. Resuspend cell pellets in 50μΙ Dulbecco's PBS.

11. Add 50μΙ of 2μΜ Sytox Blue (Molecular Probes Catalogue Number S11348) to each assay well then acquire data using the FACS Canto II (BD Biosciences) and FACSDiva 8.01 software.

12. Determine the appropriate flow cytometry parameters using an unstained sample well then apply settings to all data samples. Identify live and dead cells by Sytox blue dead cell exclusion staining.

Method 13: Open Access Analytical Size Exclusion Chromatography (aSEC)

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 μg 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.

Method 14: Functional LAG-3 assay

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 μΙ anti-LAG-3 antibody ten-point dose curve starting at 25μg/ml (or control antibody dose curve)

25 μΙ LAG 3- effector cells (4xl06; CS194801) in assay medium (RMPI 1640 medium containing 1% FBS)

25 μΙ Raji cells /SEE (a 1:1 mixture of Raji cells (2 x 106/ml; ATCC catalogue number CCL-86) in assay medium and 300 ng/ml Staphylococcal enterotoxin D, endotoxin reduced (Toxin Technology, Inc., Catalogue number DT303red).

The plates were incubated for six hour at 37°C in a C02 incubator. They were then allowed to reach room temperature and 75μΙ BioGlo™ Reagent (prepared as described in the Bio-Glo Luciferase Assay System, Promega catalogue number G7940) was added to each well.

Following incubation for 5-10 minutes, luminescence was measured in a plate reader. 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 μg/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 μg/ml, 300 s), then washed in PBSF and dipped into each clone (15 μg/ml, 60 s). This was a self binding check to ensure that no binding occurred in the absence of LAG3. After a 60 s baseline in PBSF, sensors were then dipped into huLAG3 (50 nM, 180 s) then into each other antibody (lOug/ml in PBSF), 180 s. Dissociation analysis was conducted using ForteBio Data analysis software, version 8.0. Where binding of the second antibody to LAG3 can be seen in the presence of the primary antibody, the two antibodies are non-competitive. Where no binding of the secondary antibody is seen, the two antibodies compete for binding to LAG3 and are assigned to the same bin.

Results

Clones 57B02, 57E02, 57C06 and 57H08 competed for binding to LAG3 and were assigned to the same epitope bin. They have the following properties:

Table7


CDR sequences for these clones are given in Table 8.

Table 8


57H0 GYYM H WIN PNSGGT EGPYDDDGFDY RASQSISSYLN AASSLQS QQSFPAPPY

8 (SEQ ID NYAQKFQG (SEQ I D NO: 6) (SEQ I D NO: (SEQ I D NO: T (SEQ ID

NO: 4) (SEQ I D NO: 42) 8) NO: 45)

5)

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.

Comparison of the sequences for CDRL1, permits a consensus sequence to be identified as follows: RASQX1ISSX2LX3 (SEQ I D NO: 56), wherein Xi is G or S, X2 is W, F or Y, and X3 is A or N.

Example 5: Cloning of LAG-3 mAb

The VH and VL sequences for clone 57E02 was codon optimised using an automated

optimisation script (that compares pre-optimised germline sequences as the baseline

sequences, identifies any differences between the query sequence and the optimised

germline, and then mutates the differences using the "highest frequency" codon). An Apal restriction enzyme site identified within the optimised VH sequences was removed

manually. I n-fusion oligo sequences were added to the 5' and 3' ends of the light chain sequence (5' = GGCCACCGCCACCGGTGTGCACAGC (SEQ I D NO: 48); 3' =

CGTACGGTGGCCGCCC (SEQ I D NO: 49)).

The amino acid sequence of the light chain is given by SEQ ID NO: 50

DIQMTQSPSSVSASVGDRVTITCRASQGISSWLAWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQETNSFWTFGGGTKVEI KRTVAAPSVFI FPPSDEQLKSGTASVVCLLNN FYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RG EC

The DNA sequence of the light chain is given by SEQ ID NO: 51

GACATCCAGATGACCCAGAGCCCCAGCTCAGTGAGCGCTAGCGTGGGCGACAGGGTGACCATCACC TG C AG G G C C AG C C AG G G C ATTAG CAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCC CAAGCTCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCTCCAGGTTTAGCGGCAGCGG AAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGC

CAGGAGACCAACAGCTTCTGGACCTTCGGCGGCGGCACAAAAGTCGAGATCAAGCGTACGGTGGCC

GCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTG

TGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAG

AGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAG

CACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCA

GGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC

The amino acid sequence of the heavy chain is given by SEQ I D NO: 52

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYM HWVRQAPGQGLEWMGWI NPNSGGTNYAQKFQ

GRVTMTRDTSISTAYM ELSRLRSDDTAVYYCAREGPYDDDGFDYWGQGTLVTVSSASTKGPSVFPLAPS

SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV

NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKG

QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV

DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

and the DNA sequence of the heavy chain is given by SEQ. ID NO : 53.

CAGGTGCAGCTCGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGCGCCTCTGTCAAGGTGAGCTG

CAAGGCCAGCGGCTACACCTTCACCGGCTACTACATGCACTGGGTGAGGCAGGCTCCCGGACAGGG

CCTGGAGTGGATGGGCTGGATCAACCCCAACAGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGG

CAGGGTGACCATGACCAGGGACACCAGCATCAGCACCGCCTACATGGAACTGAGCAGGCTGAGGA

GCGACGACACCGCCGTGTATTACTGCGCCAGGGAGGGACCCTACGACGACGACGGCTTCGACTACT

GGGGCCAGGGCACCCTGGTGACAGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTG

GCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTC

CCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCC

GTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGG

CACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGA

GCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGGCCGGAGCCCCC

AGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCT

GTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTG

GAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTC

CGTG CTG ACCGTG CTG C ACC AG G ATTG G CTG AACG G C AAG G AGTAC AAGTGTAAG GTGTCCAAC AA GGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGT

GTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAA

GGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACA

AGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAA

GAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCACTA

CACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG

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.

To generate the DNA encoding the 57E02x51A09-188001 mAb-dAb, the VH region in the mAb-dAb containing the 51A09-188001 dAb (generated in Example 3) 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 In Fusion reactions were transformed into NEB 5-alpha competent E.coli (High

Efficiency; Catalogue Number C2987) cells. DNA isolated from the transformed individual clones was sequenced and 250 μg of each of the heavy and light chain vectors were used for transfection and expression in HEK293-6E cells. 5 days post transfection, the culture was harvested by centrifugation and clarified culture supernatants were loaded onto pre-equilibrated 5 ml HiTrap MabSelect SuRe (5ml; GE Healthcare) connected to Akta Xpress systems (GE Healthcare) at constant flow rate (5 ml/min). Peak fractions were pooled and passed through anion exchange chromatography at pH7.5.

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:

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYM HWVRQAPGQGLEWMGWI NPNSGGTNYAQKFQ

GRVTMTRDTSISTAYM ELSRLRSDDTAVYYCAREGPYDDDGFDYWGQGTLVTVSSASTKGPSVFPLAPS

SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNV

NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKG

QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV

DKSRWQQGNVFSCSVM HEALHN HYTQKSLSLSPGKGSTGLDSPTEVQLLESGGGLVQPGGSLRLSCAA

SGFTFRTHYMVWVRQAPGKGLEWVSFIGPAGDTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA

VYYCAAYTATSGVDTYDVMGQGTLVTVSSA

The heavy chain nucleotide sequence of the 57E02x51A09-188001 mAbdAb is given by SEQ. I D NO: 55:

CAGGTGCAGCTGGTGCAGAGCGGGGCCGAGGTGAAGAAACCCGGCGCTAGCGTCAAGGTGAGCT

GCAAGGCCAGCGGGTACACCTTCACCGGCTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGG

GACTCGAGTGGATGGGGTGGATCAACCCCAACAGCGGCGGCACAAACTACGCCCAGAAGTTTCAG

GGCAGGGTGACCATGACCAGGGACACCAGCATCAGCACCGCCTACATGGAACTCAGCAGGCTGAG

GTCCGACGACACCGCCGTGTACTATTGCGCCAGGGAGGGACCATACGACGACGACGGCTTCGATTA

CTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCT

GGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACT

TCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCG

CCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTG

GGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTG

GAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGGCCGGAGCC

CCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGA

CCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGC

GTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGT

GTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAA

CAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCA

GGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGT

GAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACT

ACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGA

C AAG AG CAG ATG G CAG CAG GG CAACGTGTTC AG CTG CTCCGTG ATG C ACG AG G CCCTG CAC AATC A

CTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGATCCACCGGCCTGGACAGCCCCACCGA

GGTCCAGTTGCTGGAAAGTGGGGGAGGACTGGTGCAGCCTGGCGGAAGCCTTAGACTCAGCTGCG

CCGCTAGTGGCTTCACTTTCCGCACCCACTACATGGTGTGGGTTAGGCAGGCACCCGGAAAAGGTCT

AGAGTGGGTTAGCTTTATCGGCCCTGCCGGCGATACCACCTATTACGCCGATTCCGTGAAGGGCAG

GTTCACAATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTCAGGGCTGA

GGACACCGCGGTGTACTACTGTGCTGCCTACACCGCAACCTCAGGAGTCGATACCTACGACGTGATG

GGACAGGGCACTTTGGTTACCGTGAGTAGCGCC

Example 7: Characterisation of PD-l/LAG-3 mAbdAb

The mAb-dAb generated in Example 6 was tested in the following assays:

1. PDl+ Jurkat assay (method 8)

2. LAG3+ Jurkat assay (method 13)

3. Binding to LAG 3+ Jurkat cells (method 9)

4. Binding to human and rhesus LAG3+ cells

5. Binding to PD1+ Jurkat cells (method 9)

6. Human PD1/PDL1 Receptor Binding assay (method 3)

7. Macaque PD1/PDL1 receptor binding assay (method 3)

8. Mixed lymphocyte reaction assay (method 15)

9. HIV proliferation assay (method 16)

10. I ntracellular cytokine assay (method 17)

11. Viral Induction M LR Assay (method 18)

The results for items 1 to are shown in Table 9:

Table 9

Cell Binding - Human PD1+ Jurkat (EC50) 4 1.97 (nM) 0.7 (method 9)

Cell Binding - Human LAG 3+ Jurkat (EC50) 5 1.22 (nM) 0.94

(method 9)

Cell Binding - Human LAG 3+ HEK (EC50) 1 2.56 (nM)

Cell Binding - Rhesus LAG 3+ HEK (EC50) 1 3.15 (nM)

Functional Assay - PD1+ Jurkat Assay (method 8) 24 7.25 (nM) 8.29

Functional Assay - LAG3+ Jurkat Assay (method 10 0.46 0.15 ^g/mL) 13) ^g/mL)

Several 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.

Interestingly, it is noted that two mAbdAbs tested including the same PD-1 dAb component, but different LAG-3 mAbs exhibited differences in clearance in vivo (prior to the expected onset of anti-drug antibody-mediated clearance). This is a surprising benefit of this particular combination of PD-1 dAb and LAG-3 mAb.

Method 15: Mixed Lymphocyte Reaction Assay

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. This assay protocol was used to characterize the ability of LAG3-PD1 mAb-dAbs to enhance cytokine production from T cells.

Method: Peripheral blood mononuclear cells (PBMCs) from three HIV-infected stably antiretroviral treated donors were thawed and rested overnight in RPMI 1640 medium containing 10% FBS. The following day, 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 106/mL jn RPMI + 10% FBS. CD4 T cells were plated in triplicate with 1X105 cells in ΙΟΟμί/ννβΙΙ in tissue culture treated U-bottom plates. Serial dilutions of control or test mAbs or mAbdAbs were performed in a separate plate

generating a six-point, four-fold dilution series (20X) and ΙΟμί of each binding protein added per well and the cell binding protein 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 106/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. 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% C02 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.

Statistical Analysis: Outlier analysis was performed prior to statistical analysis. Outlier as well as statistical analyses were performed on the logio transformed scale. The outlier reduced data was averaged across technical replicates and a mixed model was fit to the data with antibody, dose and the interaction of antibody and dose as fixed effects and the interaction of donor and run as random effects. Unadjusted p-values were obtained via pairwise comparisons and a Bonferroni adjustment was applied, adjusting for 6

concentrations per antibody comparison.

Results: The results are shown in Table 10 and Figure 1. Antibodies that block PD1 inhibitory signals significantly increase IFNy production from CD4 T cells, compared to the control antibody in the MLR Assay. Statistical significance p<0.0001 was observed at all concentrations tested in the MLR Assay (Figure 1). Antagonizing LAG3 signals alone does not increase CD4 IFNy production, however, co-culture with both LAG3 and PD1 blocking antibodies, enhances cytokine production compared to PD1 blockade alone. The fold difference in cytokine production between the LAG3/PD1 mAbdAb and either the individual antibodies or combination of LAG3 and PD1 antibodies was determined. On average we observed a 1.86 to 3.38 fold increase in cytokine production from CD4 T cells co-cultured with the bispecific LAG3/PD1 versus the combination of two antibodies (Table 10). In addition, the bispecific showed a 2.08 to 4.19, and a 5.95 to 17.44 fold increase in cytokine production over PD1 and LAG3 blocking antibodies respectively. In conclusion, the

LAG3/PD1 bispecific mAbdAb facilitates significantly greater cytokine production from T cells than either LAG3, PD1 or the combination of the two antibodies.

Table 10

Method 16: HIV Specific Proliferation Assay:

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.

Method:

Serial dilutions of control or test mAbdAbs were performed in triplicate wells on a 96 well U bottom plate generating a four-point dilution series for final concentrations of 20, 2, 0.2, 0.02ug/ml of each antibody in 50 μΙ total volume. Replicate plates were made in advance and stored at -20° until use.

Peripheral blood mononuclear cells (PBMCs) 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.

PBMCs were washed, counted and resuspend cells at 5 million/ml in DPBS.

Carboxyfluorescein succinimidyl ester (CFSE) 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. CFSE labelling was performed adding 0.25 μί of diluted CFSE per 1ml of PBMCs (0.25μΜ 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 μΙ 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 0.5μβ/μΙ stock concentration. A Gag Peptide stimulation mix was made adding Gag peptides to AIMV+EFV media ^g/ml, 2X concentration).

ΙΟΟμΙ of Gag peptide mix was added to each well of the plate containing 50μΙ of labelled PBMCs and 50μΙ 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).

Plates were placed in 37°C humidified incubator with 5% C02 for 6 days.

Cells were then pelleted and stained with fluorescently labelled antibodies for 30 minutes at 4°C. Staining was performed in 96 well V bottom plates with the pelleted cells resuspended in a 50μΙ total volume of diluted commercially available antibodies to the following surface proteins: CD3(Clone: SP34-2), CD8(Clone: RPA-T8),

CD4(Clone: L200), CD27 (Clone: M-T271) from BD Biosciences; PDl(Clone: EH12) from Biolegend; CD45RO (Clone: UCHL1) from Beckman Coulter, and the Live Dead Amine Aqua Dye (from Life Technologies).

12. Cells were washed twice with PBS and the fixed with 0.5% PFA diluted in PBS in a 200μΙ volume.

13. Cells were acquired on a BD LSRII- Fortessa using the High throughput sampler

14. Samples were analyzed using Flowjo and the Average frequency of HIV Gag Specific CD8 T cells that have proliferated determined for each antibody type and dilution.

15. Proliferating CD8 T Cells are defined as: CFSE dim, Live (Amine -ve), CD8+ T cells i.e.( CD3+, CD8+, CD4).

16. The results shown are the average from two independent runs of the assay, each run performed in triplicate, from an HIV infected stably treated donor.

Statistical Analysis: Data was analysed using a mixed effects model that included antibody type, concentration the interaction of antibody and concentration as fixed effects and run as a random effect. Unadjusted p-values were obtained via pairwise comparisons and a Bonferroni adjustment was applied, adjusting for 4 concentrations per antibody comparison.

Results: Dual antagonism of LAG3 and PDl facilitated the expansion of HIV Gag specific CD8 T cells. PBMCs co-cultured with Gag peptides and the LAG3/PD1 mAbdAb (57E02-51A09-188001) demonstrated significantly greater CD8 proliferation (%CD8+CFSE-dim T cells) compared to cells cultured with a control antibody (figure 2). Statistical significance was achieved at the 0.05 Bonferroni adjusted significance level at doses ranging from 20-0.2ug/ml.

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.

Methods

1. Cryopreserved peripheral blood mononuclear cells (PBMC) from 19 HIV infected stably treated donors were thawed then rested overnight in RPMI 1640 (Invitrogen Cat. No. 22400) supplemented with 10% Premium Grade Fetal Bovine Serum

(Seradigm Cat. No. 1500-500) along with 500nM Efavirenz (R10+EFV) and absolute number of cells determined.

2. Either the LAG3/PD1 mAbdAb or a control antibody was diluted to a 4x

concentration of 8μg/mL in R10+EFV and 50μΙ added to wells of non-tissue culture- treated 96 well U bottom plates (Falcon Cat. No. 351177).

3. Rested PBMCs were resuspended at lOmillion/mL in R10+EFV containingGolgiStop protein transport inhibitor containing monensin (BD Biosciences Cat. No. 554724) at 0.7μΙ/ιηΙ-. 50μΙ of the cell culture mixture was added to the non-tissue culture- treated 96 well U bottom plates containing the corresponding 4x antibodies.

4. 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.

5. One hundred microliters of a 2x R10+EFV stimulation cocktail containing

staphylococcal enterotoxin A & B (SEB/A), each at a final concentration of 500ng/mL, (List Biologicals) was added to the corresponding wells. Bacterial antigens such as SEB/A crosslink the TCR& MHC molecules in-vitro leading to T cell activation and cytokine production.

6. A negative control or unstimulated well was set up for each donor where

spontaneous cytokine production/degranulation would be determined.

7. Plates were placed in a 37°C humidified incubator with 5% C02 for 6 hours.

8. Staining for cell surface antigens was performed in 96 well V bottom plates with the pelleted cells resuspended in 50μΙ total volume of diluted commercially available antibodies for 30 minutes at 4°C to the following surface proteins: CD3 (Clone: SP34- 2), CD8 (Clone: RPA-T8), CD4 (Clone: L200), CD27 (Clone: M-T271) from BD

Biosciences; PD1 (Clone: EH12) from Biolegend; CD45RO (Clone: UCHL1) from Beckman Coulter and the Live/Dead Amine Aqua Dye from Life Technologies.

9. Cells were washed twice in cold Pharmingen Stain Buffer (BSA) (BD Pharmingen Cat.

No. 554657) then fixed in cold 4% paraformaldehyde solution for 15 minutes at 4°C followed by permeablization in cold lx BD Perm/Wash buffer for 15 minutes at 4°C.

10. Fixed/permeabilzed cells were resuspended in 50μΙ total volume of cold lx BD

Perm/Wash buffer with diluted commercially available antibodies to the following intracellular cytokines: TNFa (Clone: Mabll), IFNy (Clone: 4S.B3) and IL2 (Clone: MQ1-17H12) from BioLegend for 30 minutes at 4°C.

11. Cells were washed twice in cold lx BD Perm/Wash buffer and then resuspended in cold Pharmingen Stain Buffer (BSA) (BD Pharmingen Cat. No. 554657) in a 200μΙ volume.

12. Cells were acquired on a BD LSRII- Fortessa using the high throughput sampler.

13. Samples were analyzed using the FlowJo software package (Treestar, Ashland, OR)

14. 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.

15. The average frequency of Memory&/effector CD4/CD8 T cells that have secreted cytokines (TNFa, IFNy, IL2) or degranulated, i.e. increased CD107a expression, was determined.

Statistical Analysis: Data was analysed on the Data0,2 transformed scale using a mixed effects model that included stimulus, antibody, and the interaction of stimulus and antibody as fixed effects and donor as a random effect. Unadjusted p-values were obtained via pairwise comparisons and a Bonferroni adjustment was applied, adjusting for 2 comparisons of interest.

Results: Dual antagonism of LAG3 and PD1 increased the functional capacity of T cells from HIV infected donors. T cells co-cultured with the LAG3/PD1 mAbdAb (57E02-51A09-188001) in the presence of SEB/SEA produced significantly greater TNFa, IFNy and IL2 compared to cells cultured with a control antibody (figure 3, A-B). Importantly, co-culture with 57E02-51A09-188001 in the absence of a stimulant (unstimulated) did not induce cytokine production. 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.

Hence we focused on the ability of CD4+ T cells to produce both IL2 and TNFa. This helper function is markedly reduced during chronic HIV infection. Using SEB/SEA to crosslink MHC-TCR we observe that LAG3/PD1 blockade significantly increased the frequency of CD4 T cells from HIV infected donors that produce IL2 and TNFa (figure 3, C). The function of CD8 T cells is also compromised during HIV Infection. A measure of cytotoxic potential is the ability to degranulate measured by CD107a expression and produce IFNy . Co-culture with the LAG3/PD1 mAbdAb significantly increased the ability of CD8 T cells from HIV Infected donors to express both CD107a and IFNy (figure 3, D). Of note 10 of the 19 donors tested increased their cytotoxic potential by approximately 1.5-3 fold in this assay in the presence of

LAG3/PD1 vs control.

Method 18: Viral Induction MLR Assay:

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. We adapted the mixed lymphocyte reaction assay using allogenic stimulation by dendritic cells to induce HIV RNA expression. We then evaluated the ability of LAG3/PD1 to enhance HIV RNA induction from CD4 T cells obtained from HIV infected donors.

Method: Peripheral blood mononuclear cells (PBMCs) from 5 HIV-infected stably antiretroviral treated donors were thawed and rested overnight in RPMI 1640 medium containing 10% FBS. The following day, 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 106/mL jn RPMI + 10% FBS. Twenty replicates of 1X105 CD4 T cells were place in tissue culture treated U-bottom plates at ΙΟΟμί/ννθΙΙ. 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 106/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. Plates were placed in a 37°C humidified incubator with 5% C02 for 5 days. On Day 6, the cells and supernatant were collected and pooled. RNA was isolated from the cell pellets using the RNeasy kit from Qjagen Catlog #74104. RNA was isolated from the supernatants by using the QJAamp Viral RNA kit from Qiagen Catalog# 52904.

To evaluate the expression Gag RNA levels in both cells and supernatant qPCR was performed with the primers and probes listed in Table 11:

Table 11


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.

Statistical Analysis: Outlier analysis was performed prior to statistical analysis. Outlier as well as statistical analyses were performed on the logio transformed scale. A fixed effects model was fit to the data with antibody as fixed effect. A separate analysis was performed for each donor. Unadjusted p-values were obtained via pairwise comparisons and contrast statements and a Bonferroni adjustment was applied, adjusting for a total of 3 comparisons.

Results:

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. As cellular activation can lead to viral reactivation, we used allogenic stimulation from dendritic cells to induce HIV RNA production in a modified MLR assay. We demonstrate that the LAG3/PD1 bispecific significantly increases HIV RNA production from CD4 T cells compared to a control antibody in 4 of the 5 donors tested (Figure 4).

Example 8: Cloning and characterisation of CTLA4/PD-1 mAbdAb

The 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

EIVLTOSPGTLSLSPGERATLSCRASOSVGSSYLAWYOOKPGOAPRLLIYGAFSRATGIPDRFSGSGSGTDF TLTISRLEPEDFAVYYCOOYGSSPWTFGOGTKVEIKRTVAAPSVFIFPPSDEOLKSGTASVVCLLNNFYPRE AKVOWKVDNALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKHKVYACEVTHOGLSSPVTKSFNRG EC

The amino acid sequence of the heavy chain is given by SEQ. ID NO: 80

QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTM HWVRQAPGKGLEWVTFISYDGNN KYYADSVKGRF

TISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSN

TKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSHEDPEVKFNWY

VDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQ

VYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVM HEALHNHYTQKSLSLSPGKGSTGLDSPTEVQLLESGGGLVQPGGSLRLSCAASGFTFR

THYMVWVRQAPGKGLEWVSFIGPAGDTTYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTAVYYCAA

YTATSGVDTYDVMGQGTLVTVSSA

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). However, the levels of cytokine production induced by the CTLA4/PD1 bispecific is comparable to the combination of CTLA4 and PD1 antibodies at 5 of 6 concentrations tested. Table shows the fold difference between CTLA/PD1 mAbdAb and the individual antibodies or combination of CTLA4 and PD1 antibodies across the six

concentrations evaluated in the MLR assay.

Table 12

Unless indicated by an ( a ) cytokine production of CTLA4/PD1 mAbdAb is significantly greater than the indicated antibody after multiplicity adjustment. Bonferroni adjusted p-values are < 0.05.

Sequence Listing

SEQ. ID NO: 1 (Heavy chain variable domain of clone 37Y056-57E02-1)

OVOLVOSGAEVKKPGASVKVSCKASGYTFTGYYMHWVROAPGOGLEWMGWINPNSGGTNYAQKFO GRVTMTRDTSISTAYMELSRLRSDDTAVYYCAREGPYDDDGFDYWGQGTLVTVSS

SEQ ID NO: 2 (Light chain variable domain of clone 37Y056-57E02-1)

DIOMTOSPSSVSASVGDRVTITCRASOGISSWLAWYOOKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQETNSFWTFGGGTKVEIK

SEQ ID NO: 3 (Heavy chain variable domain of clone 22D034-51A09-188001)

EVOLLESGGGLVOPGGSLRLSCAASGFTFRTHYMVWVRQAPGKGLEWVSFIGPAGDTTYYADSVKGRF

TISRDNSKNTLYLQMNSLRAEDTAVYYCAAYTATSGVDTYDVMGQGTLVTVSSA

SEQ ID NO: 4 (CDRHl LAG 3, Kabat definition)

GYYMH

SEQ ID NO: 5 (CDRH2 LAG 3, Kabat definition)

WINPNSGGTNYAQKFQG

SEQ ID NO: 6 (CDRH3 LAG 3, Kabat definition)

EGPYDDDGFDY

SEQ ID NO: 7 (CDRL1 LAG 3, Kabat definition)

RASQGISSWLA

SEQ ID NO: 8 (CDRL2 LAG 3, Kabat definition)

AASSLQS

SEQ ID NO: 9 (CDRL3 LAG 3, Kabat definition)

QETNSFWT

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)

FIGPAGDTTYYADSVKG

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)

YTATSGVDTYDV

SEQ ID NO: 13 (CDRH1 LAG -3, Chothia definition)

GYTFTGY

SEQ ID NO: 14 (CDRH2 LAG -3, Chothia definition)

NPNSGG

SEQ ID NO: 15 (CDRH1 PD-1, Chothia definition)

GFTFRTH

SEQ ID NO: 16 (CDRH2 PD-1, Chothia definition)

GPAGDT

SEQ ID NO: 17 (CDRH1 LAG -3, AbM definition)

GYTFTGYYMH

SEQ ID NO: 18 (CDRH2 LAG -3, AbM definition)

WINPNSGGTN

SEQ ID NO: 19 (CDRH1 PD-1, AbM definition)

GFTFRTHYMV

SEQ ID NO: 20 (CDRH2 PD-1, AbM definition)

FIGPAGDTTY

SEQ ID NO: 21 (CDRH1 LAG -3, Contact definition)

TGYYMH

SEQ ID NO: 22 (CDRH2 LAG -3, Contact definition)

WMG WINPNSGGTN

SEQ ID NO: 23 (CDRH3 LAG -3, Contact definition)

AREGPYDDDGFD

SEQ ID NO: 24 (CDRL1 LAG-3, Contact definition)

SSWLAWY

SEQ ID NO: 25 (CDRL2 LAG-3, Contact definition)

LLIYAASSLQ

SEO ID NO: 26 (CDRL3 LAG-3, Contact definition)

QETNSFW

SEO ID NO: 27 (CDRH1 PD-1, Contact definition)

RTHYMV

SEO ID NO: 28 (CDRH2 PD-1, Contact definition)

WVSFIGPAGDTTY

SEO ID NO: 29 (CDRH2 PD-1, Contact definition)

AAYTATSGVDTYDSEQ ID NO. 30 (Linker sequence)

STGLDSPT

SEO ID NO: 31 (CDRH1 (Kabat definition) of clone 22D034-51A09-163)

THYMA

SEO ID NO: 32 (CDRH2 (Kabat definition) of clones 22D034-51A09-183, 22D034-51A09-135, 22D034-51A09-110, 22D034-51A09-54, 22D034-51A09-10 and 22D034-51A09-4)

FIGPAGDFTYYADSVKG

SEO ID NO: 33 (CDRH2 (Kabat definition) of clones 22D034-51A09-163, 22D034-51A09-126 and 22D034-51A09-16)

FIGPAGDSTYYADSVKG

SEO ID NO: 34 (CDRH2 (Kabat definition) of clone 22D034-51A09-55)

FIGPAGDFTYYADSVEG

SEO ID NO: 35 (CDRH3 (Kabat definition) of clone 22D034-51A09-163)

YTATSSVDTYDV

SEO ID NO: 36 (CDRH3 (Kabat definition) of clones 22D034-51A09-140 and 22D034-51A09-10)

YTATSDVDTYDV

SEQ ID NO: 37 (CDRH3 (Kabat definition) of clone 22D034-51A09-135)

YTETSGVDTYDV

SEQ ID NO: 38 (CDRH3 (Kabat definition) of clones 22D034-51A09-126 and 22D034-51A09-55)

YTATSGFDTYDV

SEQ ID NO: 39 (CDRH3 (Kabat definition) of clone 22D034-51A09-54)

YTATSGYDTYDV

SEQ ID NO: 40 (CDRH3 (Kabat definition) of clone 22D034-51A09-16)

YTATSGFDSYDV

SEQ ID NO: 41 (CDRL1 Kabat definition) of clone 57C06)

RASQSISSFLN

SEQ ID NO: 42 (CDRL1 Kabat definition) of clone 57H08)

RASQSISSYLN

SEQ ID NO: 43 (CDRL3 Kabat definition) of clone 57B02)

QQAVDHFT

SEQ ID NO: 44 (CDRL3 Kabat definition) of clone 57C06)

QDLYSTPLT

SEQ ID NO: 45 (CDRL3 Kabat definition) of clone 57H08)

QQSFPAPPYT

SEQ ID NO: 46 (forward primer oligonucleotide)

CCCGGAAAGGGATCCACAGGACTGGACTCCCCGACAGAGGTGCAGCTGTTGGAGTCT

SEQ ID NO: 47 (reverse primer oligonucleotide)

GGGGATCTAGAATTCATCAGCTCGAGACGGTGACCAGGGTT

SEQ ID NO: 48 (forward primer oligonucleotide)

GGCCACCGCCACCGGTGTGCACAGC

SEQ I D NO: 49 (reverse primer oligonucleotide)

CGTACGGTGGCCGCCC

SEQ. I D NO: 50 (Amino acid sequence of light chain of LAG -3 mAb 57E02 and PD-l/LAG-3 mAbdAb 57E02x51A09-188001)

DIOMTOSPSSVSASVGDRVTITCRASOGISSWLAWYOOKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTD FTLTISSLOPEDFATYYCOETNSFWTFGGGTKVEI KRTVAAPSVFI FPPSDEQLKSGTASVVCLLNN FYPRE AKVOWKVDNALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFN RG EC

SEQ I D NO: 51 (DNA sequence of light chain of LAG -3 mAb 57E02 and PD-l/LAG-3 mAbdAb 57E02x51A09-188001)

GACATCCAGATGACCCAGAGCCCCAGCTCAGTGAGCGCTAGCGTGGGCGACAGGGTGACCATCACC

TG C AG G G C C AG C C AG G G C ATTAG CAGCTGGCTGGCCTGGTACCAGCAGAAGCCCGGCAAGGCCCC

CAAGCTCCTGATCTACGCCGCCAGCAGCCTGCAGAGCGGCGTGCCCTCCAGGTTTAGCGGCAGCGG

AAGCGGCACCGACTTCACCCTGACCATCAGCAGCCTGCAGCCCGAGGACTTCGCCACCTACTACTGC

CAGGAGACCAACAGCTTCTGGACCTTCGGCGGCGGCACAAAAGTCGAGATCAAGCGTACGGTGGCC

GCCCCCAGCGTGTTCATCTTCCCCCCCAGCGATGAGCAGCTGAAGAGCGGCACCGCCAGCGTGGTG

TGTCTGCTGAACAACTTCTACCCCCGGGAGGCCAAGGTGCAGTGGAAGGTGGACAATGCCCTGCAG

AGCGGCAACAGCCAGGAGAGCGTGACCGAGCAGGACAGCAAGGACTCCACCTACAGCCTGAGCAG

CACCCTGACCCTGAGCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGTGAGGTGACCCACCA

GGGCCTGTCCAGCCCCGTGACCAAGAGCTTCAACCGGGGCGAGTGC

SEQ I D NO: 52 (Amino acid sequence of heavy chain of LAG-3 mAb 57E02)

OVOLVOSGAEVKKPGASVKVSCKASGYTFTGYYM HWVROAPGOGLEWMGWI NPNSGGTNYAQKFO

GRVTMTRDTSISTAYM ELSRLRSDDTAVYYCAREGPYDDDGFDYWGQGTLVTVSSASTKGPSVFPLAPS

SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTQTYICNV

NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKG

OPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV

DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

SEQ I D NO: 53 (DNA sequence of heavy chain of LAG -3 mAb 57E02)

CAGGTGCAGCTCGTGCAGAGCGGCGCCGAGGTGAAAAAGCCCGGCGCCTCTGTCAAGGTGAGCTG

CAAGGCCAGCGGCTACACCTTCACCGGCTACTACATGCACTGGGTGAGGCAGGCTCCCGGACAGGG

CCTGGAGTGGATGGGCTGGATCAACCCCAACAGCGGCGGCACCAACTACGCCCAGAAGTTCCAGGG

CAGGGTGACCATGACCAGGGACACCAGCATCAGCACCGCCTACATGGAACTGAGCAGGCTGAGGA

GCGACGACACCGCCGTGTATTACTGCGCCAGGGAGGGACCCTACGACGACGACGGCTTCGACTACT

GGGGCCAGGGCACCCTGGTGACAGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCTG

GCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACTTC

CCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCGCC

GTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTGGG

CACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTGGA

GCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGGCCGGAGCCCCC

AGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGACCT

GTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGCGTG

GAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGTGTC

CGTG CTG ACCGTG CTG C ACC AG G ATTG G CTG AACG G C AAG G AGTAC AAGTGTAAG GTGTCCAAC AA

GGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCAGGT

GTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGTGAA

GGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACTACA

AGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGACAA

G AGCAG ATGG CAG CAGGG CAACGTGTTCAG CTGCTCCGTG ATG CACG AGG CCCTG CACAATCACTA

CACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAG

SEQ. I D NO: 54 (Amino acid sequence of heavy chain of PD-l/LAG-3 mAbdAb 57E02x51A09-188001)

OVOLVOSGAEVKKPGASVKVSCKASGYTFTGYYM HWVROAPGOGLEWMGWI NPNSGGTNYAQKFO

GRVTMTRDTSISTAYM ELSRLRSDDTAVYYCAREGPYDDDGFDYWGQGTLVTVSSASTKGPSVFPLAPS

SKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTQTYICNV

NHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

VKFNWYVDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKG

OPREPOVYTLPPSRDELTKNOVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV

DKSRWOOGNVFSCSVM HEALHN HYTOKSLSLSPGKGSTGLDSPTEVOLLESGGGLVQPGGSLRLSCAA

SGFTFRTHYMVWVROAPGKGLEWVSFIGPAGDTTYYADSVKGRFTISRDNSKNTLYLQM NSLRAEDTA

VYYCAAYTATSGVDTYDVMGQGTLVTVSSA

SEQ I D NO: 55 (DNA sequence of heavy chain of PD-l/LAG-3 mAbdAb 57E02x51A09-188001)

CAGGTGCAGCTGGTGCAGAGCGGGGCCGAGGTGAAGAAACCCGGCGCTAGCGTCAAGGTGAGCT

GCAAGGCCAGCGGGTACACCTTCACCGGCTACTACATGCACTGGGTGAGGCAGGCCCCCGGCCAGG

GACTCGAGTGGATGGGGTGGATCAACCCCAACAGCGGCGGCACAAACTACGCCCAGAAGTTTCAG

GGCAGGGTGACCATGACCAGGGACACCAGCATCAGCACCGCCTACATGGAACTCAGCAGGCTGAG

GTCCGACGACACCGCCGTGTACTATTGCGCCAGGGAGGGACCATACGACGACGACGGCTTCGATTA

CTGGGGCCAGGGCACCCTGGTGACCGTGAGCAGCGCCAGCACCAAGGGCCCCAGCGTGTTCCCCCT

GGCCCCCAGCAGCAAGAGCACCAGCGGCGGCACAGCCGCCCTGGGCTGCCTGGTGAAGGACTACT

TCCCCGAGCCCGTGACCGTGTCCTGGAACAGCGGAGCCCTGACCAGCGGCGTGCACACCTTCCCCG

CCGTGCTGCAGAGCAGCGGCCTGTACAGCCTGAGCAGCGTGGTGACCGTGCCCAGCAGCAGCCTG

GGCACCCAGACCTACATCTGTAACGTGAACCACAAGCCCAGCAACACCAAGGTGGACAAGAAGGTG

GAGCCCAAGAGCTGTGACAAGACCCACACCTGCCCCCCCTGCCCTGCCCCCGAGCTGGCCGGAGCC

CCCAGCGTGTTCCTGTTCCCCCCCAAGCCTAAGGACACCCTGATGATCAGCAGAACCCCCGAGGTGA

CCTGTGTGGTGGTGGATGTGAGCCACGAGGACCCTGAGGTGAAGTTCAACTGGTACGTGGACGGC

GTGGAGGTGCACAATGCCAAGACCAAGCCCAGGGAGGAGCAGTACAACAGCACCTACCGGGTGGT

GTCCGTGCTGACCGTGCTGCACCAGGATTGGCTGAACGGCAAGGAGTACAAGTGTAAGGTGTCCAA

CAAGGCCCTGCCTGCCCCTATCGAGAAAACCATCAGCAAGGCCAAGGGCCAGCCCAGAGAGCCCCA

GGTGTACACCCTGCCCCCTAGCAGAGATGAGCTGACCAAGAACCAGGTGTCCCTGACCTGCCTGGT

GAAGGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAACGGCCAGCCCGAGAACAACT

ACAAGACCACCCCCCCTGTGCTGGACAGCGATGGCAGCTTCTTCCTGTACAGCAAGCTGACCGTGGA

CAAGAGCAGATGGCAGCAGGGCAACGTGTTCAGCTGCTCCGTGATGCACGAGGCCCTGCACAATCA

CTACACCCAGAAGAGCCTGAGCCTGTCCCCTGGCAAGGGATCCACCGGCCTGGACAGCCCCACCGA

GGTCCAGTTGCTGGAAAGTGGGGGAGGACTGGTGCAGCCTGGCGGAAGCCTTAGACTCAGCTGCG

CCGCTAGTGGCTTCACTTTCCGCACCCACTACATGGTGTGGGTTAGGCAGGCACCCGGAAAAGGTCT

AGAGTGGGTTAGCTTTATCGGCCCTGCCGGCGATACCACCTATTACGCCGATTCCGTGAAGGGCAG

GTTCACAATCAGCAGGGACAACAGCAAGAACACCCTGTACCTGCAGATGAACTCCCTCAGGGCTGA

GGACACCGCGGTGTACTACTGTGCTGCCTACACCGCAACCTCAGGAGTCGATACCTACGACGTGATG

GGACAGGGCACTTTGGTTACCGTGAGTAGCGCC

SEQ I D NO: 56 (Generic CDRL1 LAG 3, Kabat definition)

RASQX1ISSX2LX3 (wherein Xi is G or S, X2 is W, F or Y, and X3 is A or N).

SEQ ID NO: 57 (Generic CDRH1 PD-1, Kabat definition)

THYMX4 (wherein X4 is V or A)

SEQ ID NO: 58 (Generic CDRH2 PD-1, Kabat definition)

FIGPAGDX5TYYADSVX6G (wherein X5 is T, F or S and X6 is K or E)

SEQ ID NO: 59 (Generic CDRH3 PD-1, Kabat definition)

YTXyTSXsXgDXioYDV (wherein X7 is A or E, X8 is G, S or D, X9 is V, F or Y, and Χω is T or S)

SEO ID NO: 60 (DNA sequence of HIV-1 Gag forward primer)

ATCAAG CAG CTATG C AAATGTT

SEO ID NO: 61 (DNA sequence of HIV-1 Gag probe)

ACCATCAATGAGGAAGCTGCAGAATGGGA

SEO ID NO: 62 (DNA sequence of HIV-1 Gag reverse primer)

CTGAAGGGTACTAGTAGTTCCTGCTATGTC

SEO ID NO: 63 (C-terminal extension to epitope binding domain or single variable domain when n is

0 and X is absent).

VTVS

SEO ID NO: 64 (C-terminal extension to epitope binding domain or single variable domain when n is

1 and X is absent).

VTVSS

SEO ID NO: 65 (C-terminal extension to epitope binding domain or single variable domain when n is

0 and Xn is present).

VTVSX12X13X14X15X16X17X18X19 (wherein X12, Xi3, Xi4, X15, Xie, X17, Xis orXig may represent any amino acid, wherein the sequence may terminate at position 6, 7, 8, 9 10, 11 or 12, and wherein the sequence between residues 5 to 12 may contain the sequence: A, AAA or T).

SEO ID NO: 66 (C-terminal extension to epitope binding domain or single variable domain when n is

1 and Xn is present).

VTVSSX12X13X14X15X16X17X18X19 (wherein X12, X13, Xi4, X15, Xie, X17, Xis orXig may represent any amino acid, wherein the sequence may terminate at position 7, 8, 9 10, 11, 12 or 13, and wherein the sequence between residues 6 to 13 may contain the sequence: A, AAA or T).

SEQ. ID NO: 67 (C-terminal extension to epitope binding domain or single variable domain when n is O and Xu is A).

VTVSA

SEQ ID NO: 68 (C-terminal extension to epitope binding domain or single variable domain when n is

VTVSSA

SEQ ID NO: 69 (C-terminal extension to epitope binding domain or single variable domain when n is O and Xu is AS).

VTVSAS

SEQ ID NO: 70 (C-terminal extension to epitope binding domain or single variable domain when n is l and Xu is AS).

VTVSSAS

SEQ ID NO: 71 (C-terminal extension to epitope binding domain or single variable domain when n is

VTVSAST

SEQ ID NO: 72 (C-terminal extension to epitope binding domain or single variable domain when n is

VTVSSAST

SEQ ID NO: 73 (C-terminal extension to epitope binding domain or single variable domain when n is

VTVSASTK

SEQ ID NO: 74 (C-terminal extension to epitope binding domain or single variable domain when n is

VTVSSASTK

SEQ. I D NO: 75 (C-terminal extension to epitope binding domain or single variable domain when n is

0 and Xii is ASTKG).

VTVSASTKG

SEQ I D NO: 76 (C-terminal extension to epitope binding domain or single variable domain when n is

1 and Xii is ASTKG).

VTVSSASTKG

SEQ I D NO: 77 (C-terminal extension to epitope binding domain or single variable domain when n is O and Xu is ASTKGP).

VTVSASTKG P

SEQ I D NO: 78 (C-terminal extension to epitope binding domain or single variable domain when n is l and Xu is ASTKGP).

VTVSSASTKG P

SEQ I D NO: 79 (Amino acid sequence of light chain of CTLA-4/PD-1 mAbdAb CTLA4 x51A09-188001)

EIVLTOSPGTLSLSPGERATLSCRASOSVGSSYLAWYOOKPGQAPRLLIYGAFSRATGI PDRFSGSGSGTDF TLTISRLEPEDFAVYYCOOYGSSPWTFGOGTKVEIKRTVAAPSVFI FPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALOSGNSOESVTEODSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFN RG EC

SEQ I D NO: 80 (Amino acid sequence of heavy chain of CTLA-4/PD-1 mAbdAb CTLA4 x51A09-188001)

OVOLVESGGGVVOPGRSLRLSCAASGFTFSSYTM HWVRQAPGKGLEWVTFISYDGNN KYYADSVKGRF

TISRDNSKNTLYLOMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSG

GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSN

TKVDKKVEPKSCDKTHTCPPCPAPELAGAPSVFLFPPKPKDTLM ISRTPEVTCVVVDVSH EDPEVKFNWY

VDGVEVHNAKTKPREEOYNSTYRVVSVLTVLHODWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPO

VYTLPPSRDELTKNOVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

OOGNVFSCSVMH EALHNHYTOKSLSLSPGKGSTGLDSPTEVOLLESGGGLVQPGGSLRLSCAASGFTFR

THYMVWVRQAPGKGLEWVSFIGPAGDTTYYADSVKGRFTISRDNSKNTLYLQ.M NSLRAEDTAVYYCAA YTATSGVDTYDVMGQGTLVTVSSA

SEQ ID NO: 81 (C-terminal sequence of 51A09-188001)

LVTVSSA