TW201829462A - Binding proteins - Google Patents

Abstract

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.

Description

   Binding protein   

The present invention relates to a binding protein specific for at least one immune checkpoint receptor 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 a method for preparing the binding protein, a pharmaceutical composition containing the binding protein, and medical use of the binding protein.

T-cell co-stimulation and co-suppression molecules (collectively called co-signaling molecules) play a key role in regulating T-cell activation, subpopulation differentiation, effector function and survival (Chen et al 2013, Nature Rev. Immunol. 13: 227-242). After the T-cell receptor recognizes the homologous peptide-MHC complex on the antigen-presenting cell, the co-signaling receptor co-localizes with the T-cell receptor at the immune synapse, where it Synergize with TCR signal transmission to promote or inhibit T cell activation and function (Flies et al 2011, Yale J. Biol. Med. 84: 409-421). The final immune response is regulated by the balance between co-stimulation and co-suppression signals ("immune checkpoints") (Pardoll 2012, Nature 12: 252-264). The functional systems of programmed death-1 (PD-1) and lymphocyte activation gene 3 (LAG-3) at immune checkpoints are summarized below.

    PD-1    

PD-1 (also known as CD279) is a protein receptor of 288 amino acids 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 costimulatory molecule) family of T cell co-suppression receptors (Chen et al 2013, Nat. Rev. Immunol. 13: 227-242). The main function of PD-1 is to weaken the immune response (Riley 2009, Immunol. Rev. 229: 114-125). PD-1 has two kinds of ligands, PD-ligand 1 (PD-L1) and PD-L2. PD-L1 (also known as CD274, B7H1) is widely expressed in lymphoid and non-lymphoid tissues, such as CD4 ⁺ and CD8 ⁺ T-cells, macrophage lineage cells, peripheral tissues, as well as cancer cells, virus-infected cells and autologous On immune tissue cells. PD-L2 (CD273, B7-DC) has a more restricted performance than PD-L1, and its lines are expressed on activated dendritic cells and macrophages (Dong et al 1999, Nature Med.). The PD-L1 line manifests in most human cancers, including melanoma, non-small cell lung cancer, squamous cell carcinoma of the head and neck, leukemia, pancreatic cancer, renal cell carcinoma, and hepatocellular carcinoma, and may be in almost all cancers Inducible in type (Zou and Chen 2008, Nat. Rev. Immunol. 8: 467-77).

The binding of PD-1 to its ligands (PD-L1 and PD-L2) reduces the function of T-cells, including decreased activation, decreased proliferation and altered cytokine secretion. This ability is used by chronic viral infections and tumors to avoid immune responses.

Blocking PD-1 binding to reverse immunosuppression has been studied in autoimmunity, virus and 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). Blockers of PD-1 with antagonists, including monoclonal antibodies, have been studied in the clinical treatment of cancer and chronic viral infections (Sheridan 2012, Nature Biotechnology 30: 729-730).

In the case of infection, antigen-specific CD8 + T cells initially acquire effector function but gradually become weaker during chronic infection, allowing infected cells to survive and avoid immune supervision. In-depth studies have been conducted (Wherry et al., Immunity , 27 (4): 670-684, 2007). This loss of function is also known as T cell depletion and is characterized by the possibility of hyperplasia, cytokine production, cytotoxic function, and reduced cell survival (Freeman 2008, PNAS 105: 10275-10276). Studies have shown that blocking PD-1 may reverse T cell depletion on bacterial infections, parasitic infections, viral infections, and sepsis (secondary infections).

Bacterial infections

PD-1 deficient mice have been shown to listeria monocytogenes (Yao et al., Blood, 2009, 113 (23): 5811-8) and Streptococcus pneumonia (McKay) et al., J Immunol, 2015, 194 (5): 2289-99) The bacterial infection is resistive. In addition, studies have shown that PD-1 blockade increases T cell effector function 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 infection

The role of PD-1 during malaria infection was re-examined by Wykes and Colleagues (Front Microbiol, 2014, 27 (5): 249) and suggested that it may be treated by blocking PD-1 in combination with other immune checkpoint inhibitors (see Title PD-1 / LAG-3).

Viral infection

Erikson and colleagues have pointed out that PD-1 mediated CD8 + T cell damage occurs in mice after infection with human interstitial pneumonia virus or influenza virus, and in patients with 2009 H1N1 influenza virus, respiratory fusion virus or paravirus PD1 and PDL1 appear to be upregulated in the lungs of patients with influenza virus, which shows that PD-1 blockade may represent a therapeutic target for the treatment of viral respiratory infections (Erikson et al., J Clin Invest 2012 122 (8) 2967-82).

PD-1 blockade is also considered effective in the treatment of other chronic viral infections. In a clinical trial, blocking PD-1 with anti-PD1 all-human monoclonal antibody (BMS-936558) produced persistent inhibition of HCV replication in some patients with chronic infections. Ye and his colleagues (Cell Death Dis, 2015, 6: e1694) re-examine the role of the PD-1 / PD-L1 pathway in hepatitis B infection, and conclude that although other immune checkpoint inhibitors may also be important , But it may help eliminate T cell depletion. Similarly, anti-PD-L1 antibodies also showed the proliferation of CMV (cytomegalovirus) specific T cells and increased cytokines, as well as the increase in HSV (herpes simplex virus) specific CD8 cell pairs in an animal model of HSV infection HSV peptide response. Treatment of mice injected with EBV (Eberstein-Barr virus) -infected cord blood with anti-PD-1 antibodies reduced EBV-induced lymphoma growth. HIV infection is characterized by depleted CD8 T cells, which cannot proliferate, produce cytokines and perform cytotoxic functions. This action prevented the immune system from launching an effective antiviral response. Even though existing HIV treatments can inhibit viral replication to low levels, they are not completely eliminated and the low levels of virus performance persist in tissues with poor drug penetration and / or from activated latently infected cells. Xianxin, despite antiretroviral therapy (ART), viral antigens continue to stimulate immune cells and continue to inflame HIV-infected individuals. There are several types of non-AIDS morbidity and increased mortality rates in patients, including cardiovascular disease, cognitive impairment, and frailty that are considered to be well controlled by ART (Deeks, Annu Rev Med., 2011, 62: 141-55). This increased incidence occurs in the context of elevated systemic inflammation and is related to the sustained immune 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 the elimination of antiviral responses of cells expressing viral antigens, resulting in a reduction in the incidence of immune stimulation, inflammation and non-AIDS.

The latent HIV gene has been shown to be concentrated in CD4 + memory T cells. De Fonseca et al. Pointed out that blocking the PD-1 line increases the HIV response of CD4 T cells in the presence of optimal T cell stimulation. PD-1 is also highly expressed in memory CD8 cells during HIV infection (Yamamoto et al., Blood, 117: 4805-4815, 2011). CD8 T cells may be cytotoxic and kill infected cells. PD-1 blockade has been tested in animal models of HIV-1 infection and it enhances the antiviral response. More specifically, treatment of SIV-infected viremia with anti-PD-1 antibodies increased the multifunctionality of HIV-specific CD8 + T and B cells, reducing viremia and improving overall survival. Bristol Myers Squibb Another study found that blocking the inhibition of SIV infection in rhesus ART waiting in PDL-1, resulting in half of the animals after treatment interruption reduced viremia ^{(Whitney et al., 6 th} International Workshop on HIV persistence during therapy , Miami, FL, 2009).

septicemia

Treatment of cells in patients with sepsis with anti-PD-1 or anti-PD-L1 antibodies reduces apoptosis and increases IFN-γ and IL-2 production (Chang et al., Crit Care, 2014, 18 (1): R3). In addition, compared with wild-type mice, only a few PD1 knockout mice died in the experimentally induced sepsis model (Huang et al., Proc. Natl. Acad. Sci., 2009, 106 (15): 6303-8 ). Another group found that PD-L1 expression in the liver increased in this animal model. In the same study, it was found that administration of anti-PD-L1 reduced the amount of specific pre-inflammatory cytokines (Zhu et al., Mediators Inflamm, 2013, 361501).

cancer

In addition, as mentioned above, the PD-L1 line is expressed in a wide variety of tumors and has been studied in animal models to show that PD-L1 on tumors inhibits T-cell activation and tumor cell dissolution and may increase tumor-specific T Cell death. PD-1: The PD-L1 system also plays an important role in inducing T-regulated (Treg) cell development and maintaining Treg function (Francisco et al 2010, Immunol. Rev. 236: 219-242).

Known PD-1 binding protein

PD-1 antibodies and methods for treating diseases 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 the US Publication numbers US20110008369; US20130017199; US20130022595; US20110171220; US20110171215; and US20110271358 and WO2006121168; WO20091154335; WO2012145493; WO2013014668; WO2009101611; EP2262837; and EP2504028 The combination of CTLA-4 and PD-1 antibodies is described in US Patent No. 9,084,776.

OPDIVO / nivolumab is a fully human monoclonal antibody sold by Bristol Myers Squibb that has immune enhancing activity against the negative immunomodulatory human expression receptor PD-1. Nivolumab binds to PD-1 (an Ig superfamily transmembrane protein) and blocks PD-1 activation by its ligands PD-L1 and PD-L2, making it resistant to tumor cells or pathogenic T-cells and cells -The immune response of the medium is activated. Activated PD-1 negatively regulates T-cell activation and effector functions via inhibition of P13k / Akt pathway activation. Other names for nivolumab include: BMS-936558, MDX-1106 and ONO-4538. The amino acid sequence of nivolumab and methods of use and manufacture are disclosed in US Patent No. US 8,008,449. A first-stage, double-blind, placebo-controlled, incremental single-dose study evaluating the safety and immune response of this antibody in combination with antiretroviral therapy to completely suppress HIV infection is ongoing. Nivolumab has also been used to treat 12 HIV + patients with non-small cell lung cancer. While no changes in plasma HIV viral load, CD4 or CD8 cell count were observed, the total HIV-DNA volume in a patient decreased sharply, showing a possible virus nest effect (Guihot et al., IAS Abstract A-854-0121-02601).

KEYTRUDA / pembrolizumab is an anti-PD-1 antibody sold by Merck for the treatment of lung cancer. The amino acid sequence and method of use of pamluzumab is disclosed in US Patent No. 8,168,757. A multi-center study (Uldrick, Clinical Trial Design Considerations: Leveraging Cancer Immunotherapy Studies to Evaluate HIV Endpoints, presentation at the International AIDS Symposium in Paris, 2017) has been proposed to evaluate pamluzumab in HIV and relapsed refractory cancer patients.

The anti-PD-L1 antibody BMS-936559 also targets the PD1-PDL1 axis. In a clinical study of 8 HIV-infected participants who received a BMS-936559 infusion, the HIV-1 Gag-specific CD8 + T cell response increased within 28 days after infusion, including increased CD107a performance, which is related to CD8 + The reverse of T cell depletion (Eron et al., Safety, Immunologic and Virologic Activity of Anti-PD-L1 in HIV-1 participants on ART, Abstract No. 25, Conference on Retroviruses and Opportunistic Infections, February 22-25 2016 , Boston).

The ability to block PD-1 to reduce immunosuppression or eliminate T-cell depletion has been shown to be useful as an adjunct therapy for cancer and infectious diseases. For example, several clinical trials have been conducted to detect the combination therapy of PD-1 antibody and cancer vaccine. In these vaccines, the PD-1 antibody can be regarded as an adjuvant.

LAG-3

LAG-3 (also known as CD223) is a member of an immunoglobulin supergene family and is a membrane protein that is structurally and genetically related to CD4. There are several cell types that express LAG-3. For example, the LAG-3 line shows infiltration of activated CD4 ⁺ and CD8 ⁺ T cells, killer (NK) cells, plasmacytoid dendritic cells (DC), and tumor-infiltrating lymphocytes, such as head and neck squamous cell carcinoma (HNSCC) Lymphocytes. LAG-3 is also expressed in highly inhibitory-induced and natural Treg. For example, highly inhibited FoxP3 + nTreg and FoxP3-iTreg 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).

The ligands of LAG-3 include, for example, MHC type 2 and L-SECtin. Blocking LAG-3 can restore the activity of effector cells and reduce the activity of inhibitors of Treg. For example, in vitro studies of antigen-specific T cell responses have shown that the addition of anti-LAG-3 antibodies increases T cell proliferation, higher expression of activated antigens, such as CD25, and higher concentrations of cytokines, such as interferon -γ and interleukin-4, which shows that LAG-3 blockade down-regulates antigen-dependent CD4 ⁺ T lymphocyte stimulation (Huard et al. (1994) Eur. J. Immunol. 24: 3216-3221). LAG-3 blockade in tumor autoantigen (Gross et al. (2007) J Clin Invest. 117: 3383-3392) and virus model (Blackburn et al. (2009) Nat. Immunol. 10: 29-37) It also showed that CD8 ⁺ lymphocytes were recovered. Furthermore, CD4 ⁺ CD25 ⁺ regulatory T cells (Treg) have been shown to express LAG-3 after activation and antibodies to LAG-3 inhibit the suppression induced by Treg cells in vitro and in vivo, which shows that LAG contributes Inhibitor 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 signal transmission and function and causes the depletion of T cells during chronic viral infections (including HIV), parasitic infections, sepsis, and cancer.

Viral infection

While the size and operation of T cell responses in LAG-3-deficient mice are similar to those of wild-type animals, it has recently been found that CD8 ( +) T cells show a slight decrease in the rate of cell division (Cook et al., 2016, J Immunol., 197 (1): 119-127). Erikson et al., 2016, J Immunol., 197 (1): 233-43 pointed out that blocking LAG-3 in PD-1 deficient mice infected with human interstitial pneumonia virus caused increased CD8 effector function (compared to In mice not treated with anti-LAG-3 mAb).

Especially in the case of HIV, Fromentin and colleagues identified HIV individuals controlled by antiretroviral therapy, and the LAG-3 expression on CD4 + T cells is related to the integrated HIV gene.) Induction for expression of HIV gene (Fromentin et al., PLoS Pathog., 2016, 12 (7): e1005761). Tian and colleagues (J. Immunol. 194: 3873-3882, 2015) pointed out that treatment of peripheral blood mononuclear cells derived from HIV-infected individuals without antiretroviral treatment with LAG-3-Fc chimera increased Hyperplasia and cytokines produced by CD4 + and CD8 + cells in response to B-Gag stimulation.

Antibodies to LAG-3 and methods for treating diseases are described in US20110150892, US20150259420 and US Patent No. 6,143,273. Several anti-LAG-3 monoclonal antibodies BMS-986016 (BMS) and LAG525 (Novartis) have also entered clinical trials for cancer treatment.

Parasitic infection

Doe et al. (2016, Microbiol. Immunol., 60 (2): 121-131) pointed out that CD4 + T cells of mice infected with Plasmodium express LAG-3. Blocking PD-L1 and LAG-3 in vivo restores the function of CD4 (+) T cells, increases follicular helper T cells, germinal center B cells and plasmablasts, enhances protective antibodies and quickly clears blood from mice Stage malaria (Butler et al., 2011, Nat Immunol., 13 (2): 188-95).

septicemia

Boomer et al., 2012, Crit Care, 16 (3): R112 pointed out that the amount of LAG3 (and Tim 3) on CD4 + cells in hospitalized patients with sepsis increased during 7 days.

cancer

Grosso and colleagues (J Clin Invest., 2007, 117: 3383-3392) pointed out that the antigen-specific organs or tumor-specific antigen-specific CD8 ⁺ T cells showed an increase in the amount of LAG-3 protein. LAG-3 blockade or gene excision causes accumulation of antigen-specific CD8 ⁺ T cells in organs expressing their cognate antigens and increased effector function, and the combination of LAG-3 blockade and specific anti-tumor vaccine causes intratumoral Activated CD8 ⁺ T cells are significantly increased and the tumor parenchyma is destroyed.

    Known LAG-3 combinatorial protein    

Antibodies to LAG-3 and methods for treating diseases are described in US20110150892, US20150259420 and US Patent No. 6,143,273. Several anti-LAG-3 monoclonal antibodies, including BMS-986016 (BMS) and LAG525 (Novartis) have also entered clinical trials for cancer treatment.

PD-1 / LAG-3

However, LAG-3 is typically not limited to co-presentation on PD-1 ⁺ cells. Although the co-expression of LAG-3 and PD-1 can be found on functional T cells, co-expression is an indicator of compromised function in the case of chronic infection and cancer (Nguyen and Ohashi, (2015) Nat. Rev. Immunol. 15: 45-56). For example, the latent HIV genome has been shown to be concentrated in CD4 + memory T cells expressing PD1 and LAG3 (Fromentin et.al PLOS Pathogens 2016). In particular, as far as HIV is concerned, LAG3 and PD1 performance during the acute phase of HIV infection is related to clinical disease progression (Hoffman M PLOS Pathogens 2016 Jul 14; 12 (7)). In addition, the extent of CD8 ⁺ T cell depletion, expressed as the percentage of dual IFNγ / TNFα-producing cells, is associated with the number of expressed inhibitory receptors (Blackburn et al. (2009) Nat. Immunol. 10 (1 ): 29-37). At the subcellular level, both PD-1 and LAG-3 bind to T cell receptor complexes after conjugation with their cognate ligands (Yokosuka et al., (2012) J. Exp. Med. 209: 1201 -1217; Hannier and Triebel, (1999), International Immunology 11: 1745-1752). In melanoma, high PD-1 / LAG-3 expression is associated with T cell infiltration. Blocking LAG-3 with anti-PD-1 or PD-L1 in the latency model may result in tumor suppressive activity, for example, in SalN fibrosarcoma and MC38 colorectal cancer models, anti-LAG-3 and anti-PD-1 Blocking display utility (Woo et al. (2012) Cancer Res. 72 (4): 917-27). In the lymphocytic choriomeningitis virus (LCMV) model, PD-1 / LAG-3 blockade is also effective. During chronic LCMV infection, PD-L1 plus LAG-3 blockade enhances the antiviral CD8 + T cell response (Blackburn et al. (2009) Nat. Immunol. 10 (1): 29-37). In the case of a murine model of Plasmodium infection that triggers T cell depletion, antagonizing LAG-3 and PD-1 restores CD4 +, CD8 + and Tfh functions and causes the elimination of Plasmodium infection in the blood (Butler et al. Immunol. 13 (2): 188-95, 2011).

Several anti-LAG-3 monoclonal antibodies (LAG525, RGN3767, IMP321, and BMS 986016 combined with anti-PD-1 antibodies (PDR001, REGN2810, pamluzumab, and nivolumab) were used to treat solid tumors, melanoma, and glial Clinical trials for blastoma, colorectal cancer, virus-related tumors, and hematological tumors are underway. Furthermore, Macrogenics is conducting MGD013, a tetravalent dual specificity (PD-1 x LAG3) with Fc DART protein and human IgG4 skeleton, Phase I trial for the treatment of solid tumors and hematological tumors (Motte-Mohs, MGD013, a Bispecific PD-1 x LAG-3 Dual Affinity Re-Targeting (DART) Protein with T-cell Immuuomodulatory Activity for Cancer Treatment, Poster 3217, presented at the 2016 American Association for Cancer Research Annual Meeting, April 16-20, 2016, New Orleans, Louisiana). There are also reports that TESARO uses dual-specific anti-LAG-3 in preclinical development / PD-1 antibody is used in cancer.

Other immune checkpoints

Inhibition pathways have different mechanisms of inhibition, so targeting multiple immune checkpoints should increase the frequency with which patients respond to treatment. Tumor-specific CD8 T cells showed high amounts of PD-1, but also showed CTLA-4 and other inhibitory receptors (Ahmadzadeh et al., Blood, 2009, 114: 1537-1544). Therefore, clinical trials combining PD-1 and CTLA-4 block have higher response rates than previously proposed single treatment of melanoma patients (Wolchok et al., N Engl J Med., 2013, 369: 122-133 ).

Several other inhibitory receptors are currently aimed at improving T cell responses. In animal models, it has been shown that the combination of blocking Tim-3 and blocking the PD-1 pathway can further enhance T cell rescue (Sakuishi Ket al., J Exp Med., 2010, 207: 2187-2194; Jin et al. Proc Natl Acad Sci US 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) pointed out that anti-Tim in combination with anti-PD-1 in a mixed lymphocyte reaction increases IL-2 secretion and reduces the effective EC50 of each single agent.

In the first aspect, the present invention provides a binding protein specific for human LAG-3 and human PD-1, which includes an antibody specific for human LAG-3 through a linker and one or more pairs The human PD-1 specific epitope binding regions are connected, and the anti-human LAG-3 specific resistance system includes one or more CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: CDRH1 shown in SEQ ID NO: 1 and CDRH1 different from CDRH1 shown in SEQ ID NO: 1 by adding or deleting or substituting 1, 2 or 3 amino acids, where CDRH2 is in the group consisting of Select: CDRH2 as shown in SEQ ID NO: 1 and CDRH2 different from CDRH2 as shown in SEQ ID NO: 1 by adding or deleting or substituting 1, 2 or 3 amino acids, and CDRH3 is composed of the following From the group consisting of: CDRH3 shown in SEQ ID NO: 1 and CDRH3 different from CDRH3 shown in SEQ ID NO: 1 by adding or deleting or substituting 1, 2 or 3 amino acids; wherein Human LAG-3 specific resistance system includes one or more CDRL1, CDRL2 and CDRL3, of which CDRL1 is selected from the group consisting of : CDRL1 as shown in SEQ ID NO: 2 and CDRL1 different from CDRL1 as shown in SEQ ID NO: 2 by adding or deleting or substituting 1, 2 or 3 amino acids, where CDRL2 is composed of the following Selected from the group: CDRL2 as shown in SEQ ID NO: 2 and CDRL2 different from CDRL2 as shown in SEQ ID NO: 2 by adding or deleting or replacing 1, 2 or 3 amino acids, and the CDRL3 Select from the group consisting of: CDRL3 as shown in SEQ ID NO: 2 and CDRL3 different from CDRL3 as shown in SEQ ID NO: 2 by adding or deleting or replacing 1, 2 or 3 amino acids; The one or more epitope binding regions specific for human PD-1 include one or more CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: as shown in SEQ ID NO: 3 CDRH1 and CDRH1 different from CDRH1 shown in SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids, wherein CDRH2 is selected from the group consisting of: as SEQ ID NO: CDRH2 shown in 3 and CDRH2 different from CDRH2 shown in SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids, and CDRH3 is selected from the group consisting of: CDRH3 as shown in SEQ ID NQ: 3 and CDRH3 as shown in SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids CDRH3; and the linker is selected from the group consisting of a bond or a peptide linker.

In one embodiment, the binding protein specific for human LAG-3 and human PD-1 has> 50% inhibition of LAG3-MHCII interaction in competitive flow cytometry analysis and in PD-1 / PDL- 1 IC50 of less than or equal to 5 nM in the competition analysis.

In a more specific aspect, the present invention provides a binding protein having the general formula (I) specific for human LAG-3 and human PD-1: Wherein: H (LAG-3) is an IgG antibody heavy chain including CDRH1, CDRH2 and CDRH3, wherein the CDRH1 is selected from: CDRH1 shown in SEQ ID NO: 1, and by adding or deleting or replacing 1, 2 or 3 amino acids and CDRH1 different from CDRH1 shown in SEQ ID NO: 1; wherein CDRH2 is selected from: CDRH2 shown in SEQ ID NO: 1 and by adding or deleting or replacing 1, 2 or 3 Amino acids and CDRH2 different from CDRH2 shown in SEQ ID NO: 1; and CDRH3 is selected from: CDRH3 shown in SEQ ID NO: 1 and by adding, deleting or replacing 1, 2 or 3 Amino acid and CDRH3 different from CDRH3 shown in SEQ ID NO: 1; L (LAG-3) is an IgG antibody light chain including CDRL1 and CDRL2, wherein the CDRL1 is selected from: SEQ ID NO: 2 The CDRL1 shown and the CDRL1 different from the CDRL1 shown in SEQ ID NO: 2 by adding or deleting or substituting 1, 2 or 3 amino acids; and the CDRL2 is selected from: SEQ ID NO: 2 CDRL2 and CDRL2 different from CDRL2 shown in SEQ ID NO: 1 by adding or deleting or replacing 1, 2, or 3 amino acids; n is an integer selected from 2, 4, and 11; A is a bond or peptide linker; and V _H (PD-1) is There are antibody heavy chain variable regions of CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from: CDRH1 as shown in SEQ ID NO: 3, and by adding or deleting or replacing 1, 2 or 3 amino acids CDRH1 different from CDRH1 shown in ID NO: 3; wherein CDRH2 is selected from: CDRH2 shown in SEQ ID NO: 3 and is different from SEQ ID NO by adding, deleting or replacing 1, 2 or 3 amino acids CDRH2 shown in 3 is different from CDRH2; and CDRH3 is selected from: CDRH3 shown in SEQ ID NO: 3 and by adding or deleting or substituting 1, 2 or 3 amino acids to be the same as SEQ ID NO: 3 The CDRH3 shown is different from the CDRH3.

In one embodiment, the binding protein of formula (I) specific for human LAG-3 and human PD-1 has> 50% inhibition of LAG3-MHCII interaction in competitive flow cytometry analysis, and in PD- 1 / PDL-1 competition analysis has IC50 less than or equal to 5nM.

In the second aspect, the present invention provides a binding protein comprising one or more CDRH1, CDRH2 and CDRH3 specific for human PD-1, wherein CDRH1 is selected from the group consisting of: as shown in SEQ ID NO: 3 CDRH1 and CDRH1 different from CDRH1 shown in SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids, wherein CDRH2 is selected from the group consisting of: as SEQ ID NO: CDRH2 shown in 3 and CDRH2 different from CDRH2 shown in SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids, and CDRH3 among them are selected from the group consisting of: CDRH3 shown in SEQ ID NO: 3 and CDRH3 different from CDRH3 shown in SEQ ID NO: 3 by adding, deleting, or replacing 1, 2, or 3 amino acids. In an embodiment. This binding protein has an IC50 of less than or equal to 5 nM in the PD-1 / PDL-1 competition analysis.

In a more specific aspect, a binding protein specific for human PD-1 can additionally neutralize human checkpoint inhibitors other than PD-1. In the context of the present invention, a human checkpoint inhibitor is a human protein that restricts the function of human immune cells by inhibiting the signaling cascade that the immune cells regulate activation, proliferation, cytokine production or function after stimulation. Neutralizing the human checkpoint inhibitor blocks the biological activity of the human checkpoint inhibitor.

In a third aspect, the present invention provides a binding protein specific for human LAG-3, which includes: one or more CDRH1, CDRH2, and CDRH3, wherein CDRH1 is selected from the group consisting of: SEQ ID NO : CDRH1 shown in 1: and CDRH1 different from CDRH1 shown in SEQ ID NO: 1 by adding or deleting or replacing 1, 2 or 3 amino acids, where CDRH2 is selected from the group consisting of: CDRH2 shown in SEQ ID NO: 1 and CDRH2 different from CDRH2 shown in SEQ ID NO: 1 by adding or deleting or replacing 1, 2 or 3 amino acids, and CDRH3 is a group consisting of Selected from: CDRH3 as shown in SEQ ID NO: 1 and different from CDRH3 as shown in SEQ ID NO: 1 by adding, deleting or replacing 1, 2 or 3 amino acids; and one or more CDRL1 , CDRL2 and CDRL3, of which CDRL1 is selected from the group consisting of: CDRL1 as shown in SEQ ID NO: 2 and with SEQ ID NO: 2 by adding or deleting or replacing 1, 2 or 3 amino acids The CDRL1 shown is different from the CDRL1, where CDRL2 is selected from the group consisting of: CDRL2 as shown in SEQ ID NO: 2 and by adding or deleting or taking The CDRL2 which is different from the CDRL2 shown in SEQ ID NO: 2 by substituting 1, 2 or 3 amino acids, and the CDRL3 is selected from the group consisting of: the CDRL3 shown in SEQ ID NO: 2 and by Add or delete or replace 1, 2 or 3 amino acids and CDRL3 different from CDRL3 shown in SEQ ID NO: 2.

In one embodiment, a binding protein specific for human LAG-3 has> 50% inhibition of LAG3-MHCII interaction in competitive flow cytometry analysis.

In a more specific aspect, binding proteins specific for human LAG-3 can additionally neutralize human checkpoint inhibitors other than LAG-3. As explained above, human checkpoint inhibitors are human proteins that limit the function of human immune cells by inhibiting the signaling cascade that the immune cells modulate activation, proliferation, cytokine production, or function after stimulation. Neutralizing the human checkpoint inhibitor blocks the biological activity of the human checkpoint inhibitor.

In another aspect, the present invention provides encoding proteins of the present invention (ie, binding proteins specific for human LAG-3 and human PD-1, and binding proteins specific for human PD-1 (and additional testing if necessary) Point inhibitors) and binding proteins specific for human LAG-3 (and additional checkpoint inhibitors if necessary)) vectors containing the isolated nucleic acids encoding these binding proteins, and vectors containing these vectors Of the host cell. For example, the present invention provides isolated nucleic acids encoding HAV _H (PD-1), H, and L, where H, A, V _H (PD-1), and L are as defined above. The present invention also provides a method for manufacturing the binding protein of the present invention, which includes culturing a host cell containing an appropriate carrier under conditions suitable for protein expression, and isolating the binding protein.

In one aspect, the invention provides a pharmaceutical composition comprising the binding protein of the invention and a pharmaceutically acceptable excipient. The present invention also provides a method for treating diseases using the binding protein of the present invention or the pharmaceutical composition of the present invention. In particular, the present invention provides a method for treating cancer and a method for treating and curing HIV.

Fig. 1 is a bar graph showing that it is combined with isolated CD4 T cells, dendritic cell-derived monocytes (MDDC) and LAG3 / PD1 bispecific 57E02x51A09-188001, LAG3 or PD1 monovalent antagonists or combinations thereof After 5 days of cultivation, the average IFNγ produced in the mixed lymphocyte reaction analysis. The bar graph shows the double-repetition analysis, in which the average and standard deviation of three replicates for each analysis. Anti-systems were tested at concentrations ranging from 0.195-200nM. Compared to LAG3 or PD1 monovalent antagonists or combinations thereof, the LAG3 / PD1 dual specificity 57E02x51A09-188001 causes CD4 + T cells to produce more IFNγ.

Figure 2 is a bar graph showing the proliferation of (CFSEdim) HIV-specific CD8 T during 6 days of culture in the presence of LAG3 / PD1 mAbdAb 57E02-51A09-188001 (black bar) or control antibody (gray bar) The weight of the cell. The bar graph shows the average and standard deviation of 6 replicates with Bonferroni corrected p-values from the pairwise comparison shown in the bar graph above. Please note that it is incorrect to refer to the LAG3 / PD1 mAbdAb as 57E02-51A09-188 in Figure 2 (the correct name is 57E02-51A09-188001).

Figure 3 is a scatter diagram showing the cellular components that produce specific cytokines derived from 19 HIV-infected stable ART-treated donors, stimulated or not stimulated by SEB & SEA, with or without LAG3 / PD1 mAbdAb 57E02 -51A09-188001 or control antibody: A) the amount of CD4 memory & / or effector T cells producing IFNγ, IL2 and TNFα, B) the amount of CD8 memory & / effector T cells producing IFNγ, IL2 and TNFα , C) The component of CD4 memory & / effector T cells producing IL2 and TNFα, and D) The component of CD8 memory & / effector T cells that dually express IFNγ and CD107. In each case, statistical significance was determined by a mixed effect model followed by a pairwise comparison. Bonferroni corrected p-values are shown in the figure. It should be noted that the data analysis system analyzes the magnitude of the ^0.2 transformation of the data to make the variation more homogeneous.

Figure 4 is a scatter diagram showing the amount of HIV Gag RNA per 1 million CD4 T cells from 5 HIV-infected (ST) donors who were treated stably. Cultivate CD4 T cells alone (CD4 round & triangle) or in the presence of monocytes derived from dendritic cells (mDDC-star & square) in LAG3 / PD1 bispecific antibody 57E02-51A09-188001 Or culture in the presence of control antibody VHDUM. The P-values shown in the figure are obtained by comparing antibodies in pairs, and corrected by Bonferroni after multiple comparisons. The data is analyzed in log ₁₀ transform magnitudes, and each donor is analyzed individually.

    Binding protein    

Several different binding proteins are provided. These can be identified by targeting their combination. In individual aspects, the present invention provides binding proteins specific for human PD-1 and human LAG-3, binding proteins specific for human PD-1 (and other checkpoint inhibitors as needed) and for humans LAG-3 (and other checkpoint inhibitors as needed) are specific binding proteins.

In the first aspect, the present invention provides a binding protein, which comprises an antibody specific for human LAG-3 and is linked to one or more epitope binding regions specific for human PD-1 through a linker , Where the anti-human LAG-3 specific resistance system includes one or more CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: CDRH1 as shown in SEQ ID NO: 1 and by adding Or delete or replace 1, 2 or 3 amino acids and CDRH1 different from CDRH1 shown in SEQ ID NO: 1, CDRH2 is selected from the group consisting of: CDRH2 shown in SEQ ID NO: 1 and CDRH2 different from CDRH2 shown in SEQ ID NO: 1 by adding or deleting or replacing 1, 2 or 3 amino acids, and CDRH3 is selected from the group consisting of: as shown in SEQ ID NO: 1 The CDRH3 shown and the CDRH3 different from the CDRH3 shown in SEQ ID NO: 1 by adding or deleting or replacing 1, 2, or 3 amino acids; wherein the resistance system specific to human LAG-3 includes one or Multiple CDRL1, CDRL2 and CDRL3, of which CDRL1 is selected from the group consisting of: CDRL1 as shown in SEQ ID NO: 2 and CDRL1 different from CDRL1 shown in SEQ ID NO: 1 by adding or deleting or substituting 1, 2 or 3 amino acids, where CDRL2 is selected from the group consisting of: as shown in SEQ ID NO: 2 CDRL2 and CDRL2 different from CDRL2 shown in SEQ ID NO: 2 by adding or deleting or replacing 1, 2 or 3 amino acids, and CDRL3 is selected from the group consisting of: SEQ ID NO : CDRL3 shown in 2 and CDRL3 different from CDRL3 shown in SEQ ID NO: 2 by adding or deleting or replacing 1, 2 or 3 amino acids; wherein the one or more are specific to human PD1 The epitope binding region includes one or more CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: CDRH1 as shown in SEQ ID NO: 3 and by addition or deletion or substitution 1, 2 or 3 amino acids and CDRH1 different from CDRH1 shown in SEQ ID NO: 3, wherein CDRH2 is selected from the group consisting of: CDRH2 shown in SEQ ID NO: 3 and by addition or deletion or substitution 1 , 2 or 3 amino acids and CDRH2 different from CDRH2 shown in SEQ ID NO: 3, and CDRH3 is selected from the group consisting of: CDRH3 shown in SEQ ID NO: 3 and CDRH3 different from CDRH3 shown in SEQ ID NO: 3 by adding or deleting or substituting 1, 2 or 3 amino acids; and wherein the linker consists of one bond Or selected from the group consisting of a peptide linker. This term defines a binding protein specific for human PD-1 and human LAG-3.

In one embodiment, a binding protein specific for human PD-1 and human LAG-3 has> 50% inhibition of LAG3-MHCII interaction in competitive flow cytometry analysis and in PD-1 / PDL 1 IC50 of less than or equal to 5 nM in the competition analysis.

In the context of the present invention, the antibody is an IgA or IgG heterotetrameric glycoprotein composed of two heavy chains and two light chains, wherein the heavy chain system includes VH and CH1 regions, and the light chain system includes VL and CL Area. More specifically, this antibody is an IgG antibody. In one embodiment, this anti-system is selected from the IgG1, IgG2, IgG3, IgG4, and IgG4PE subclasses. In a more specific embodiment, the antibody system is selected from the IgG1 subclass, and even more specifically, the antibody has an IgG1 invalid isotype (LAGA).

Alternatively, the antibody is an IgA or IgG glycoprotein composed of two heavy chains and two light chains, wherein the heavy chain system includes VH and CH1 regions, and the light chain system includes VL and CL regions. More specifically, this antibody is an IgG antibody. In one embodiment, this anti-system is selected from the IgG1, IgG2, IgG3, IgG4, and IgG4PE subclasses. In a more specific embodiment, the antibody system is selected from the IgG1 subclass, and even more specifically, the antibody has an IgG1 invalid isotype (LAGA).

In one embodiment, the heavy chain system includes CH1, CH2 and VH regions and the light chain system includes CL and VL regions. In a more specific embodiment, the heavy chain system contains CH1, CH2, CH3 and VH regions and the light chain system contains CL and VL regions.

The CH1, CH2, CH3, VH, CL and VL regions referred to above may be complete regions or modified regions truncated or containing N or C-terminal extensions, or one or more of the ring systems may not have the indicated Replacement of the sequence of regional features. Also includes other modifications, such as adding other post-translational modifications, such as phosphorylation, deamidation, oxidation, disulfide bond recombination, isomerization, C-terminal amino acid truncation, and N-terminal glutamic acid cyclization Or include one or more unnatural amino acids. For the avoidance of doubt, antibodies are glycoproteins and the above regions can be glycosylated.

In the context of the present invention, the term epitope binding region specific for human PD1 refers to a folded protein structure, which retains the tertiary structure independent of the remaining regions of the binding protein. In one embodiment, the epitope binding region specific for human PD1 has CDRs that allow the binding protein to bind to human PD-1. In a more specific embodiment, an epitope binding region specific for human PD1 has a CDR in the PD-1 / PDL-1 competition analysis to cause the binding protein to exhibit an IC50 of less than or equal to 5 nM. In order to bind to PD-1, it is clear that the CDR region of the epitope binding region must maintain the proper configuration by the protein scaffold. In one embodiment, the epitope binding region specific for human PD1 is a single variable region of an antibody (in other words, the protein scaffold is an immunoglobulin scaffold).

In one embodiment, the single variable region may be a complete antibody variable region, such as VH, VHH and VL or a modified region truncated or containing an N or C-terminal extension, or one or more loop systems Replaced by sequences that do not have the characteristics of the indicated area. In a particular embodiment, the single variable region may be a VH region or a modified VH region truncated or containing N or C-terminal extensions, or one or more of the ring systems may not have the characteristics of the indicated region Sequence substitution. Other modifications can also be included, such as adding other post-translational modifications, such as phosphorylation, deamidation, oxidation, disulfide bond recombination, isomerization, C-terminal amino acid truncation, and N-terminal glutamic acid ring Or include one or more unnatural amino acids. In an embodiment, the single variable region may be a VH region.

In other embodiments, the epitope binding region specific for human PD1 is a non-immunoglobulin scaffold with a loop connecting element of secondary structure, which can be engineered to include a CDR region. Non-immunoglobulin scaffolds include CTLA-4 (Evibodies; Journal Immunological Methods 248 (1-2): 31-45, 2001), lipocalin, protein A-derived molecules such as the Z-region of protein A (Affibodies, Protein Eng Des Sel 17 : 455-462, 2004 and EP1641818), A-region (Avimer / Maxibody), heat shock proteins, such as GroEI and GroES, transferrin (trans-body), ankyrin repeat protein (ankyrin repeat protein) ( DARPin), peptide aptamer, C-type lectin region (Tetranectin), human γ-crystallin and human ubiquitin (affilins), PDZ region, human protease inhibitor scorpion toxin kunitz-type region and fiber Protein (fibronectin) / adnectin. These scaffolds were protein engineered to arrange the CDRs in a functional manner (see Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No 9, 1126-1136 for a summary of alternative antibody models).

In one embodiment, an antibody specific for LAG-3 (which forms part of a binding protein specific for human PD-1 and human LAG-3) includes CDRH1, CDRH2, and CDRH3, wherein the CDRH1 is Selected from: CDRH1 shown in SEQ ID NO: 1, and CDRH1 different from CDRH1 shown in SEQ ID NO: 1 by adding, deleting or replacing 1, 2 or 3 amino acids; wherein CDRH2 is selected from : CDRH2 shown in SEQ ID NO: 1 and CDRH2 different from CDRH2 shown in SEQ ID NO: 1 by adding or deleting or replacing 1, 2 or 3 amino acids; and CDRH3 is selected from: CDRH3 shown in SEQ ID NO: 1 and CDRH3 different from CDRH3 shown in SEQ ID NO: 1 by adding, deleting, or replacing 1, 2, or 3 amino acids.

In another embodiment, the antibody specific for LAG-3 (which forms part of a binding protein specific for human PD-1 and human LAG-3) includes CDRL1 and CDRL2, wherein the CDRL1 is selected From: CDRL1 shown in SEQ ID NO: 2 and CDRL1 different from CDRL1 shown in SEQ ID NO: 2 by adding or deleting or substituting 1, 2 or 3 amino acids; and CDRL2 is selected from: CDRL2 shown in SEQ ID NO: 2 and CDRL2 different from CDRL2 shown in SEQ ID NO: 1 by adding or deleting or replacing 1, 2 or 3 amino acids.

In yet another embodiment, the antibody specific for LAG-3 (which forms part of a binding protein specific for human PD-1 and human LAG-3) includes CDRL1 and CDRL2, wherein the CDRL1 is Selected from: CDRL1 shown in SEQ ID NO: 2 and CDRL1 different from CDRL1 shown in SEQ ID NO: 2 by adding, deleting or substituting 1, 2 or 3 amino acids; and wherein CDRL2 is shown as SEQ ID NO: 2 shows.

In another embodiment, the antibody specific for LAG-3 (which forms part of a binding protein specific for human PD-1 and human LAG-3) includes CDRL1, CDRL2, and CDRL3, wherein the CDRL1 Is selected from: CDRL1 shown in SEQ ID NO: 2 and CDRL1 different from CDRL1 shown in SEQ ID NO: 2 by adding or deleting or replacing 1, 2 or 3 amino acids; wherein CDRL2 is selected from : CDRL2 shown in SEQ ID NO: 2 and CDRL2 different from CDRL2 shown in SEQ ID NO: 1 by adding or deleting or replacing 1, 2 or 3 amino acids; and CDRL3 is selected from: CDRL3 shown in SEQ ID NO: 2 and CDRL3 different from CDRL3 shown in SEQ ID NO: 1 by adding, deleting, or replacing 1, 2, or 3 amino acids.

In another embodiment, the antibody specific for LAG-3 (which forms part of a binding protein specific for human PD-1 and human LAG-3) includes CDRL1, CDRL2, and CDRL3, wherein the CDRL1 It is selected from: CDRL1 shown in SEQ ID NO: 2 and CDRL1 different from CDRL1 shown in SEQ ID NO: 2 by adding, deleting or replacing 1, 2 or 3 amino acids; wherein CDRL2 is like SEQ ID NO: 2; and CDRL3 is selected from: CDRL3 as shown in SEQ ID NO: 2 and shown as SEQ ID NO: 1 by adding, deleting, or replacing 1, 2 or 3 amino acids The CDRL3 is different from the CDRL3.

In yet another embodiment, one or more epitope binding regions specific for PD1 (which form part of a binding protein specific for human PD-1 and human LAG-3) include CDRH1, CDRH2 And CDRH3, wherein the CDRH1 is selected from: CDRH1 shown in SEQ ID NO: 3, and is different from CDRH1 shown in SEQ ID NO: 3 by adding, deleting, or replacing 1, 2, or 3 amino acids CDRH1; wherein CDRH2 is selected from: CDRH2 shown in SEQ ID NO: 3 and CDRH2 different from CDRH2 shown in SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids; and CDRH3 is selected from: CDRH3 as shown in SEQ ID NO: 3 and CDRH3 different from CDRH3 as shown in SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids.

In yet another embodiment, one or more epitope binding regions specific for PD1 (which form part of a binding protein specific for human PD-1 and human LAG-3) include CDRH1, CDRH2 And CDRH3, wherein the CDRH1 is selected from: CDRH1 shown in SEQ ID NO: 3, and CDRH1 different from CDRH1 shown in SEQ ID NO: 3 by adding or deleting or replacing one amino acid; wherein CDRH2 It is selected from: CDRH2 shown in SEQ ID NO: 3 and CDRH2 different from CDRH2 shown in SEQ ID NO: 3 by adding or deleting or substituting 1 or 2 amino acids; and CDRH3 is selected from: CDRH3 shown in SEQ ID NO: 3 and CDRH3 different from CDRH3 shown in SEQ ID NO: 3 by adding or deleting or substituting 1 or 2 amino acids.

In the foregoing embodiments, the complementarity determining region or CDR of an antibody specific for LAG-3 can be defined by any numbering convention, such as Kabat, Chothia, AbM, and Contact conventions. The series of definition methods of CDR regions of SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO3 are listed in Table 1.

As mentioned above, each CDR may be substituted, deleted, or added with 1, 2, or 3 amino acids to form a CDR variant. In a specific embodiment in which the binding protein specific for human PD-1 and human LAG-3 contains one or more CDR variants, the retention of the biological activity of the binding protein is defined as analysis by competitive flow cytometry It appears that> 50% inhibits LAG3-MHCII interaction and has an IC50 of less than or equal to 5 nM in the PD-1 / PDL-1 competition analysis. Those skilled in the art should understand that a single CDR variant can contain any combination of substitutions, additions, or deletions compared to unmodified CDR amino acid sequences.

Typically, the modification is substitution. In one embodiment, the CDR is modified by substituting 1, 2, or 3 amino acids. More specifically, this modification is a conservative substitution in which amino acids with side chains of similar properties are substituted. In this regard, those skilled in the art should understand that amino acids can be classified as hydrophobic, neutral hydrophilic, acidic, basic, residues that affect chain orientation, or aromaticity as shown in Table 2 below. A conservative substitution is that one amino acid residue is replaced by another residue of the same group.

In one embodiment, an antibody specific for LAG-3 (which forms part of a binding protein specific for human PD-1 and human LAG-3) includes RASQX ₁ ISSX ₂ LX ₃ (SEQ ID NO: 56) CDRL1 of the sequence, wherein X ₁ is G or S, X ₂ is W, F or Y, and X ₃ is A or N.

In another embodiment, one or more epitope binding regions specific for PD1 (which form part of a binding protein specific for human PD-1 and human LAG-3) have CDRH1, CDRH2 and CDRH3 , Where CDRH1 has the sequence of THYMX ₄ (SEQ ID NO: 57), where X ₄ is V or A, where CDRH2 has the sequence of FIGPAGDX ₅ TYYADSVX ₆ G (SEQ ID NO: 58), where X ₅ is T, F, or S X ₆ is K or E, and CDRH3 is YTX ₇ TSX ₈ X ₉ DX ₁₀ YDV (SEQ ID NO: 59) sequence, where X ₇ is A or E, X ₈ is G, S or D, X ₉ is V, F or Y, and X ₁₀ is T or S.

In one embodiment, the antibody specific for LAG-3 (which forms part of a binding protein specific for human PD-1 and human LAG-3) includes CDRH1 as shown in SEQ ID NO.1 , CDRH2 and CDRH3. CDR can be defined by any numbering convention. In a more specific embodiment, CDR is defined by Kabat numbering convention, such that CDRH1 has the sequence defined by SEQ ID NO: 4, CDRH2 has the sequence defined by SEQ ID NO: 5, and CDRH3 has SEQ ID NO: 6 The sequence defined.

In one embodiment, the antibody specific for LAG (which forms part of a binding protein specific for human PD-1 and human LAG-3) includes CDRL1 and CDRL2 as shown in SEQ ID NO. 2 . CDR can be defined by any numbering convention. In a more specific embodiment, CDR is defined by the Kabat numbering convention, such that CDRL1 has the sequence defined by SEQ ID NO: 7, CDRL2 has the sequence defined by SEQ ID NO: 8, and CDRL3 has SEQ ID NO: 9 defined sequence.

In one embodiment, the antibody specific for LAG (which forms part of a binding protein specific for human PD-1 and human LAG-3) includes CDRL1 and CDRL2 as shown in SEQ ID NO. 2 And CDRL3. CDR can be defined by any numbering convention. In a more specific embodiment, CDR is defined by the Kabat numbering convention, such that CDRL1 has the sequence defined by SEQ ID NO: 7, CDRL2 has the sequence defined by SEQ ID NO: 8, and CDRL3 has SEQ ID NO: 9 defined sequence.

In one embodiment, one or more epitope binding regions specific for PD1 (which form part of a binding protein specific for human PD-1 and human LAG-3) have the sequence as SEQ ID NO. 3 CDRH1, CDRH2 and CDRH3 shown. CDR can be defined by any numbering convention. In a more specific embodiment, the CDR is defined by the Kabat numbering convention, such that CDRH1 has the sequence of SEQ ID NO: 10, CDRH2 has the sequence of SEQ ID NO: 11, and CDRH3 has the sequence of SEQ ID NO: 12. Defined sequence.

In one embodiment, an antibody specific for human LAG-3 (which forms part of a binding protein specific for human PD-1 and human LAG-3) includes two identical heavy chains, the heavy chain It includes a sequence as defined in SEQ ID NO. 1 or a variant of SEQ ID NO. 1 with up to 10 amino acid additions, deletions or substitutions. In a more specific embodiment, the heavy chain CDR is defined as described in any of the above embodiments, and up to 10 amino acid additions, deletions, or substitutions are not within the CDR region.

In another embodiment, an antibody specific for human LAG-3 (which forms part of a binding protein specific for human PD-1 and human LAG-3) includes two identical light chains, the light The chain system includes a sequence as defined in SEQ ID NO. 2 or a variant of SEQ ID NO. 2 with a difference of up to 10 amino acid additions, deletions, or substitutions. In a more specific embodiment, the light chain CDR is defined as described in any of the above embodiments, and up to 10 amino acid additions, deletions, or substitutions are not within the CDR region.

In another embodiment, one or more epitope binding regions specific for human PD1 (which form part of a binding protein specific for human PD-1 and human LAG-3) include, for example, SEQ ID The sequence defined by NO. 3 or a variant of SEQ ID NO. 3 with up to 10 amino acid addition, deletion or substitution differences. In a more specific embodiment, the CDR is defined as described in any of the above embodiments, and up to 10 amino acid additions, deletions, or substitutions are not within the CDR region.

Those skilled in the art should understand that the variable light chain, variable heavy chain or epitope binding region sequence may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, Add or delete any combination. Typically, this modification is a substitution. In one embodiment, the sequences are substituted with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids. In a more specific embodiment, this modification is a conservative substitution (one amino acid residue is replaced by another residue of the same group in Table 2).

In one embodiment, an antibody specific for human LAG-3 (which forms part of a binding protein specific for human PD-1 and human LAG-3) includes two identical heavy chains, the heavy chain It includes a sequence as defined in SEQ ID NO.1 or a sequence having at least 90% sequence identity with the sequence of SEQ ID NO.1.

In another embodiment, an antibody specific for human LAG-3 (which forms part of a binding protein specific for human PD-1 and human LAG-3) includes two identical light chains, the light The tether system includes a sequence as defined in SEQ ID NO. 2 or a sequence having at least 90% sequence identity with the sequence of SEQ ID NO.

In another embodiment, one or more epitope binding regions specific for human PD1 (which form part of a binding protein specific for human PD-1 and human LAG-3) include, for example, SEQ ID The sequence defined by NO.3 or a sequence having at least 90% sequence identity with the sequence of SEQ ID NO.3.

The percentage of identity between a query amino acid sequence and a main amino acid sequence is the same degree value expressed as a percentage, which is after pairwise BLASTP alignment, when the main amino acid sequence has 100% coverage of the query amine The base acid sequence is calculated by the BLASTP algorithm. The pairwise BLASTP alignment between the main amino acid sequence and the query amino acid sequence is achieved by using the default settings of the BLASTP algorithm available on the website of the National Center for Biotechnology Institute. Turn off the low complexity area.

The percent identity can determine the entire full-length query sequence (including CDR). Alternatively, the percentage of identity can exclude CDRs. For example, the CDR is 100% identical to the host sequence and the variation of the percentage of identity is in the rest of the query gene, so that the CDR sequence is fixed / complete.

In a particular embodiment, an antibody specific for human LAG-3 (which forms part of a binding protein specific for human PD-1 and human LAG-3) and one or more specific antibodies for human PD1 The heavy and light chains of the specific epitope binding region (which forms part of the binding protein specific for human PD-1 and human LAG-3) have a CDR sequence as defined in any of the above examples and The framework of SEQ ID NO.1 (for the heavy chain), SEQ ID NO.2 (for the light chain) and SEQ ID NO.3 (for the epitope binding region) is at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% the same frame.

In one embodiment, an antibody specific for human LAG-3 (which forms part of a binding protein specific for human PD-1 and human LAG-3) includes two identical heavy chains, the heavy chain It includes the sequence as defined in SEQ ID NO.1.

In another embodiment, an antibody specific for human LAG-3 (which forms part of a binding protein specific for human PD-1 and human LAG-3) includes two identical light chains, the light The tether system includes the sequence as defined in SEQ ID NO.2.

In yet another embodiment, one or more epitope binding regions specific for human PD1 (which form part of a binding protein specific for human PD-1 and human LAG-3) include, for example, SEQ The sequence defined by ID NO.3.

In one embodiment, one or more epitope binding regions specific for human PD1 (which form part of a binding protein specific for human PD-1 and human LAG-3) are compared to equivalent Unmodified molecules have reduced binding capacity to earlier existing antibody (ADA) lines. With regard to reduced binding capacity, it is meant to modify the molecular system to bind to the pre-existing ADA with reduced affinity or reduced affinity. The one or more epitope binding regions specific for human PD1 include one or more modifications selected from: (a) C-terminal addition, extension, deletion or labeling, and / or (b) one or Multiple amino acid framework substitutions.

In one embodiment, one or more modified epitope binding regions specific for human PD1 include: a) a C-terminal sequence composed of the VTVS (S) nX ₁₁ sequence (this general sequence example is based on SEQ ID NO: 63-SEQ ID NO: 78 represents); and if necessary b) compared to the human germ cell framework sequence, one or more amino acids at position 14, 41, 108, 110 or 112 are substituted for: n represents an integer independently selected from 0 or 1; X ₁₁ may be present or absent, and if present represents an amino acid extension of 1 to 8 amino acid residues. In one embodiment, X does not exist. In another embodiment, X is present and is an extension of 1 to 8 amino acids, in particular, 1 to 8 amino acid extensions, which include a single alanine residue, such as a single alanine Extension, or AS, AST, ASTK, ASTKG, ASTKGP extension. In another embodiment, X is present and is 1 to 8 amino acid extensions including A, AAA, or T extensions.

In one embodiment, one or more modified epitope binding regions specific for human PD1 include one or more amino acid substitutions, wherein the one or more amino acid substitutions are in the group consisting of Selected: P14A substitution, P41A substitution, L108A substitution, T110A substitution, S112A substitution, P14K substitution, P14Q substitution and P14T substitution.

In one embodiment, one or more epitope binding regions are connected to the C-terminus of the heavy chain of the antibody specific for human LAG-3 via a linker. In a more specific embodiment, there are two epitope binding regions, one connected to the C-terminus of two heavy chains of an antibody specific for LAG-3.

The linker may be a one-button or a peptide linker. In one embodiment, the linker is a peptide linker with a length of 1 to 100 amino acids, more specifically, a length of 1 to 50 or 1 to 10 amino acids. Suitable linkers are described in the published PCT patent application WO2010 / 136483. In a more specific embodiment, the linker has the sequence listed in SEQ ID NO. 30 (STGLDSPT).

In another aspect, the present invention provides a binding protein having the general formula (I): Wherein: H (LAG-3) is an IgG antibody heavy chain including CDRH1, CDRH2 and CDRH3, wherein the CDRH1 is selected from CDRH1 shown in SEQ ID NO: 1, and is added or deleted or substituted 1, 2 Or 3 amino acids and CDRH1 different from CDRH1 shown in SEQ ID NO: 1; wherein CDRH2 is selected from: CDRH2 shown in SEQ ID NO: 1 and 1, 2 or 3 are added or deleted or substituted Amino acid and CDRH2 different from CDRH2 shown in SEQ ID NO: 1; and CDRH3 is selected from: CDRH3 shown in SEQ ID NO: 1 and 1, 2 or 3 amines are added or deleted or substituted CDRH3 which is different from the CDRH3 shown in SEQ ID NO: 1 based on acid; L (LAG-3) is an IgG antibody light chain including CDRL1 and CDRL2, wherein the CDRL1 is selected from: SEQ ID NO: 2 CDRL1 shown, and CDRL1 different from CDRL1 shown in SEQ ID NO: 2 by adding or deleting or replacing 1, 2 or 3 amino acids; and CDRL2 is selected from: SEQ ID NO: 2 CDRL2 and CDRL2 different from CDRL2 shown in SEQ ID NO: 2 by adding or deleting or substituting 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 V _H (PD-1 ) Is an antibody heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1 is selected from: CDRH1 shown in SEQ ID NO: 3, and by adding or deleting or replacing 1, 2 or 3 amino acids And CDRH1 different from CDRH1 shown in SEQ ID NO: 3; wherein CDRH2 is selected from: CDRH2 shown in SEQ ID NO: 3 and by adding or deleting or replacing 1, 2 or 3 amino acids IDRH: 3 is different from CDRH2; and CDRH3 is selected from: CDRH3 as shown in SEQ ID NO: 3 and by adding or deleting or substituting 1, 2 or 3 amino acids to match with SEQ ID CDRH3 different from CDRH3 shown in NO: 3.

The binding protein of formula (I) is a binding protein specific for human PD1 and human LAG-3.

In one embodiment, the binding protein of formula (I) has> 50% inhibition of LAG3-MHCII interaction in competitive flow cytometry analysis and less than or equal to 5 nM in PD-1 / PDL-1 competitive analysis Of IC50.

In one embodiment, the H series includes V _H and CH1 regions, and the L series includes CL and V _L regions. In a more specific embodiment, the H series includes CH1, CH2, and VH regions and the L series includes CL and VL regions. Even more specifically, the H line contains CH1, CH2, CH3 and V _H regions and the L line contains CL and V _L regions.

Referred to in the above _{CH1, CH2, CH3, V H,} CL and V _L, may be the entire area or region containing a modified or truncated region extends the N-or C- terminal, or one or more ring system by having no Replaced by the sequence of features of the indicated area. Also includes other modifications, such as adding other post-translational modifications, such as phosphorylation, deamidation, oxidation, disulfide bond recombination, isomerization, C-terminal amino acid truncation, and N-terminal glutamic acid cyclization Or include one or more unnatural amino acids. For the avoidance of doubt, antibodies are glycoproteins and the above regions can be glycosylated.

In one embodiment, H is a heavy chain of IgG1, IgG2, IgG3, IgG4 or IgG4 PE subclass. When H is the heavy chain of IgG1 and IgG4 subclasses, n is 2. When H is the heavy chain of IgG2 subclass, n is 4. When H is the heavy chain of the IgG3 subclass, n is 11. In a more specific embodiment, the antibody system is selected from the IgG1 subclass, and even more specifically, the antibody has an IgG1 invalid isotype (LAGA).

V _H (PD-1) may be a complete antibody variable region or a modified region truncated or containing an N or C-terminal extension, or one or more loop systems are replaced by a sequence that does not have the characteristics of the indicated region. Also includes other modifications, such as adding other post-translational modifications, such as phosphorylation, deamidation, oxidation, disulfide bond recombination, isomerization, C-terminal amino acid truncation, and N-terminal glutamic acid cyclization Or include one or more unnatural amino acids.

The CDRs of H, L, and V _H (PD-1) can be defined by any coding convention, such as Kabat, Chothia, AbM, and contact conventions. The series of CDR regions of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO 3 defined by each method are listed in Table 1 above.

As mentioned above, each CDR may be substituted, deleted, or added with 1, 2, or 3 amino acids to form a CDR variant. In specific embodiments, the binding protein of formula (I) containing one or more CDR variants retains biological activity, which is defined as exhibiting> 50% inhibition of LAG3-MHCII interaction in competitive flow cytometry analysis, and In the PD-1 / PDL-1 competition analysis, the IC50 is less than or equal to 5nM. Those skilled in the art should understand that a single CDR variant can contain any combination of substitutions, additions, or deletions compared to unmodified CDR amino acid sequences.

Typically, the modification is substitution. In one embodiment, the CDR is modified by substituting 1, 2, or 3 amino acids. More specifically, this modification is a conservative substitution (monoamino acid residues are substituted with additional residues of the same groups in Table 2).

In an embodiment of the binding protein of formula (I), H (LAG-3) includes CDRH1, CDRH2, and CDRH3, wherein the CDRH1 is selected from: CDRH1 shown in SEQ ID NO: 1, and by addition or deletion Or replace 1, 2, or 3 amino acids with CDRH1 different from CDRH1 shown in SEQ ID NO: 1; wherein CDRH2 is selected from: CDRH2 shown in SEQ ID NO: 1 and by addition or deletion or substitution 1 , 2 or 3 amino acids and CDRH2 different from CDRH2 shown in SEQ ID NO: 1; and CDRH3 is selected from: CDRH3 shown in SEQ ID NO: 1 and by adding or deleting or replacing 1, 2 or 3 amino acids which are different from CDRH3 shown in SEQ ID NO: 1.

In an embodiment of the binding protein of formula (I), L (LAG-3) includes CDRL1 and CDRL2, wherein the CDRL1 is selected from: CDRL1 shown in SEQ ID NO: 2, and by addition or deletion or substitution 1, 2 or 3 amino acids and CDRL1 different from CDRL1 shown in SEQ ID NO: 2; and CDRL2 is selected from: CDRL2 shown in SEQ ID NO: 2 and by adding or deleting or replacing 1, 2 or 3 amino acids which are different from CDRL2 shown in SEQ ID NO: 2.

In another embodiment of the binding protein of formula (I), L (LAG-3) includes CDRL1 and CDRL2, wherein the CDRL1 is selected from: CDRL1 shown in SEQ ID NO: 2, and by addition or deletion Or replace 1, 2, or 3 amino acids and CDRL1 different from CDRL1 shown in SEQ ID NO: 2; and among them CDRL2 is shown in SEQ ID NO: 2.

In another embodiment of the binding protein of formula (I), L (LAG-3) includes CDRL1, CDRL2 and CDRL3, wherein the CDRL1 is selected from: CDRL1 shown in SEQ ID NO: 2, and by adding or Delete or replace 1, 2, or 3 amino acids from CDRL1 different from CDRL1 shown in SEQ ID NO: 2; wherein CDRL2 is selected from: CDRL2 shown in SEQ ID NO: 2 and by addition or deletion or substitution 1, 2 or 3 amino acids and CDRL2 different from CDRL2 shown in SEQ ID NO: 2; and CDRL3 is selected from: CDRL3 shown in SEQ ID NO: 2 and by adding or deleting or replacing 1, 2 or 3 amino acids which are different from CDRL3 shown in SEQ ID NO: 2.

In another embodiment of the binding protein of formula (I), L (LAG-3) includes CDRL1, CDRL2 and CDRL3, wherein the CDRL1 is selected from: CDRL1 shown in SEQ ID NO: 2, and by adding or Delete or replace 1, 2, or 3 amino acids and CDRL1 different from CDRL1 shown in SEQ ID NO: 2; wherein CDRL2 is shown in SEQ ID NO: 2; and in which CDRL3 is selected from: as SEQ ID NO : CDRL3 shown in 2: and CDRL3 different from the CDRL3 shown in SEQ ID NO: 2 by adding or deleting or substituting 1, 2 or 3 amino acids.

In an embodiment of the binding protein of formula (I), L (LAG-3) includes CDRL1 having the sequence RASQX ₁ ISSX ₂ LX ₃ (SEQ ID NO: 56), where X ₁ is G or S, and X ₂ is W, F or Y, and X ₃ is A or N.

In one embodiment, V _H (PD-1) includes CDRH1, CDRH2 and CDRH3, wherein the CDRH1 is selected from: CDRH1 shown in SEQ ID NO: 3, and by adding or deleting or replacing 1, 2 or 3 amino acids and CDRH1 different from CDRH1 shown in SEQ ID NO: 3; wherein CDRH2 is selected from: CDRH2 shown in SEQ ID NO: 3 and 1, 2 or 3 amines are added or deleted or substituted The basic acid is different from CDRH2 shown in SEQ ID NO: 3; and CDRH3 is selected from: CDRH3 shown in SEQ ID NO: 3 and by adding or deleting or replacing 1, 2 or 3 amine groups Acid is different from CDRH3 shown in SEQ ID NO: 3.

In yet another embodiment, V _H (PD-1) includes CDRH1, CDRH2 and CDRH3, wherein the CDRH1 is selected from: CDRH1 shown in SEQ ID NO: 3, and by adding or deleting or replacing one Amino acid and CDRH1 different from CDRH1 shown in SEQ ID NO: 3; wherein CDRH2 is selected from: CDRH2 shown in SEQ ID NO: 3 and by adding or deleting or replacing 1 or 2 amino acids and The CDRH2 different from the CDRH2 shown in SEQ ID NO: 3; and the CDRH3 in it is selected from: CDRH3 shown in SEQ ID NO: 3 and is the same as SEQ ID NO by adding or deleting or replacing 1 or 2 amino acids : CDRH3 different from CDRH3 shown in 3.

In another embodiment, the V _H (PD-1) line includes CDRH1, CDRH2, and CDRH3, wherein CDRH1 has the sequence of THYMX ₄ (SEQ ID NO: 57), where X ₄ is V or A, and CDRH2 has FIGPAGDX ₅ TYYADSVX ₆ G (SEQ ID NO: 58) sequence, wherein X ₅ is T, F or S and X ₆ is K or E, and CDRH3 is YTX ₇ TSX ₈ X ₉ DX ₁₀ YDV (SEQ ID NO: 59) sequence, Where X ₇ is A or E, X ₈ is G, S or D, X ₉ is V, F or Y, and X ₁₀ is T or S.

In one embodiment, H (LAG-3) includes CDRH1, CDRH2 and CDRH3 shown in SEQ ID NO.1. CDR can be defined by any numbering convention. In a more specific embodiment, the CDR is defined by the Kabat numbering convention such that CDRH1 has the sequence defined by SEQ ID NO: 4, CDRH2 has the sequence defined by SEQ ID NO: 5, and CDRH3 has the following The sequence defined by SEQ ID NO: 6.

In another embodiment, L (LAG-3) includes CDRL1 and CDRL2 as shown in SEQ ID NO.2. CDR can be defined by any numbering convention. In a more specific embodiment, the CDR is defined by the Kabat numbering convention such that CDRL1 has the sequence defined by SEQ ID NO: 7, and CDRL2 has the sequence defined by SEQ ID NO: 8.

In another embodiment, L (LAG-3) includes CDRL1, CDRL2, and CDRL3 as shown in SEQ ID NO.2. CDR can be defined by any numbering convention. In a more specific embodiment, the CDR is defined by the Kabat numbering convention such that CDRL1 has the sequence defined by SEQ ID NO: 7, CDRL2 has the sequence defined by SEQ ID NO: 8, and CDRL3 has the following The sequence defined by SEQ ID NO: 9.

In one embodiment, V _H (PD-1) has CDRH1, CDRH2, and CDRH3 as shown in SEQ ID NO. CDR can be defined by any numbering convention. In a more specific embodiment, CDR is defined by Kabat numbering convention such that CDRH1 has the sequence defined by SEQ ID NO: 10, CDRH2 has the sequence defined by SEQ ID NO: 11, and CDRH3 has the following The sequence defined by SEQ ID NO: 12.

In one embodiment, _VH has a sequence as defined in SEQ ID NO. 1 or a variant of SEQ ID NO. 1 with up to 10 amino acid addition, deletion, or substitution differences. In a more specific embodiment, the CDR is defined as described in any of the above embodiments, and up to 10 amino acid additions, deletions, or substitutions are not within the CDR region.

In further embodiments, V _L having a sequence of SEQ ID NO.2 defined as having a high or 10 amino acids added, deleted or substituted differences of SEQ ID NO.2 variants. In a more specific embodiment, the CDR is defined as described in any of the above embodiments, and up to 10 amino acid additions, deletions, or substitutions are not within the CDR region.

In further embodiments, V _H (PD-1) has a sequence as defined in SEQ ID NO. 3 or a variant of SEQ ID NO. 3 with up to 10 amino acid additions, deletions, or substitutions. In a more specific embodiment, the CDR is defined as described in any of the above embodiments, and up to 10 amino acid additions, deletions, or substitutions are not within the CDR region.

Those skilled in the art should understand that V _H , V _L and V _H (PD-1) may contain 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions and additions Or delete any combination. Typically, this modification is a substitution. In one embodiment, the sequences are modified with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In a more specific embodiment, this modification is a conservative substitution (one amino acid residue is replaced by another residue of the same group in Table 2).

In one embodiment, _VH has a sequence as defined in SEQ ID NO. 1 or a sequence having at least 90% sequence identity with the SEQ ID NO. 1 sequence.

In further embodiments, V _L having a sequence as defined in of SEQ ID NO.2 and SEQ ID NO.2 or a sequence having at least 90% identical degree of sequence.

In another embodiment, V _H (PD-1) has a sequence as defined in SEQ ID NO. 3 or a sequence having at least 90% sequence identity with the SEQ ID NO. 3 sequence.

The percentage of identity between a query amino acid sequence and a main amino acid sequence is the same degree value expressed as a percentage, which is after pairwise BLASTP alignment, when the main amino acid sequence has 100% coverage of the query amine The base acid sequence is calculated by the BLASTP algorithm. The pairwise BLASTP alignment between the main amino acid sequence and the query amino acid sequence is achieved by using the default settings of the BLASTP algorithm available on the website of the National Center for Biotechnology Institute. Turn off the low complexity area.

The percent identity can determine the entire full-length query sequence (including CDR). Alternatively, the percentage of identity can exclude CDRs. For example, the CDR is 100% identical to the host sequence and the variation of the percentage of identity is in the rest of the query gene, so that the CDR sequence is fixed / complete.

In a specific embodiment, V _H , V _L and V _H (PD-1) have a CDR sequence as defined in any of the above embodiments and are in accordance with SEQ ID NO. 1 (as far as V _H ), SEQ ID NO. 2 (In terms of V _L ) and SEQ ID NO. 3 (in terms of V _H (PD-1)) are at least 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% identical.

In one embodiment, V _H has the sequence defined by SEQ ID NO.1.

In further embodiments, V _L having a sequence of SEQ ID NO.2 as defined.

In yet another embodiment, V _H (PD-1) has a sequence as defined in SEQ ID NO.3.

In one embodiment, A is a peptide linker of 1 to 100 amino acids in length, more specifically, 1 to 50 or 1 to 10 amino acids in length. Suitable linkers are described in the published PCT patent application WO2010 / 136483. In a more specific embodiment, the linker has the sequence listed in SEQ ID NO. 30 (STGLDSPT).

The 57E02x51A09-188001 mAbdAb described in the examples is a binding protein of formula (I) specific for human PD-1 and human LAG-3. It suppresses negative signals from PD-1 and LAG-3 receptors by preventing interaction with their individual ligands. Because of this, it enhances T cell activation verified by NFAT signaling in reporter analysis and enhances cytokine production in mixed lymphocyte response analysis. 57E02x51A09-188001 mAbdAbs have been shown to promote T cell activation and HIV RNA production in CD4 T cells from HIV donors, and the functional capabilities of HIV donor CD8 T cells, which supports the use in the treatment of HIV. Xianxin's effect of T cell activation and proliferation is caused by blocking PD-1 and LAG-3 signal transmission. Therefore, it is reasonable to believe that this mAbdAb has the potential to treat other medical indications (disclosed in the section on medical uses) while having the principle of PD-1 / LAG-3 blocking for treatment.

Please also note that the example discloses 16 PD-1 dAb (mAbdAb mode) with CDR sequences related to PD-1 dAb 51A09-188001 shown in the exemplary mAbdAb. As measured by reporter gene analysis, these variant dAbs also block PD-1 signaling. It is reasonable to expect that when the LAG-3 mAb is formatted in mAbdAb mode, these and other variants with associated CDRs should be able to block PD-1 signal transmission, and therefore have the uses disclosed in the medical uses section. Likewise, the example reveals 4 LAG-3 mAb with CDR sequences related to LAG-3 mAb 57E02 shown in the exemplified mAbdAb. As measured by reporter gene analysis, these variant mAbs also blocked LAG-3 signaling. It is reasonable to expect that when the PD-1 dAb is formatted in mAbdAb mode, these and other variants with related CDRs should be able to block LAG-3 signal transmission, and therefore have the uses disclosed in the medical uses section.

The example specificity 57E02x51A09-188001 mAbdAb advantageously shows better T cell enhancement than the corresponding combination of LAG-3 mAb (57E02) and PD-1 dAb (mAbdAb mode with anti-RSV mAb; RSV-51A09-188001) activation. Manufacture other PD-1 / LAG-3 mAbdAbs, some of which share the same PD-1 dAb epitope binding region. However, among these tested antibodies, 57E02x51A09-188001 mAbdAb (which was shown to promote T cell activation more than the combination of component binding proteins) showed the best pharmacokinetics (lowest clearance) before the occurrence of anti-drug antibodies in cynomolgus monkeys Rate), followed by rapid clearance (when the antibody administered to the cynomolgus monkey is a human antibody, which may be considered as foreign by the cynomolgus monkey, it is expected to be an anti-drug antibody).

Based on all the above information and reasonable expectations, the advantageous properties observed for 57E02x51A09-188001 mAbdAb should also be observed in the relevant binding proteins with variant CDRs and framework regions as disclosed in the above examples.

In a second aspect, the present invention provides a binding protein specific for human PD1, which includes one or more CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: SEQ ID NO: 3 CDRH1, and CDRH1 different from CDRH1 shown in SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids; wherein CDRH2 is selected from the group consisting of: SEQ ID NO : CDRH2 shown in 3 and CDRH2 different from CDRH2 shown in SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids; and CDRH3 is selected from the group consisting of: CDRH3 shown in SEQ ID NO: 3 and CDRH3 different from the CDRH3 shown in SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids. In one embodiment, a binding protein specific for human PD1 has an IC50 of less than or equal to 5 nM in the PD-1 / PDL-1 competition analysis.

In order to bind to PD-1, it is obvious that the CDR region of the binding protein specific for human PD1 must be maintained in proper configuration by a protein scaffold. In one embodiment, the binding protein specific for human PD1 is a single variable region of an antibody, which contains its tertiary structure in the absence of other antibody regions (in other words, the protein scaffold is an immunoglobulin scaffold). The single variable region may be a complete antibody variable region, such as VH, VHH, and VL or a modified region truncated or containing an N or C-terminal extension, or one or more loop systems may not have the characteristics of the indicated region Of the sequence. In an embodiment, the single variable region may be a VH region or a modified VH region truncated or containing an N or C-terminal extension, or one or more loops may have a sequence that does not have the characteristics of the indicated region Replace. Also includes other modifications, such as adding other post-translational modifications, such as phosphorylation, deamidation, oxidation, disulfide bond recombination, isomerization, C-terminal amino acid truncation, and N-terminal glutamic acid cyclization Or include one or more unnatural amino acids.

In other embodiments, the binding protein specific for human PD1 has a non-immunoglobulin scaffold, which is a loop connecting element with a secondary structure that can be engineered to include the CDR region. Non-immunoglobulin scaffold systems include CTLA-4 (Evibodies; Journal Immunological Methods 248 (1-2): 31-45, 2001), lipocalin, protein A derived molecules such as the Z-region of protein A (Affibodies , Protein Eng Des Sel 17: 455-462, 2004 and EP1641818), A-region (Avimer / Maxibody), heat shock proteins, such as GroEI and GroES, trans-body, ankyrin repeat sequence (ankyrin repeat protein) (DARPin), peptide aptamer, C-type lectin (Tetranectin), human γ-crystallin and human ubiquitin (PDilin), PDZ, human protease inhibitor scorpion toxin kunitz-type And fibronectin (fibronectin) / adnectin. These scaffolds were engineered to arrange the CDRs in a functional manner (see Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No 9, 1126-1136 for a summary of alternative antibody models).

In a specific embodiment, the binding protein specific for human PD-1 has the CDR sequences listed above in any specific embodiment of the epitope binding region specific for PD-1. In the embodiment in which the binding protein specific for human PD-1 contains one or more CDR variants, those skilled in the art should understand that in a specific embodiment, the pair containing one or more CDR variants The specific binding protein of human PD-1 retains biological activity and is defined as an IC50 of less than or equal to 5 nM in the PD-1 / PDL-1 competition analysis.

In a specific embodiment, as described above for the corresponding embodiment of an epitope binding region specific for human PD-1, the binding protein specific for human PD-1 includes as defined in SEQ ID: NO.3 Or a variant of SEQ ID NO. 3 with a difference of up to 10 amino acid additions, deletions or substitutions, or a sequence having at least 90% sequence identity with the SEQ ID NO. 3 sequence.

In one embodiment, a binding protein specific for human PD-1 can additionally neutralize human checkpoint inhibitors other than PD-1, making this binding protein specific for human PD-1 and other checkpoint inhibitors Sex. As mentioned above, neutralizing a human checkpoint inhibitor refers to blocking the biological activity of the human checkpoint inhibitor. Because the biological activity of human checkpoint inhibitors inhibits the signaling cascade that regulates the activation, proliferation or cytokine production of immune cells after stimulation, neutralizing human checkpoint inhibitors increases or restores the biological activity of immune cells after stimulation .

The production of cytokines by immune cells after stimulation is complex, where depending on the type of immune cells and the nature of the stimulation, the performance of specific cytokines is increased while others are decreased. In specific cell types, the effects of specific stimuli on cytokine production are fully documented and known in the art (for example, IL2, IFNγ, and TNFα are known to be infected by CD4 + or CD8 + T cells from bacterial toxins, such as Staphylococcus Enterotoxin SEB / SEA produced after stimulation). Neutralization checkpoint inhibitors should be able to normally downregulate these cytokines after stimulation, and reduce the performance of these cytokines after stimulation. The amount of cytokine production can be measured by ELISA, ELISPOT or by intracellular staining of cells and obtained on a flow cytometer. These are standard techniques and some suitable methods are outlined in Chapter 24, HIV Protocols, Second Ed., Vol. 485, 2009. ELISA can be used to measure the amount of cytokines released by immune cells. It is usually performed on blood samples or supernatants of cultured cells. ELISPOT analysis can be performed on monocytes or CD4 + or CD8 + T cell populations, and when mixed cell populations (such as peripheral blood mononuclear cells) are used, cytokine flow cytometry can of course provide individual CD4 + and CD8 + cells Information.

In terms of cytokine production, normal production increases after stimulation, and cytokine production increase refers to a situation in which the average amount of cytokine determined by at least three samples in the presence of binding protein after stimulation is higher than when no binding protein is present. In one embodiment, increased cytokine production refers to cells in which the average amount of cytokines measured in the presence of binding protein after stimulation is greater than or equal to cells measured in the absence of binding protein in at least three samples The average amount of hormone plus one standard deviation (the larger standard deviation used with or without binding protein). In a more specific embodiment, increased cytokine production refers to the average amount of cytokine measured by at least three samples in the presence of binding protein after stimulation is greater than or equal to at least three samples without binding protein The measured average amount of cytokines plus two standard deviations (larger standard deviations used with or without binding protein). Even more specifically, increased cytokine production refers to cells in which the average amount of cytokines measured in the presence of binding protein after stimulation is greater than or equal to the cells measured in the absence of binding protein in at least three samples The average amount of hormone plus three standard deviations (the larger standard deviation used with or without binding protein). In one embodiment, reduced cytokine production refers to cells in which the average amount of cytokines measured in the presence of binding protein after stimulation is higher than the cells measured in the absence of binding protein from at least three samples The average amount of hormone is 3, 20 or 50 times.

With regard to the production of cytokines, the cytokine production normally decreases after stimulation. The decrease in cytokine production refers to a situation in which the average amount of cytokines measured by at least three samples in the presence of binding protein after stimulation is lower than when no binding protein is present. In one embodiment, the decrease in cytokine production refers to an average amount of cytokine measured by at least three samples in the presence of binding protein after stimulation is lower than or equal to that determined by at least three samples without binding protein The average amount of cytokines minus one standard deviation (the larger standard deviation used with or without binding protein). In a more specific embodiment, the decrease in cytokine production refers to the case where the average amount of cytokine determined by at least three samples in the presence of binding protein after stimulation is lower than or equal to at least three samples when there is no binding protein The measured average amount of cytokines minus two standard deviations (using the larger standard deviation used with or without binding protein). Even more specifically, the decrease in cytokine production refers to the average amount of cytokines measured by at least three samples in the presence of binding protein after stimulation is lower than or equal to that measured by at least three samples without binding protein The average amount of cytokines minus three standard deviations (larger standard deviations used with or without binding protein). In one embodiment, the decreased cytokine production refers to the average amount of cytokines measured by at least three samples in the presence of binding protein after stimulation is lower than the cytokines measured by at least three samples without binding protein The average volume is 10%, 20% or 50%.

Normal proliferation of immune cells is suppressed by human checkpoint inhibitors. Therefore, blocking human checkpoint inhibitors should increase immune cell proliferation. Basically, as described in Chapter 19, Methods in Cell Biology, Vol. 75, 2004, the proliferation after in vitro stimulation can be achieved by using the technique of carboxyfluorescein diacetate succinimide (CSFE). The cytometer monitors the cell penetration staining of cell division. In one embodiment, the increase in hyperplasia refers to the average percentage of divided immune cells measured from at least three samples (calculated by determining the percentage of diluted CSFE after subtracting the background division, which is divided in the negative control The percentage of cells in the presence of binding protein is higher than in the absence of binding protein. In one embodiment, the increase in proliferation refers to the average percentage of divided cells measured by at least three samples in the presence of binding protein, which is equal to or higher than that determined by at least three samples in the absence of binding protein The average percentage of divided cells plus one standard deviation (the larger standard deviation used with or without binding protein). In a more specific embodiment, the increase in hyperplasia refers to the average percentage of divided cells determined by at least three samples in the presence of binding protein, which is equal to or higher than that of at least three samples without binding protein The measured average percentage of divided cells plus two standard deviations (using the larger standard deviation used with or without binding protein). Even more specifically, the increase in hyperplasia refers to the average percentage of divided cells determined by at least three samples in the presence of binding protein, which is equal to or higher than that determined by at least three samples in the absence of binding protein The average percentage of divided cells plus three standard deviations (the larger standard deviation used with or without binding protein). In one embodiment, the increase in hyperplasia refers to where the average percentage of divided cells determined by at least three samples in the presence of binding protein is greater than that determined by at least three samples without binding protein at the beginning of the treatment period The average percentage of divided cells is 1.2, 1.5, 5 or 20 times.

The normal activation of immune cells is inhibited by human checkpoint inhibitors. Therefore, blocking human checkpoint inhibitors should increase activation. Immune cell activation routinely is determined by measuring changes in transcription factors or upregulation of cell surface proteins. It should be understood that the properties of transcription factors and cell surface proteins after activation differ according to the nature of immune cells. However, changes in the expression of transcription factors and cell surface proteins after activation in specific cell types have been fully documented and known in the art.

Changes in transcription factors can be monitored in vitro (activation after known stimulation) or in vivo. Changes in transcription factors such as AKT, NFAT, or NFkB can be monitored using standard protein techniques, such as Western blots or using reporter gene systems. Luciferase reporter subsystems are commonly used, where T cell lines, B cell lines, or monocyte cell lines are engineered with luciferase expression under the control of relevant transcription factor promoters. In the examples, the binding protein production was determined in PD-1 expressing Jurkat T cells to dose-dependently increase NFAT-driven luciferase expressing ability.

A transcription factor whose production, activation or phosphorylation status normally increases after stimulation. Increased activation refers to the transcription factor determined from at least three samples, the activity or the average amount of downstream reporter gene products in the presence of the binding protein It is higher than when there is no binding protein. In one embodiment, increased activation refers to a transcription factor determined from at least three samples, the activity or the average amount of downstream reporter gene products in the presence of binding protein is greater than or equal to that from at least three samples without binding When the corresponding value of the protein is measured plus one standard deviation (use the larger standard deviation used with or without binding protein). In a more specific embodiment, increased activation refers to a transcription factor determined from at least three samples whose activity or the average amount of downstream reporter gene products is greater than or equal to at least three samples in the presence of the binding protein The case where the corresponding value measured when there is no binding protein plus two standard deviations (using the larger standard deviation used with or without binding protein). Even more specifically, increased activation refers to a transcription factor determined from at least three samples whose activity or the average amount of downstream reporter gene products in the presence of binding protein is greater than or equal to that from at least three samples without binding When the corresponding value of the protein is measured plus three standard deviations (the larger standard deviation used with or without binding protein) is used. In one embodiment, increased activation refers to the transcription factor determined from at least three samples, the activity or the average amount of downstream reporter gene products in the presence of binding protein is higher than that from at least three samples without binding protein When the corresponding value measured is 1.5, 15 or 50 times.

A transcription factor that normally declines after stimulation in terms of its production, activation, or phosphorylation status. Decrease in activation refers to the transcription factor measured from at least three samples, the activity or the average amount of downstream reporter gene products in the presence of the binding protein It is lower than when there is no binding protein. In one embodiment, the decrease in activation refers to a transcription factor determined from at least three samples whose activity or the average amount of downstream reporter gene products in the presence of the binding protein is less than or equal to that from at least three samples to no The case where the corresponding value measured when binding the protein minus one standard deviation (use the larger standard deviation used with or without binding protein). In a more specific embodiment, the decrease in activation refers to a transcription factor determined from at least three samples whose activity or the average amount of downstream reporter gene products in the presence of the binding protein is less than or equal to at least three The corresponding value of the sample measured without binding protein minus two standard deviations (using the larger standard deviation used with or without binding protein). Even more specifically, the decrease in activation refers to the transcription factor determined from at least three samples whose activity or the average amount of downstream reporter gene products in the presence of the binding protein is lower than or equal to that from at least three samples to no The case where the corresponding value measured when binding the protein minus three standard deviations (using the larger standard deviation used with or without the binding protein). In one embodiment, the decrease in activation refers to the transcription factor determined from at least three samples, the activity or the average amount of downstream reporter gene products in the presence of binding protein is lower than that from at least three samples without binding protein When the corresponding value measured is 10%, 20% or 50%.

In the embodiment where the activation is measured by measuring changes in the production, activation or phosphorylation status of a specific transcription factor, it is obvious how to evaluate whether the activation is normally manifested in activated or non-activated immunity according to the transcription factor referred to Depending on the cell.

Several fully characterized cell surface proteins are up-regulated in activated immune cells; these include CD69 on T cells, B cells and NK cells, CD80 and CD86 on antigen presenting cells, and HLADR and CD38 on T cells . The performance of these cell surface proteins (or activation markers) on monocytes can be monitored using flow cytometry.

In one embodiment, the increase in immune cell activation refers to a situation in which the average frequency of immune cells exhibiting specific activation markers measured by at least three samples or the expression amount of the marker increases in the presence of the binding protein than in the absence of the binding protein .

In one embodiment, increased activation refers to the average frequency in which immune cells measured from at least three samples in the presence of a binding protein exhibit a specific activation marker, or where the amount of expression of the marker is compared to when no binding protein is present Corresponding values measured in at least three samples plus a standard deviation (the larger standard deviation used with or without binding protein) is an increase. In one embodiment, increased activation refers to the average frequency in which immune cells measured from at least three samples in the presence of the binding protein exhibit specific activation markers, or in which at least three samples are measured in the presence of the binding protein The performance of the marker is higher than or equal to the corresponding value measured by at least three samples when there is no binding protein plus a standard deviation (using the larger standard deviation used with or without binding protein) . In a more specific embodiment, increased activation or differentiation refers to the average frequency in which immune cells measured from at least three samples in the presence of the binding protein exhibit specific activation markers, or in the presence of at least three in the presence of the binding protein The performance of the marker measured by each sample is higher than or equal to the corresponding value measured by at least three samples when there is no binding protein plus two standard deviations (using the comparison method used with or without binding protein Large standard deviation). Even more specifically, increased activation refers to the average frequency in which immune cells measured from at least three samples in the presence of the binding protein exhibit specific activation markers, or in which at least three samples are measured in the presence of the binding protein The performance of the marker is higher than or equal to the corresponding value measured by at least three samples when there is no binding protein plus three standard deviations (using the larger standard deviation used with or without binding protein) Happening. In one embodiment, increased activation refers to the average frequency in which immune cells measured from at least three samples in the presence of the binding protein exhibit specific activation markers, or in which at least three samples are measured in the presence of the binding protein The performance of the marker is increased by 1.5 times, 5 times, 10 times, 20 times or 50 times compared to the corresponding values measured by at least three samples without binding protein.

In one embodiment, human checkpoint inhibitors other than PD-1 are selected from the group consisting of: CTLA-4, TIM-3, CD160, and TIGIT. In a more specific embodiment, the binding protein includes a region specific for human PD-1 (which can be any of the foregoing embodiments) through a linker and one or more pairs of PD-1 Of human checkpoint inhibitors are connected to specific regions. In a more specific embodiment, the region specific for binding to human PD-1 is linked by a linker to the C-terminus of the antibody heavy chain specific for human checkpoint inhibitors other than PD-1 (In this context, the antibody system is defined as an immunoglobulin including two heavy chains and two light chains, wherein each heavy chain system includes a variable region and one or more constant regions and wherein each light chain system includes a variable region and A constant area). In a more specific embodiment, there are two binding regions specific for human PD-1, one region and two heavy chains C for each antibody specific for human checkpoint inhibitors other than PD-1端 连接 连接。 End connected.

This linker may be a one-button or a peptide linker. In one embodiment, the linker is a peptide linker with a length of 1 to 100 amino acids, more specifically, a length of 1 to 50 or 1 to 10 amino acids. Suitable linkers are described in the published PCT patent application WO2010 / 136483. In a more specific embodiment, the linker has the sequence listed in SEQ ID NO. 30 (STGLDSPT).

In a third aspect, the present invention provides a binding protein of general formula (II): Among them: H (CPI) is the heavy chain of IgG antibody and L (CPI) is the light chain of IgG antibody; so that H (CPI) and L (CPI) together form a human checkpoint inhibitor other than PD-1 Specific antibodies; n is an integer selected from 2, 4, and 11; A is a bond or a peptide linker; and V _H (PD-1) is an antibody heavy chain variable region with CDRH1, CDRH2, and CDRH3 , Wherein the CDRH1 is selected from: CDRH1 shown in SEQ ID NO: 3, and CDRH1 different from CDRH1 shown in SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids; CDRH2 is selected from: CDRH2 shown in SEQ ID NO: 3 and CDRH2 different from CDRH2 shown in SEQ ID NO: 3 by adding or deleting or substituting 1, 2 or 3 amino acids; and CDRH3 It is selected from: CDRH3 shown in SEQ ID NO: 3 and CDRH3 different from CDRH3 shown in SEQ ID NO: 3 by adding, deleting or substituting 1, 2 or 3 amino acids.

The binding protein of formula (II) is a binding protein specific for human PD-1 and other human checkpoint inhibitors.

In one embodiment, the binding protein of formula (II) has an IC50 of less than or equal to 5 nM and a human checkpoint inhibitor other than neutralizing PD-1 in the PD-1 / PDL-1 competition analysis.

In one embodiment, H (CPI) system comprising V _H and CH1 regions and L (CPI) lines CL and V _L, including regions. In a more specific embodiment, the H (CPI) system includes CH1, CH2, and VH regions and the L (CPI) system includes CL and VL regions. Even more specifically, H (CPI) contains CH1, CH2, CH3 and V _H regions and L (CPI) contains CL and V _L regions.

Referred to in the above _{CH1, CH2, CH3, V H,} CL and V _L, the entire region or regions may be truncated or contain a modified region extending in the N-or C- terminal, or one or more ring system by having no Replaced by the sequence of features of the indicated area. Also includes other modifications, such as adding other post-translational modifications, such as phosphorylation, deamidation, oxidation, disulfide bond recombination, isomerization, C-terminal amino acid truncation, and N-terminal glutamic acid cyclization Or include one or more unnatural amino acids. For the avoidance of doubt, antibodies are glycoproteins and the above regions can be glycosylated.

In one embodiment, H (CPI) is a heavy chain of IgG1, IgG2, IgG3, IgG4, or IgG4PE subclass. When H (CPI) is the heavy chain of IgG1 and IgG4 subclasses, n is 2. When H (CPI) is the heavy chain of IgG2 subclass, n is 4. When H (CPI) is the heavy chain of IgG3 subclass, n is 11. In a more specific embodiment, the antibody system is selected from the IgG1 (PE) subclass, and even more specifically, the antibody has an IgG1 invalid isotype (LAGA).

V _H (PD-1) is as defined in relation to the binding protein of formula (I) in any of the above embodiments. In one embodiment, the binding protein of formula (II) containing one or more CDR variants in V _H (PD-1) retains biological activity, which is defined as having less than Or IC50 equal to 5nM.

A is as defined in relation to the binding protein of formula (I) in any of the above embodiments.

In anti-RSV-PD1 mAbdAb (RSV-51A09-188001) LAG-3 / PD-1 mAbdAb (57E02x51A09-188001) and anti-CTLA-4 mAb mAbdAb (CTLA4-51A09-188001, which was determined in human PD1-PDL1 In the inhibition analysis, the PD-1 dAb 51A09-188001 can act to block PD-1 signal transmission in the context of mAbdAb as shown in its function in the IC50 of 0.24 (n = 1) and 0.29 (n = 2). This duality of CTLA4 / PD1 also helps cytokine production in mixed lymphocyte response analysis. Based on this, it is reasonable to expect that it can form the mAbdAb effect of formula (II) with other mAbs on other checkpoint inhibitors (including but not limited to CTLA4, OX40, TIM-3, CD160 and TIGIT). The mAbs of these antibodies are known in the art.

As explained above, when the anti-RSV mAb is formatted as mAbdAb, the 16 variant dAbs with CDRs associated with 51A09-188001 also block PD-1 signaling as measured by reporter gene analysis. It is reasonable to expect that when other checkpoint inhibitors are formatted as mAbdAbs of formula (II), these and other variants with related CDRs should be able to block PD-1 signaling. Changing the frame area expectations should also be tolerable.

In a fourth aspect, the present invention provides a binding protein specific for human LAG-3, which includes: one or more CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: SEQ ID NO : CDRH1 shown in 1: and CDRH1 different from CDRH1 shown in SEQ ID NO: 1 by adding or deleting or replacing 1, 2 or 3 amino acids, where CDRH2 is selected from the group consisting of: CDRH2 shown in SEQ ID NO: 1 and CDRH2 different from CDRH2 shown in SEQ ID NO: 1 by adding or deleting or replacing 1, 2 or 3 amino acids, and CDRH3 is a group consisting of Choose from: CDRH3 as shown in SEQ ID NO: 1 and CDRH3 different from CDRH3 as shown in SEQ ID NO: 1 by adding or deleting or replacing 1, 2 or 3 amino acids; and one or more CDRL1, CDRL2 and CDRL3, wherein CDRL1 is selected from the group consisting of: CDRL1 as shown in SEQ ID NO: 2 and SEQ ID NO by adding or deleting or substituting 1, 2 or 3 amino acids: CDRL1 shown in 1 is different from CDRL1, where CDRL2 is selected from the group consisting of: CDRL2 as shown in SEQ ID NO: 2 and by Add or delete or replace 1, 2 or 3 amino acids and CDRL2 different from CDRL2 shown in SEQ ID NO: 2, and CDRL3 among them is selected from the group consisting of: as shown in SEQ ID NO: 2 CDRL3 and CDRL3 different from CDRL3 shown in SEQ ID NO: 2 by adding or deleting or replacing 1, 2, or 3 amino acids.

In one embodiment, a binding protein specific for human LAG-3 has> 50% inhibition of LAG3-MHCII interaction in competitive flow cytometry analysis.

In one embodiment, the binding protein specific for human LAG-3 is an antibody. In the context of this example, the antibody is an IgA or IgG heterotetrameric glycoprotein composed of two heavy chains and two light chains, wherein the heavy chain system includes VH and CH1 regions, and the light chain system includes VL And CL area. More specifically, this antibody is an IgG antibody. In one embodiment, this anti-system is selected from the IgG1, IgG2, IgG3, IgG4, and IgG4PE subclasses. In a more specific embodiment, the antibody system is selected from the IgG1 subclass, and even more specifically, the antibody has an IgG1 invalid isotype (LAGA). Alternatively, the antibody is an IgA or IgG glycoprotein composed of two heavy chains and two light chains, wherein the heavy chain system includes VH and CH1 regions, and the light chain system includes VL and CL regions. More specifically, this antibody is an IgG antibody. In one embodiment, this anti-system is selected from the IgG1, IgG2, IgG3, IgG4, and IgG4PE subclasses. In a more specific embodiment, the antibody system is selected from the IgG1 subclass, and even more specifically, the antibody has an IgG1 invalid isotype (LAGA).

In an example of a pair of binding proteins specific for human LAG-3, the heavy chain system includes CH1, CH2 and VH regions and the light chain system includes CL and VL regions. In a more specific embodiment, the heavy chain system contains CH1, CH2, CH3 and VH regions and the light chain system contains CL and VL regions.

The CH1, CH2, CH3, VH, CL and VL regions referred to above may be complete regions or modified regions truncated or containing N or C-terminal extensions, or one or more of the ring systems may not have the indicated Replacement of the sequence of regional features. Also includes other modifications, such as adding other post-translational modifications, such as phosphorylation, deamidation, oxidation, disulfide bond recombination, isomerization, C-terminal amino acid truncation, and N-terminal glutamic acid cyclization Or include one or more unnatural amino acids. For the avoidance of doubt, antibodies are glycoproteins and the above regions can be glycosylated.

In other embodiments, the binding protein specific for human LAG-3 is a non-immunoglobulin scaffold with a loop connection element of secondary structure, which can be engineered to include a CDR region. Non-immunoglobulin scaffolds include CTLA-4 (Evibodies; Journal Immunological Methods 248 (1-2): 31-45, 2001), lipocalin, such as protein A derived molecules such as the Z-region of protein A (Affibodies, Protein Eng Des Sel 17: 455-462, 2004 and EP1641818), A-region (Avimer / Maxibody), heat shock proteins, such as GroEI and GroES, trans-body, ankyrin repeat protein (DARPin) , Peptide aptamer, C-type lectin region (Tetranectin), human γ-crystallin and human ubiquitin (affilins), PDZ region, human protease inhibitor scorpion toxin kunitz-type region and fibronectin (fibronectin) / adnectin. These scaffolds were engineered to arrange the CDRs in a functional manner (see Holliger and Hudson, Nature Biotechnology, 2005, Vol 23, No 9, 1126-1136 for a summary of alternative antibody models).

In one embodiment, the binding protein specific for human LAG-3 has the specificity for human LAG-3 as shown in the binding proteins specific for human PD-1 and human LAG-3 listed above CDR sequences in any specific embodiment of an antibody. In the embodiment in which the binding protein specific for human LAG-3 contains one or more CDR variants, those skilled in the art should understand that, in a particular embodiment, one or more CDR variants Binding proteins specific for human LAG-3 retain biological activity and are defined as> 50% inhibition of LAG3-MHCII interaction in competitive flow cytometry analysis.

In specific embodiments, as described above for the corresponding examples of antibodies specific for human LAG-3, the binding protein specific for human LAG-3 is an antibody that includes two identical heavy chains. It includes a sequence as defined in SEQ ID NO. 1 or a variant of SEQ ID NO. 1 with a difference of up to 10 amino acid additions, deletions or substitutions, and / or an antibody that includes two identical light chains. The chain system includes a sequence as defined in SEQ ID NO. 2 or a variant of SEQ ID NO. 2 with a difference of up to 10 amino acid additions, deletions, or substitutions.

In a specific embodiment, as described above for the binding protein specific for human PD-1 and human LAG-3, the corresponding embodiment of the antibody specific for human LAG-3, for human LAG-3 The specific binding protein is an antibody having two identical heavy chains, the heavy chain comprising the sequence defined by SEQ ID NO.1 or a sequence having at least 90% sequence identity with the sequence of SEQ ID NO.1, and And / or two identical light chains, the light chain comprising the sequence as defined in SEQ ID NO. 2 or a sequence having at least 90% sequence identity with the sequence of SEQ ID NO. 2.

In one embodiment, a binding protein specific for human LAG-3 can additionally neutralize human checkpoint inhibitors other than LAG-3. As mentioned above, neutralizing a human checkpoint inhibitor refers to blocking the biological activity of the human checkpoint inhibitor. Because the biological activity of human checkpoint inhibitors inhibits the signaling cascade that regulates the activation, proliferation or cytokine production of immune cells after stimulation, neutralizing human checkpoint inhibitors increases or restores the biological activity of immune cells after stimulation . The methods for measuring the activation, proliferation or cytokine production of immune cells to assess neutralization are outlined above (for neutralizing human checkpoint inhibitors other than PD-1).

In one embodiment, human checkpoint inhibitors other than LAG-3 are selected from the group consisting of: CTLA-4, TIM-3, CD160, and TIGIT. In a more specific embodiment, the binding protein includes an antibody specific for binding to human LAG-3 (which can be any of the foregoing embodiments) by using a linker and one or more pairs Human checkpoint inhibitors other than LAG-3 are connected to specific areas. In a more specific embodiment, the region specific for binding to another human checkpoint inhibitor is linked to the C-terminus of the heavy chain of the antibody specific for human LAG-3 via a linker. In a more specific embodiment, there are two binding regions specific for human checkpoint inhibitors other than LAG-3, and one is the C-terminus of the two heavy chains of the antibody specific for binding human LAG-3 Connected.

The linker may be a one-button or a peptide linker. In one embodiment, the linker is a peptide linker with a length of 1 to 100 amino acids, more specifically, a length of 1 to 50 or 1 to 10 amino acids. Suitable linkers are described in the published PCT patent application WO2010 / 136483. In a more specific embodiment, the linker has the sequence listed in SEQ ID NO. 30 (STGLDSPT).

In a fifth aspect, the present invention provides a binding protein of general formula (III): Wherein: H (LAG-3) is an IgG antibody heavy chain including CDRH1, CDRH2 and CDRH3, wherein the CDRH1 is selected from: CDRH1 shown in SEQ ID NO: 1, and by adding or deleting or replacing 1, 2 or 3 amino acids and CDRH1 different from CDRH1 shown in SEQ ID NO: 1; wherein CDRH2 is selected from: CDRH2 shown in SEQ ID NO: 1 and by adding or deleting or replacing 1, 2 or 3 Amino acids and CDRH2 different from CDRH2 shown in SEQ ID NO: 1; and CDRH3 is selected from: CDRH3 shown in SEQ ID NO: 1 and by adding, deleting or replacing 1, 2 or 3 Amino acid and CDRH3 different from CDRH3 shown in SEQ ID NO: 1; L (LAG-3) is an IgG antibody light chain including CDRL1 and CDRL2, wherein the CDRL1 is selected from: SEQ ID NO: 2 The CDRL1 shown and the CDRL1 different from the CDRL1 shown in SEQ ID NO: 2 by adding or deleting or substituting 1, 2 or 3 amino acids; and the CDRL2 is selected from: SEQ ID NO: 2 CDRL2 and CDRL2 different from CDRL2 shown in SEQ ID NO: 1 by adding or deleting or substituting 1, 2 or 3 amino acids; n is an integer selected from 2, 4 and 11; A is a single bond or Peptide linker; and CPI can bind Areas of human checkpoint inhibitors other than LAG-3.

The binding protein of formula (III) is a binding protein specific for human LAG-3 and other checkpoint inhibitors.

In one embodiment, the binding protein of formula (III) has> 50% inhibition of LAG3-MHCII interaction in competitive flow cytometry analysis and exhibits neutralization of human checkpoint inhibitors other than LAG-3.

H (LAG-3) and L (LAG-3) are as defined above in any of the embodiments related to the 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 and is defined as a competitive flow cytometer In the analysis, the line had> 50% inhibition of LAG3-MHCII interaction.

CPI is a single variable trend, which may be a complete antibody variable region, such as VH, VHH, and VL or a modified region truncated or containing an N or C-terminal extension, or one or more of the loop systems does not have Replaced by the sequence of features of the indicated area. Also includes other modifications, such as adding other post-translational modifications, such as phosphorylation, deamidation, oxidation, disulfide bond recombination, isomerization, C-terminal amino acid truncation, and N-terminal glutamic acid cyclization Or include one or more unnatural amino acids.

In other embodiments, the CPI may include a non-immunoglobulin scaffold with a loop connection element of secondary structure, which may be engineered to include a CDR region. Non-immunoglobulin scaffolds include CTLA-4 (Evibodies; Journal Immunological Methods 248 (1-2): 31-45, 2001), lipocalin, protein A-derived molecules such as the Z-region of protein A (Affibodies, Protein Eng Des Sel 17 : 455-462, 2004 and EP1641818), A-region (Avimer / Maxibody), heat shock proteins, such as GroEI and GroES, transferrin (trans-body), ankyrin repeat protein (ankyrin repeat protein) ( DARPin), peptide aptamer, C-type lectin region (Tetranectin), human γ-crystallin and human ubiquitin (affilins), PDZ region, human protease inhibitor scorpion toxin kunitz-type region and fiber Protein (fibronectin) / adnectin. These scaffolds were engineered to arrange the CDRs in a functional manner (see Holliger and Hudson, Nature Biotechnology, 2005, Vol23, No 9, 1126-1136 for a summary of alternative antibody models).

A is as defined in any of the above examples regarding the binding protein of formula (I).

Medical use

As discussed in the section of the prior art of the present invention, PD-1 and / or LAG-3 blockade reduces immunosuppression and reverses T cell depletion and is believed to be useful in the treatment of infectious diseases and cancer.

Therefore, in one embodiment, the present invention provides a method of treating an infectious disease, which comprises administering to a human in need thereof a therapeutically effective amount of the binding protein of the present invention. Infectious diseases that can be treated include bacterial infections (such as tuberculosis, Listeria sp. , Streptococcus sp. And Salmonella sp. ), Parasitic infections (such as malaria and leishmania) Protozoal diseases), viral infections (including respiratory infections, such as influenza virus, respiratory fusion virus or parainfluenza virus, and chronic viral infections, including hepatitis B virus, hepatitis C virus, cytomegalovirus, herpes simplex) Rash virus, human papilloma virus, Ebola virus, Ebstein-Barr virus and human immunodeficiency virus) and sepsis.

Especially with regard to HIV, latently infected CD4 T cells expressing LAG-3 and PD-1 and inhibiting the interaction between PD-1 and PD-L1 / 2 have been shown to increase the production of HIV by CD4 + cells. This action allows the virus nest (cells infected with HIV) to be "seen" by the immune system. In parallel, LAG-3 and PD-1 blockade has been shown to increase HIV-specific CD8 + T cell responses. This can result in the immune clearance of the currently visible virus dens. This method of depleting the virus dens is commonly referred to as "knockout and killing" (kick the virus den into expressing viral antigens, and then kill the infected cells-immune Clear). Therefore, the present invention assumes that blocking both receptors may provide a synergistic effect in treating or curing HIV infection.

In one embodiment, the present 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 present invention. The treatment period is expected to be 3-12 months.

In the context of the present invention, the cure for HIV refers to, for example, the continuous initiation and maintenance of virus control of human immunodeficiency virus without any treatment (undetectable plasma virus measured by analysis that can detect a single copy of HIV type-1 RNA) Hememia) for at least two years. Any analysis with suitable sensitivity can be used. In one embodiment, a single copy analysis (Single Copy Assay) described in Palmer et al. (J. Clin. Microbiol., 2003, 41 (10): 4531-4536) is used. In other words, after the treatment period, HIV was not detected in the plasma for at least two years, and no other anti-HIV treatment was given during this period.

In another embodiment, the present 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 present invention.

At the population level, treatment of HIV with the binding protein of the present invention can reduce the incidence and mortality of several types of non-AIDS. At the level of individual patients, treatment of HIV with the binding protein of the present invention can reduce inflammation, decrease infectious fossa during treatment, or increase HIV-specific T cell function. For readers who are familiar with the technology, it should be obvious, and binding protein therapy may be uninterrupted, but for the purpose of evaluating the reduction of inflammation, the reduction of infectious fossa, or the increase of specific T cell function, the treatment period must be specified.

Therefore, in one embodiment, HIV treatment refers to treatment that causes a reduction in inflammation during treatment. The treatment period can be 3-12 months. The reduction in inflammation can be determined by measuring the amount of soluble CD163 or soluble CD14, the decrease in the performance of the main inflammation markers of cardiovascular disease (CVD) risk, or the decrease in vascular inflammation, as measured by the decrease in activated monocytes. Markers of CVD risk include high-sensitivity C-reactive protein (hsCRP). Interleukin-6 (IL-6) and D-dimer. The degree of vascular inflammation can be measured by arterial fluorodeoxyglucose (FDG) absorption.

During the inflammatory reaction, monocytes remove the clearer receptors CD163 and CD14 (a receptor involved in sensing bacterial products) into the plasma. The amount of soluble CD163 or soluble CD14 can be measured by ELISA in plasma. A decrease in the amount of soluble CD163 or soluble CD14 refers to a situation where the average amount of soluble CD163 or soluble CD14 measured from at least three plasma samples at the end of the treatment period is lower than at the beginning. In one embodiment, a decrease in the amount of soluble CD163 or soluble CD14 means that the average amount of soluble CD163 or soluble CD14 measured from at least three plasma samples at the end of the treatment period is lower than or equal to the average at the beginning of the treatment period The amount of soluble CD163 or soluble CD14 minus one standard deviation (use a larger standard deviation before / after treatment). In a more specific embodiment, the decrease in the amount of soluble CD163 or soluble CD14 means that the average amount of soluble CD163 or soluble CD14 measured from at least three plasma samples at the end of the treatment period is lower than or equal to the beginning of the treatment period The average amount of soluble CD163 or soluble CD14 at that time minus two standard deviations (use a larger standard deviation before / after treatment). Even more specifically, a decrease in the amount of soluble CD163 or soluble CD14 means where the average amount of soluble CD163 or soluble CD14 measured from at least three plasma samples at the end of the treatment period is lower than or equal to the average at the beginning of the treatment period The amount of soluble CD163 or soluble CD14 minus three standard deviations (using a larger pre-treatment / post-treatment standard deviation). In one embodiment, a decrease in the amount of soluble CD163 or soluble CD14 means that the average amount of soluble CD163 or soluble CD14 measured from at least three plasma samples at the end of the treatment period is less than or equal to 2 times the beginning of the treatment period Situation. In one embodiment, a decrease in the amount of soluble CD163 or soluble CD14 refers to a case where the average amount of soluble CD163 or soluble CD14 measured from at least three plasma samples at the end of the treatment period is five times that at the beginning of the treatment period.

In one embodiment, HIV treatment refers to reducing the performance of high-sensitivity C-reactive protein (hsCRP), interleukin-6 (IL-6), or D-dimer. CVD risk markers can be measured with any biological sample. In one embodiment, the amount of these markers is measured in plasma by any suitable method. In one embodiment, the reduced CVD risk marker amount refers to a situation where the average marker amount measured from at least three samples at the end of the treatment period is lower than or equal to the average marker amount at the beginning. In one embodiment, the reduction in CVD risk markers refers to where the average marker amount measured from at least three samples at the end of the treatment period is less than or equal to the average marker amount at the beginning minus one standard deviation (use a larger Standard deviation before / after treatment). In a more specific embodiment, the reduction in CVD risk markers refers to where the average marker amount measured from at least three samples at the end of the treatment period is less than or equal to the average marker amount at the beginning minus two standard deviations (Use a larger standard deviation before / after treatment). Even more specifically, a reduction in the amount of CVD risk markers means that the average marker amount measured from at least three samples at the end of the treatment period is less than or equal to the average marker amount at the beginning minus three standard deviations (use Large standard deviation before / after treatment). In one embodiment, a reduction in the amount of CVD risk markers refers to a situation where the average marker amount measured from at least three samples at the end of the treatment period is less than 2 times the beginning. In one embodiment, a reduction in the amount of CVD risk marker refers to where the average marker amount measured from at least three samples at the end of the treatment period is 5 times lower than at the beginning.

In one embodiment, HIV treatment refers to reduced vascular inflammation as measured by decreased arterial fluorodeoxyglucose (FDG) absorption. In one embodiment, FDG absorption can be evaluated by FDG-PET / CT. In one embodiment, the decrease in FDG absorption refers to where the average FDG absorption measured from at least three experiments at the end of the treatment period is lower than or equal to the average FDG-absorption measured from at least three experiments at the beginning of the treatment period Situation. In one embodiment, the decrease in FDG absorption refers to where the average FDG absorption measured from at least three experiments at the end of the treatment period is lower than or equal to the average FDG-absorption measured from at least three experiments at the beginning of the treatment period Subtract one standard deviation (use a larger standard deviation before / after treatment). In a more specific embodiment, a decrease in FDG absorption refers to a system in which the average FDG absorption measured from at least three experiments at the end of the treatment period is lower than or equal to the average measured from at least three experiments at the beginning of the treatment period FDG-absorption minus two standard deviations (use larger pre- / post-treatment standard deviations). Even more specifically, the decrease in FDG-absorption refers to where the average FDG absorption measured from at least three experiments at the end of the treatment period is lower than or equal to the average FDG-measured from at least three experiments at the beginning of the treatment period Absorption minus three standard deviations (use larger pre- / post-treatment standard deviations). In one embodiment, a decrease in FDG absorption refers to a situation where the average FDG absorption measured from at least three experiments at the end of the treatment period is less than 2 times the beginning of the treatment period. In one embodiment, the FDG-absorption decrease refers to an average FDG absorption system measured from at least three experiments at the end of the treatment period that is 5 times lower than at the beginning of the treatment period.

In another embodiment, HIV treatment refers to treatment that lowers the infectious fossa during treatment. The specific CD4 + T cells (which account for most of the HIV infection nests) have a half-life of 6 months. In this embodiment, the appropriate treatment period should not be less than 6 months, for example, 6-24 months.

The size of the HIV-infected litter can be determined by quantifying cell-linked 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 (eg, lymph node mononuclear cell suspensions derived from inguinal lymph node sections) are used as the cell source. In particular, CD4 + T cells or a subset of CD4 + T cells are used. Methods for obtaining these cell populations are well known. After separating the cells, nucleic acids can be extracted by conventional techniques. Any suitable technique can be used to quantify HIV RNA. In one embodiment, can be used Amplicor Monitor Assay (operating according to the manufacturer's instructions) (Note - When the "per milliliter replicate" value of ⁶ is more than 10, should be appropriately diluted fresh RNA extracts were repeated quantified; if When the copy number / ml value is less than 50, the single copy number analysis described above can be used instead to quantify). A mathematical conversion can be used to convert the number of copies / ml to the number of copies / mg, where the tissue weight (mg) is measured, the total RNA extracted in a specific volume (ng) is quantified, and the number of RNA copies / ng is measured. In one embodiment, quantitative PCR technology can be used to quantify the integrated HIV DNA (eg quantitative reverse PCR quantitative or real-time PCR using Applied Biosystems' 7500 real-time PCR system). Alternatively, when PCR incorporates a ³² P-calibrated nucleotide, the PCR product can be quantified using a phosphorus imager and suitable software (eg, IMAGEQUANT, Molecular Dynamics). An alternative method for measuring HIV virus nests in CD4 + T cells is the quantitative virus growth assay (QVOA) described for the first time in Chun et al. (Nature, 1997, 387: 183-8), and its well-known variation method. This analysis measures the amount of HIV virus capable of replicating per 1 million HIV units infected with CD4 + T cells. This method requires approximately 50 million dormant CD4 T cells and is therefore usually a white blood cell separation blood sample. Repetitive CD4 T cells were implanted in a limiting dilution manner: PHA was used to maximize activated cells, and PBMC and IL2 from seronegative donors were irradiated with heterologous radiation for 24 hours. Wash the culture and co-culture with target cells that are susceptible to HIV infection and replication. After 2 weeks of culture, the supernatant was collected and analyzed for viral protein products. The number of chronic wells at each cell dilution was determined and the maximum likelihood method was used to estimate the number of dormant CD4 ⁺ T cells per million infected units.

Viral pit decline refers to the situation in which the average amount of replicated virus measured by QVOA or the average HIV gene mass measured by cell-linked HIV DNA and / or RNA at the end of the treatment period is lower than at the beginning. In one embodiment, the average amount of replication-competent virus or HIV gene mass from at least three samples / experiment at the end of treatment is equal to or lower than the average replication-competence from at least three samples / experiment at the beginning of treatment The amount of viral capacity or the quality of HIV genes minus one standard deviation (use a larger standard deviation before / after treatment). In a more specific embodiment, the average amount of replicable virus or HIV gene mass from at least three samples / experiment at the end of treatment is equal to or lower than that from at least three samples / experiment at the beginning of treatment The average amount of virus with replication capacity or HIV gene material minus two standard deviations (use a larger standard deviation before / after treatment). Even more specifically, the average reproducible viral volume or HIV genetic material from at least three samples / experiment at the end of treatment is equal to or lower than the average reproductive capacity from at least three samples / experiment at the beginning of treatment The amount of virus or HIV genetic material minus three standard deviations (use a larger standard deviation before / after treatment). At the end of treatment, the average amount of replicable virus from at least three samples / experiment was 10%, 20%, or 50% lower than the average amount of virus from at least three samples / experiment. In one embodiment, treatment at the end of the experiment from at least three samples / experimental average mass of HIV genetic material is replicated 0.5log ₁₀ per ¹⁰⁶ cells, from at least three lines below the sample at the start of treatment / The average amount of HIV genetic material.

In another embodiment, HIV treatment refers to treatment that increases the function of HIV-specific T cells after antigen stimulation. HIV-specific T-cell function can be measured by cytokine production, increased cytotoxic potential, or T cell proliferation. In this embodiment, the treatment period may be 3-12 months.

Cytokine production and increased cytotoxic potential can be measured by ELISA, ELISPOT or by intracellular staining of cells and obtained on a flow cytometer. These standard techniques and some suitable methods are outlined in Chapter 24 of HIV Protocols, Volume 485, Second Edition, 2009. ELISA can be performed on blood samples to measure the amount of cytotoxin or cytotoxin products of immune cells. ELISPOT analysis can be performed on peripheral blood mononuclear cells or CD4 + or CD8 + T cell populations. When using mixed cell populations (eg peripheral blood mononuclear cells), cytokine flow cytometry can of course provide CD4 + and CD8 + cells On individual information. Various HIV peptides can be used for antigen stimulation, including gag, pol, env, nef, or pooled peptides. One or more cytokines (eg IFN-γ, IL-2, TNF) or cytotoxic products (eg CD107a, perforin or granzyme) produced after antigen stimulation can be monitored. Those skilled in the art should understand that cytokines / cytotoxic products should be measured for different cell types. For CD4 cells, suitable cytokines include IFN-γ, IL-2, and TNFα. For CD8 cells, suitable cytokines include IFN-γ, IL-2, TNFα and suitable cytotoxic products include CD107a. In one embodiment, increased production of cytokines or cytotoxic products refers to a situation where the average amount of cytokines / cytotoxic products measured from at least three samples is higher at the end of the treatment period than at the beginning. In one embodiment, increased cytokine production or increased cytotoxic product release means wherein the average amount of cytokine / cytotoxic product measured by at least three samples at the end of the treatment period is equal to or higher than that at the beginning of treatment The average amount of cytokines / cytotoxic products measured in at least three samples plus a standard deviation (use a larger standard deviation before / after treatment). In a more specific embodiment, increased cytokine production or increased cytotoxic product release means wherein the average amount of cytokine or cytotoxic product measured by at least three samples at the end of the treatment period is equal to or higher than the treatment At the beginning, the average amount of cytokines / cytotoxic products measured from at least three samples plus two standard deviations (using a larger pre-treatment / post-treatment standard deviation). Even more specifically, increased cytokine production or increased cytotoxic product release means where the average amount of cytokine or cytotoxic product measured by at least three samples at the end of the treatment period is equal to or higher than that at the beginning of treatment The average amount of cytokines / cytotoxic products measured in at least three samples plus three standard deviations (using a larger pre-treatment / post-treatment standard deviation). In one embodiment, increased cytokine production or increased cytotoxic product release means wherein the average amount of cytokine or cytotoxic product measured by at least three samples at the end of the treatment period is higher than at the beginning of treatment by at least three The average amount of cytokines / cytotoxic products measured in each sample is 5%, 15%, 50% or 100%.

T cell proliferation after antigen stimulation can be basically as described in 2004, Methods in Cell Biology, Volume 75, Chapter 19, by using carboxyfluorescein diacetate succinimide (CSFE), by flow cytometry The instrument monitors cell division by cell penetration staining technique to measure. Peripheral blood monocytes can be used in this method and various HIV peptides can be used for antigen stimulation, including gag, pol, env, nef, or pooled peptides. In one embodiment, the increase in hyperplasia refers to the average percentage of divided antigen-specific T cells (CD4 + and / or CD8 +) determined from at least three samples (by subtracting the background division to determine the percentage of diluted CSFE The calculated, that is, the percentage of dividing T cells in the negative control is higher at the end of the treatment period than at the beginning. In one embodiment, the increase in hyperplasia refers to where the average percentage of divided antigen-specific T cells measured by at least three samples at the end of the treatment period is equal to or higher than that determined by at least three samples at the beginning of the treatment period The measured average percentage of split antigen-specific T cells plus a standard deviation. In a more specific embodiment, the increase in hyperplasia refers to wherein the average percentage of divided antigen-specific T cells measured by at least three samples at the end of the treatment period is equal to or higher than that at the beginning of the treatment period by at least three The average percentage of the divided antigen-specific T cells measured in two samples plus two standard deviations. Even more specifically, the increase in hyperplasia means that the average percentage of divided antigen-specific T cells measured by at least three samples at the end of the treatment period is equal to or higher than that measured by at least three samples at the beginning of the treatment period The measured average percentage of divided antigen-specific T cells plus three standard deviations. In one embodiment, the increase in hyperplasia refers to wherein the average percentage of divided antigen-specific T cells measured by at least three samples at the end of the treatment period is greater than that determined by at least three samples at the beginning of the treatment period The average percentage of split antigen-specific T cells is 1.2, 1.5, 2, 5, or 20 times.

In another aspect, the present invention provides a method of treating cancer, which comprises administering a therapeutically effective amount of the binding protein of the present invention to a human in need thereof.

As used herein, the term "cancer" refers to a host organism that has undergone malignant transformation and causes disease. The term cancer refers to the primary cancer, which can be easily distinguished from non-cancer cells by the histological examination and cancer metastasis of the cancer by metabolically established technology. In the context of cancer treatment, the term "treatment" refers to: (1) improving or preventing the symptoms or biological characterization of one or more symptoms, (2) alleviating one or more symptoms, effects or side effects related to the symptoms or treatment, Or (3) Delay the progression of symptoms or biological characterization of one or more symptoms.

In one embodiment, the cancer is a solid tumor selected from the group consisting of brain cancer (glioma), glioblastoma, astrocytoma, glioblastoma multiforme, Bannayan-Zonana syndrome, Cowden disease, Lhermitte-Duclos disease, breast cancer, inflammatory breast cancer, Wilm's tumor, Ewing's sarcoma, rhabdomyosarcoma, intraventricular ependymoma, medulloblastoma, colorectal cancer, colorectal cancer , Head and neck cancer (including squamous cell carcinoma of the head and neck), kidney cancer, lung cancer (including squamous cell lung cancer, lung adenocarcinoma, small cell lung cancer and non-small cell lung cancer), liver cancer (including hepatocellular carcinoma), melanoma, ovarian Cancer, pancreatic cancer (including squamous cell pancreatic cancer), prostate cancer, sarcoma, osteosarcoma, giant cell tumor of bone, thyroid cancer, neuroblastoma, bladder cancer, urothelial cancer, lung cancer, vulvar cancer, Cervical cancer, uterine cancer, endometrial cancer, renal cancer (including renal clear cell carcinoma, renal papillary carcinoma, renal cell carcinoma), stromal tumor, esophageal cancer, salivary gland cancer, gastric cancer, nasopharyngeal cancer, buccal cancer, Oral cancer, gastric cancer, GIST (gastrointestinal Stromal tumor) and testicular cancer.

In other embodiments, the cancer is a hematological cancer (liquid tumor), including lymphoid and bone marrow malignancies. Bone marrow tumors include (but are not limited to) acute bone marrow (or bone marrow cells or bone marrow or bone marrow mother cells) leukemia (undifferentiated or differentiated), acute pre-marrow (or pre-marrow cells or pre-marrow or pre-marrow mother cells) Leukemia, acute bone marrow mononuclear cell (or bone marrow mononuclear cell) leukemia, acute mononuclear cell (or mononuclear cell) leukemia, erythrocyte leukemia, and megakaryocyte (or megakaryocyte) leukemia. These leukemias can be collectively referred to as acute bone marrow (or bone marrow cell or bone marrow) leukemia (AML). Bone marrow malignant tumor fluids include myeloproliferative disorders (MPD), which include (but are not limited to) chronic myelogenous (or bone marrow) leukemia (CML), chronic myelomonocytic leukemia (CMML), primary thrombocythemia (or Thrombocytosis) and polycythemia vera (PCV). Bone marrow malignancies also include myeloid metaplasia (or myelodysplastic syndrome or MDS), which can be referred to as refractory anemia (RA), refractory anemia with excessive germ cell (RAEB) and refractory anemia with mother Increased cell transformation (RAEBT); and myelofibrosis (MFS) with or without idiopathic extramedullary metaplasia.

Hematopoietic cancer also includes marrow malignancies, which can occur in lymph nodes, spleen, bone marrow, peripheral blood, and / or extranodal locations. Lymphatic cancers include B-cell malignancies, which include (but are not limited to) B-cell Hodgkin's lymphoma (B-NHL). B-NH can be mild (or low malignant), moderately malignant (or aggressive) or highly malignant (very aggressive). Mild B-cell lymphoma includes follicular lymphoma (FL); small lymphocytic lymphoma (SLL); marginal zone lymphoma (MZL) includes nodular MZL, extranodal MZL, splenic MZL, and villous lymph Splenic bulb MZL; lymphoplasma cell lymphoma (LPL); and mucosa-associated lymphoid tissue (MALT or extranodal marginal zone) lymphoma. Moderately malignant B-NHL includes mantle cell lymphoma (MCL) diffuse large cell lymphoma (DLBCL), follicular large cell (or grade 3 or 3B) lymphoma, and primary mediastinum with or without leukemia involvement Lymphoma (PML). High malignant B-NHL includes 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 humoral lymphoma, HIV-related (or AIDS-related) lymphoma, and post-transplant lymphoproliferative disorder (PTLD) or lymphoma. B-cell malignancies also include (but are not limited to) chronic lymphocytic leukemia (CLL), juvenile lymphocytic leukemia (PLL), Fahrenheit macroglobulinemia (WM), hairy cell leukemia (HCL), large granular lymphoma Cellular (LGL) leukemia, acute lymphatic (or lymphocyte or lymphoblast) leukemia, and Castleman's disease. NHL can also include T-cell non-Hodgkin's lymphoma (T-NHL), which includes (but is not limited to) T-cell non-Hodgkin's lymphoma (NOS), peripheral T-cells that are not otherwise specified Lymphoma (PTCL), degenerative large cell lymphoma (ALCL), angioimmunoblastic lymphopathy (AILD), nasal natural killer (NK) cell / T-cell lymphoma, γ / δ lymphoma, cutaneous T cell lymphoma Tumors, mycosis fungoides, Sezary syndrome, lymphoblast T cell leukemia, acute lymphoblast T cell leukemia, and lymphoblast T cell lymphoma.

Hematopoietic cancer also includes Hodgkin's lymphoma (or disease), which includes typical Hodgkin's lymphoma, tuberous sclerosis type Hodgkin's lymphoma, mixed cell type Hodgkin's lymphoma, lymphocytes Type (LP) Hodgkin's lymphoma, nodular LP Hodgkin's lymphoma and lymphocytopenic Hodgkin's lymphoma. Hematopoietic cancers also include plasma cell diseases or cancers, such as multiple myeloma (MM) including burnt MM, unknown (or unknown or unclear) single-strain gamma globulinopathy (MGUS), plasmacytoma (skeletal , Extramedullary), lymphoplasma cell lymphoma (LPL), Fahrenheit macroglobulinemia, plasma cell leukemia, primary amyloidosis (AL) and multiple myeloma megakaryoblastic leukemia. Hematopoietic cancer may also include other cancers of hematopoietic cells, including polymorphonuclear leukocytes (or neutrophils, such as chronic neutrophil leukemia), basophils, eosinophils, dendrites Shaped cells, platelets, red blood cells and natural killer cells. For example, hematopoietic cancer lines include plasmacytoma, immunoblastic large cell leukemia, mantle cell leukemia, acute megakaryocytic leukemia, promyelocytic leukemia, erythrocyte leukemia, and follicular lymphoma.

In a more specific embodiment, the cancer is a solid tumor. At this time, the body tumor can be selected from glioma, head and neck cancer (including squamous cell carcinoma of the head and neck), gastric cancer, melanoma, renal cell carcinoma (RCC), esophageal cancer, lung squamous cell carcinoma, lung adenocarcinoma, small cell lung cancer , Non-small cell lung cancer, hepatocellular carcinoma, renal clear cell carcinoma, renal papillary carcinoma, prostate cancer, esophageal cancer, colorectal cancer, breast cancer, bladder cancer, cervical cancer, endometrial cancer, ovarian cancer and pancreas. In another aspect, this human line has liquid tumors, such as diffuse large B-cell lymphoma (DLBCL), multiple myeloma, chronic lymphoblastic leukemia (CLL), follicular lymphoma, acute myeloid leukemia, and chronic myelogenous leukemia.

combination

When used to treat infectious diseases other than HIV, the binding protein can be co-administered with one or more epigenetic modifiers, immune checkpoint agonists or antagonists, or immunomodulators. Epigenetic modifiers include (but are not limited to) tissue protein deacetylase inhibitors (HDACi), bromodomain inhibitors (BETi), protein kinase C (PKC) agonists, PTEFb activators, Tissue protein methyltransferase inhibitors (HMTi) and cytokines (eg IL21). Immune checkpoint agonists or antagonists include antibodies against CTLA-4, TIM-3, CD160, TIGIT, OX40, and ICOS. Immunomodulators include indoleamine 2,3-dioxygenase-1 (IDO-1) inhibitors.

When the binding protein is desired to treat or cure HIV, the binding protein of the present invention can be used alone or in combination with other therapeutic agents. . Therefore, in other embodiments, the method of treating or curing HIV infection in a subject may include administering one or more additional pharmaceutical agents that can be used to treat or cure HIV in addition to the binding protein. Examples of such agents include: nucleotide reverse transcriptase inhibitors such as zidovudine, didanosine, tenofovir, lamivudine, Zalcitabine, abacavir, stavudine adefovir (stavudine adefovir), adefovir (adefovir dipivoxil), fozivudine (tozoxudine), todoxil, emtricitab Emtricitabine, alovudine, amdoxovir, elvucitabine, and similar agents; non-nucleotide reverse transcriptase inhibitors (including agents with antioxidant activity, (E.g. immunocal, oltipraz, etc.), such as nevirapine, delavirdine, efavirenz, loviride, loviride Immunocal, oltipraz, capravirine, lersivirine, GSK2248761, TMC-278, TMC-125, etravirine and similar agents ; Protease inhibitors , such as saquinavir (saquinavir), ritonavir (ritonavir), indinavir (indinavir) vir), 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; HIV entry inhibitors : These agents inhibit viral adhesion, 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 (Maraviroc) (UK 427,857), TAK449 and these are disclosed in WO 02/74769, PCT / US03 / 39644, PCT / US03 / 39975, PCT / US03 / 39619, PCT / US03 / 39618, PC T / US03 / 39740 and PCT / US03 / 39732, and similar agents.

Table 3 lists several FDA approved antiretroviral treatments. Highly effective antiretroviral therapy (HAART) generally includes a combination of drugs from several different classes, such as reverse transcriptase inhibitors, integrase inhibitors, and protease inhibitors.

Therefore, in one embodiment, the binding protein is co-administered with one or more antiretroviral agents, such as one or more agents selected from the group consisting of: reverse transcriptase inhibitors, integrase inhibitors and Protease inhibitor. In another embodiment, the binding protein is co-administered with a reverse transcriptase inhibitor, integrase inhibitor, and protease inhibitor. Any of the reverse transcriptase inhibitors, integrase inhibitors, and protease inhibitors listed in Table 3 can be used in these examples.

The binding proteins of the invention can be used in combination with one or more pharmacological enhancers, and with or without additional compounds used to treat or cure HIV. Examples of such pharmacological enhancers (or pharmacokinetic enhancers) include (but are not limited to) ritonavir, GS-9350 and SPI-452.

Ritonavir is 10-hydroxy-2-methyll-5- (1-methylethyl) -1-1 [2- (1-methylethyl) -4-thiazolyl] -3,6 -Dioxo-8,11-bis (phenylmethyl) -2,4,7,12-tetraazatridec-13-acid 5-thiazolylmethyl ester [5S- (5S *, 8R * , 10R *, 11R *)] and available from Abbott Laboratories of Abbott park, Illinois is NORVIR. Ritonavir is an HIV protease inhibitor and other antiretroviral agents suitable for the treatment of HIV infection. Ritonavir also inhibits P450-mediated drug metabolism and P-glycoprotein (Pgp) cell transport system, thereby increasing the concentration of active compounds in the organism.

GS-9350 is a compound developed by Gilead Sciences of Foster City, California as a pharmacological enhancement.

SPI-452 is a compound developed by Sequoia Pharmaceuticals of Gaithersburg, Maryland as a pharmacological enhancer.

When used to treat or cure HIV, the binding protein or binding protein / antiretroviral combination may be co-administered with one or more latency reversal agents, immune checkpoint agonists or antagonists, or immunomodulators. Latency reversal agents include (but are not limited to) tissue protein deacetylase inhibitors (HDACi), bromodomain inhibitors (BETi), protein kinase C (PKC) agonists, PTEFb activators, tissue protein methyltransferases Inhibitors (HMTi) and cytokines (eg IL21). Immune checkpoint agonists or antagonists include antibodies against CTLA-4, TIM-3, CD160, TIGIT, OX40, and ICOS. Immunomodulators include indoleamine 2,3-dioxygenase-1 (IDO-1) inhibitors.

When used to treat or cure HIV, the binding protein of the present invention and any other pharmaceutically active agent may be administered together or separately and, when administered separately, they may be administered simultaneously or sequentially in any order. The amount and activity of the binding protein and other pharmaceutical agents of the present invention and the related timing of administration will be selected to achieve the desired combination therapeutic effect. The administration of the binding protein and other therapeutic agents may be accompanied by: (1) a unit pharmaceutical composition comprising two pharmaceutically active agents; or (2) a separate pharmaceutical composition each comprising a pharmaceutically active agent medicine. Alternatively, the composition can be administered separately in a sequential manner, where one therapeutic agent is administered first and the other is administered second or vice versa. This sequential administration may be close in time or far apart in time.

When the binding protein is desired to treat cancer, the binding protein can be used in combination with at least one anti-tumor agent.

When used to treat cancer, this binding protein or binding protein / antitumor agent combination may be co-administered with one or more epigenetic modifiers, immune checkpoint agonists or antagonists, or immunomodulators. Epigenetic modifiers include (but are not limited to) tissue protein deacetylase inhibitors (HDACi), bromodomain inhibitors (BETi), protein kinase C (PKC) agonists, PTEFb activators, tissue protein methyl groups Transferase inhibitors (HMTi) and cytokines (eg IL21). Immune checkpoint agonists or antagonists include antibodies against CTLA-4, TIM-3, CD160, TIGIT, OX40, and ICOS. Immunomodulators include indoleamine 2,3-dioxygenase-1 (IDO-1) inhibitors.

The binding protein of the present invention can be used to improve immunosuppression in the tumor microenvironment. In one embodiment, the binding protein can be combined with adoptive cell therapy (ACT) including engineered cytotoxic cells, such as natural or non-naturally produced T cells that exhibit antigen receptors (CAR) or T cell receptors (TCR), Natural killer (NK) cells or cytotoxic T cells or NK cell strains are administered in combination. In another embodiment, the present invention provides engineered cytotoxic cells capable of expressing binding proteins and CAR or TCR.

CAR or TCR lines are specific for tumor-specific antigen (TSA) or tumor-associated antigen (TAA) present on tumor cells. Tumor antigens are well known in the art. Non-limiting examples of TSA or TAA include the following: differentiation antigens, such as MART-1 / MelanA (MART-1), gp100 (Pmet 17), tyrosinase, TRP-1, TRP-2, and tumor-specific multi-lineage antigens , Such as MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15; overexpressed embryonic antigens, such as CEA; overexpressed oncogenes and mutated tumor suppressor genes, such as p53, Ras, HER- 2 / neu; unique tumor antigens caused by chromosomal translocations; such as BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR; and viral antigens, such as Epstein Barr virus Antigen EBVA and human papilloma virus (HPV) are resistant to E6 and E7. Other large protein-based antigens, including TSP-180, MAGE-4, MAGE-5, MAGE-6, RAGE, NY-ESO, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, beta-Catenin, CDK4, Mum-1, p15, p16, 43-9F, 5T4, 791Tgp72, alpha fetal protein, beta-HCG, BCA225 , BTAA, CA 125, CA 15-3 \ CA27.29 \ 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-related protein, TAAL6, TAG72, TLP and TPS . In one embodiment, the cytotoxic cell is a human cell and CAR or TCR is a human antigen.

When faced with combination therapy, the active agents can be administered simultaneously, separately, or sequentially in one or more pharmaceutical compositions.

Pharmaceutical composition

The binding protein of the present invention is conveniently presented and used in the form of a pharmaceutical formulation, and therefore a pharmaceutical formulation comprising the binding protein and a pharmaceutically acceptable excipient includes another aspect of the present invention.

The pharmaceutical composition can be administered to the patient by any convenient route. Specific pharmaceutical compositions are those suitable for intravenous or subcutaneous injection. Pharmaceutical compositions suitable for intravenous or subcutaneous administration include aqueous and non-aqueous sterile injectable solutions (which may contain antioxidants, buffers, bacteriostatic agents, and solutes that make the composition isotonic with the blood of the intended recipient) and aqueous and Non-aqueous sterile suspension (which may include suspending agents and thickening agents). The composition can be presented in unit-dose or multi-dose containers, such as sealed ampoules and vials, and can be stored in a freeze-dried (lyophilized) state, which requires only the addition of a sterile liquid carrier, such as water for injection, immediately before use. Extemporaneous injection solutions and suspensions can be prepared from sterile powders, granules and lozenges.

In one embodiment, the pharmaceutical composition is administered via intravenous injection every two weeks or monthly. In other embodiments, the pharmaceutical composition is administered via subcutaneous injection every two weeks or monthly.

The present invention also provides engineered human cytotoxic cells (e.g., naturally or non-naturally produced T cells, natural killer (NK) cells, or cytotoxic T cells or cells that can express the binding proteins described herein and human CAR or human TCR NK cell line). In one aspect, the present invention provides a method of treating cancer in a human patient. The method includes administering an effective amount of the pharmaceutical composition to the patient.

Manufacturing

The invention also provides an isolated nucleic acid encoding this binding protein. As for the binding protein of formula (I), it is a polynucleotide sequence encoding an amino acid sequence represented by HAV _H (PD-1) and L. With respect to the binding protein of formula (II), it is a polynucleotide sequence encoding an amino acid sequence represented by H (CPI) -AV _H (PD-1) and L (CPI). For other binding proteins, these sequences will be different (for example, based on the intention of one or more epitope binding regions), but those skilled in the art will be able to easily identify them. It should be understood that these polynucleotide sequences can be cloned into a vector. In one embodiment, such polynucleotides can be cloned into expression vectors.

Expression vectors can be manufactured by placing polynucleotide coding sequences operatively linked to conventional regulatory control sequences that can control replication and expression and / or secretion from the host cell. Regulatory sequences include promoter sequences, such as the CMV promoter and signal sequences that can be derived from other known antibodies. In some embodiments, each polynucleotide line is cloned into a separate expression vector. In a specific embodiment, the expression vectors are the same, except for the coding sequence and selectable markers. The use of different selectable marker systems ensures that, whenever possible, each peptide chain is functionally functional. Alternatively, all coding sequences can reside on a single vector, such as individual expression cassettes in the same vector.

The selected host cells are co-transfected by conventional techniques to create all expression vectors required for the host cells transfected by the present invention. The present invention therefore provides host cells capable of expressing the binding proteins of the present invention. In one embodiment, the present invention provides a transfected host cell that includes an expression vector, and the expression vector includes one or more polynucleotides encoding the binding proteins described herein.

Host cells or cell lines suitable for expressing the binding protein of the present invention include mammalian cells such as NS0, Sp2 / 0, CHO (eg DG44), COS, HEK, fibroblasts (eg 3T3) and myeloma cells, such as Expressed in CHO or myeloma cells. Human cells can be used so that this molecule can be modified in a human glycosylation pattern. Alternatively, other prokaryotic cell lines can be used. The selection of suitable mammalian host cells and methods for transformation, culture, amplification, screening, and product manufacturing and purification are known in the art.

Bacterial cells may prove effective as host cells suitable for expressing the binding protein of the present invention (see, for example, Plückthun, A., Immunol. Rev., 130: 151-188 (1992)). However, because proteins expressed in bacterial cells tend to be unfolded or improperly folded or unglycosylated, any binding protein produced in the bacterial cell must be screened for retention of biological activity. If the molecule expressed by the bacterial cell is produced in a properly folded form, the bacterial cell should be the desired host, or in alternative embodiments the molecule can be expressed in the bacterial host and then subsequently folded. For example, various E. coli strains used for expression are host cells well known in the field of biotechnology. Various B. subtilis, Streptomyces, other Bacillus strains, etc. can also be used in this method.

When desired, yeast cell strains known to those skilled in the art are also available host cells, as well as insect cells, such as Drosophila and Lepidoptera and virus expression systems. See, for example, Miller et al., Genetic Engineering, 8: 277-298, Plenum Press (1986) and references therein.

The present invention further provides a method for producing any of the binding proteins described herein, the method comprising culturing the host cells described herein and recovering the produced binding protein. Typically, the culture method of the present invention is a serum-free culture method, usually by culturing cells by serum-free suspension. Similarly, once produced, the antigen-binding structure of the present invention can be purified from cell culture contents according to standard procedures of this technology, including ammonium sulfide precipitation, affinity column, column chromatography, gel electrophoresis, and the like. These techniques are well known in the art.

The present invention provides a method for manufacturing engineered cytotoxic cells capable of co-expressing the binding protein and CAR or TCR described in the article. The method includes introducing all expression vectors required for expressing the binding protein and CAR or TCR into cytotoxic cells (For example, natural or non-naturally produced T cells, natural killer (NK) cells or cytotoxic T cells or NK cell strains). In one embodiment, the one or more expression vectors are viral vectors selected from the group consisting of adeno-associated viral vectors, adenoviral vectors, lentiviral vectors, and retroviral vectors.

Other embodiments

Specific examples of binding proteins specific for human PD-1 are stated in the following numbered examples:

Example 1. A binding protein specific for human PD-1, which comprises one or more CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: as shown in SEQ ID NO: 3 CDRH1 and CDRH1 different from CDRH1 shown in SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids, wherein CDRH2 is selected from the group consisting of: as SEQ ID NO: 3 The shown CDRH2 and the CDRH2 different from the CDRH2 shown in SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids, and the CDRH3 among them is selected from the group consisting of: as SEQ CDRH3 shown in ID NO: 3 and CDRH3 different from the CDRH3 shown in SEQ ID NO: 3 by adding or deleting or substituting 1, 2 or 3 amino acids.

Example 2. The binding protein according to Example 1, which is a single variable region.

Embodiment 3. The binding protein according to embodiment 2, wherein CDRH1 is THYMX ₄ , wherein X ₄ is V or A; wherein CDRH2 is FIGPAGDX ₅ TYYADSVX ₆ G, where X ₅ is T, F or S and X ₆ is K or E; and CDRH3 is YTX ₇ TSX ₈ X ₉ DX ₁₀ YDV, where X ₇ is A or E, X ₈ is G, S or D, X ₉ is V, F or Y, and X ₁₀ is T or S.

Embodiment 4. The binding protein according to embodiment 3, wherein CDRH1, CDRH2 and CDRH3 are shown in SEQ ID NO.3.

Embodiment 5. The binding protein according to embodiment 4, wherein CDRH1 has the sequence defined by SEQ ID NO: 10, CDRH2 has the sequence defined by SEQ ID NO: 11, and CDRH3 has the sequence defined by SEQ ID NO: 12. Of sequence.

Embodiment 6. The binding protein according to any one of embodiments 2 to 5, which comprises the sequence as defined in SEQ ID NO. 3 or SEQ ID NO with up to 10 amino acid addition, deletion or substitution differences. 3 variants.

Embodiment 7. The binding protein according to embodiment 6, wherein up to 10 amino acid additions, deletions or substitutions are not within the CDR region.

Embodiment 8. The binding protein according to any one of embodiments 2 to 5, which comprises a sequence as defined in SEQ ID NO. 3 or a sequence having 90% sequence identity with the sequence of SEQ ID NO. 3.

Embodiment 9. The binding protein according to embodiment 8, wherein the variation occurs outside the CDR region.

Embodiment 10. The binding protein according to any one of embodiments 2 to 9, which comprises the sequence as defined in SEQ ID NO.3.

Embodiment 11. The binding protein according to any of the preceding embodiments, which has an IC50 of less than or equal to 5 nM in the PD-1 / PDL-1 competition analysis.

Embodiment 12. The binding protein according to any of the preceding embodiments, which includes a region specific for human PD-1 by a linker and one or more specific for human checkpoint inhibitors other than PD-1 The area is connected.

Embodiment 13. The binding protein according to embodiment 12, wherein the binding protein includes a region specific for human PD-1 by a linker and specific for human checkpoint inhibitors other than PD-1 The C-terminus of the antibody heavy chain is connected.

Embodiment 14. The binding protein according to embodiment 13, wherein there are two regions specific for human PD-1, and one region is two-fold each of antibodies specific for human checkpoint inhibitors other than PD-1 The C end of the chain is connected.

Embodiment 15. The binding protein according to any one of embodiments 12 to 14, which can neutralize human checkpoint inhibitors other than the PD-1.

Example 16. A binding protein having the general formula (II): Among them, H (CPI) is the heavy chain of IgG antibody and L (CPI) is the light chain of IgG antibody, which makes H (CPI) and L (CPI) together form a specificity for human checkpoint inhibitors other than PD-1 Antibody; n is an integer selected from 2, 4, and 11; A is a bond or a peptide linker; V _H (PD-1) is an antibody heavy chain variable region having CDRH1, CDRH2, and CDRH3, wherein The CDRH1 is selected from: CDRH1 shown in SEQ ID NO: 3, and CDRH1 different from CDRH1 shown in SEQ ID NO: 3 by adding or deleting or substituting 1, 2 or 3 amino acids; wherein CDRH2 It is selected from: CDRH2 shown in SEQ ID NO: 3 and CDRH2 different from CDRH2 shown in SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids; and CDRH3 is selected From: CDRH3 shown in SEQ ID NO: 3 and CDRH3 different from CDRH3 shown in SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids.

Embodiment 17. The binding protein according to embodiment 16, wherein CDRH1 of V _H (PD-1) is THYMX ₄ , where X ₄ is V or A; wherein CDRH2 of V _H (PD-1) is FIGPAGDX ₅ TYYADSVX ₆ G Where X ₅ is T, F or S and X ₆ is K or E; and CDRH3 of V _H (PD-1) is YTX ₇ TSX ₈ X ₉ DX ₁₀ YDV, where X ₇ is A or E and X ₈ is G, S or D, X ₉ is V, F or Y, and X ₁₀ is T or S.

Embodiment 18. The binding protein according to Embodiment 16 or Embodiment 17, wherein V _H (PD-1) has CDRH1, CDRH2 and CDRH3 as shown in SEQ ID NO.3.

Embodiment 19. The binding protein according to embodiment 18, wherein CDRH1 has the sequence defined by SEQ ID NO: 10, CDRH2 has the sequence defined by SEQ ID NO: 11, and CDRH3 has the sequence defined by SEQ ID NO: 12. Of sequence.

Embodiment 20. The binding protein according to any one of embodiments 16 to 19, wherein V _H (PD-1) comprises the sequence as defined in SEQ ID NO. 3 or has up to 10 amino acid additions, deletions or A variant of SEQ ID NO. 3 that replaces the difference.

Embodiment 21. The binding protein according to embodiment 20, wherein up to 10 amino acid additions, deletions or substitutions are not within the CDR region.

Embodiment 22. The binding protein according to any one of embodiments 16 to 21, wherein V _H (PD-1) comprises the sequence as defined in SEQ ID NO. 3 or has a 90% sequence from the sequence of SEQ ID NO. 3 Sequence of the same degree.

Embodiment 23. The binding protein according to embodiment 22, wherein the variation occurs outside the CDR region.

Embodiment 24. The binding protein according to any one of embodiments 16 to 21, wherein V _H (PD-1) comprises the sequence as defined in SEQ ID NO. 3.

Embodiment 25. The binding protein according to any one of embodiments 16 to 21, wherein the linker or A is a peptide linker.

Embodiment 26. The binding protein according to embodiment 25, wherein the linker or A line has the sequence of SEQ ID NO.30.

Embodiment 27. The binding protein according to any one of embodiments 16 to 26, which has an IC50 of less than or equal to 5 nM in PD-1 / PDL-1 competition analysis and exhibits human examinations other than neutralizing PD-1 Point inhibitor.

Embodiment 28. An isolated nucleic acid encoding a binding protein as defined in any one of embodiments 1 to 15.

Embodiment 29. An isolated nucleic acid encoding H (CPI) -AV _H (PD-1), wherein H (CPI), A and V _H (PD-1) are as described in any one of embodiments 16 to 27 definition.

Embodiment 30. An isolated nucleic acid encoding L (CPI), wherein L (CPI) is as defined in any one of embodiments 16 to 27.

Embodiment 31. A vector comprising the nucleic acid as defined in Embodiment 28, 29 or 30.

Embodiment 32. The carrier according to embodiment 31, which is a performance carrier.

Embodiment 33. A host cell comprising the vector according to Embodiment 32.

Embodiment 34. A method of manufacturing a binding protein as defined in any one of Embodiments 1 to 26, which comprises culturing the host cell according to Embodiment 33 and isolating the binding protein under conditions suitable for protein expression, wherein the host cell One or more expression vectors contain the nucleic acids defined in Example 28 or contain the nucleic acids defined in Examples 29 and 30.

Embodiment 35. A pharmaceutical composition comprising the binding protein according to any one of embodiments 1 to 16 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. The method for treating an infectious disease according to embodiment 37, wherein the infectious disease is bacterial infection, parasitic infection, 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. The method of treating HIV according to embodiment 39, further comprising administering one or more antiretroviral 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. The method of curing HIV according to Embodiment 41, further comprising administering one or more antiretroviral agents.

Embodiment 43. The binding protein as defined in any one of Embodiments 1 to 26 is used as medicine.

Embodiment 44. The binding protein as defined in any one of Embodiments 1 to 26 is for the treatment of cancer.

Embodiment 45. The binding protein as defined in any one of Embodiments 1 to 26 is for the treatment of infectious diseases.

Embodiment 46. The binding protein used according to embodiment 45, wherein the infectious disease is bacterial infection, parasitic infection, viral infection, or sepsis.

Embodiment 47. The binding protein as defined in any one of embodiments 1 to 26 is for the treatment of HIV.

Embodiment 48. The binding protein as defined in any one of embodiments 1 to 26 is used to cure HIV.

Embodiment 49. The binding protein and one or more antiretroviral agents as defined in any one of Embodiments 1 to 26 are for individual simultaneous or sequential use for the treatment of HIV.

Embodiment 50. The binding protein and one or more antiretroviral agents as defined in any one of Embodiments 1 to 26 are for individual simultaneous or sequential use to cure HIV.

Embodiment 51. The use of a binding protein as defined in any one of Embodiments 1 to 26 for the manufacture of medicines for the treatment of cancer.

Embodiment 52. The use of a binding protein as defined in any one of Embodiments 1 to 26 for the manufacture of pharmaceutical products for the treatment of infectious diseases.

Embodiment 53. Use of the binding protein according to embodiment 52, wherein the infectious disease is bacterial infection, parasitic infection, viral infection or sepsis.

Embodiment 54. The use of a binding protein as defined in any one of Embodiments 1 to 26 for the manufacture of pharmaceutical products for the treatment of HIV.

Embodiment 55. The use of a binding protein as defined in any one of Embodiments 1 to 26 for the manufacture of medicines for the cure of HIV.

Embodiment 56. The use of a binding protein as defined in any one of Embodiments 1 to 26 and one or more antiretroviral agents for the manufacture of pharmaceutical products for the treatment of HIV simultaneously or sequentially.

Embodiment 57. The use of a binding protein as defined in any one of embodiments 1 to 26 and one or more antiretroviral agents for the manufacture of pharmaceutical products for individual or simultaneous use to cure HIV.

Specific examples of binding proteins specific for human LAG-3 are stated in the following numbered examples:

Example 1. A binding protein specific for human LAG-3, which comprises: one or more CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: as shown in SEQ ID NO: 1 CDRH1 and CDRH1 different from CDRH1 shown in SEQ ID NO: 1 by adding or deleting or replacing 1, 2 or 3 amino acids, wherein CDRH2 is selected from the group consisting of: as SEQ ID NO: CDRH2 shown in 1 and CDRH2 different from CDRH2 shown in SEQ ID NO: 1 by adding or deleting or replacing 1, 2 or 3 amino acids, and CDRH3 among them are selected from the group consisting of: CDRH3 shown in SEQ ID NO: 1 and CDRH3 different from CDRH3 shown in SEQ ID NO: 1 by adding or deleting or replacing 1, 2 or 3 amino acids; and one or more CDRL1, CDRL2 and CDRL3, where CDRL1 is selected from the group consisting of: CDRL1 as shown in SEQ ID NO: 2 and as shown in SEQ ID NO: 2 by adding or deleting or replacing 1, 2 or 3 amino acids CDRL1 is different from CDRL1, where CDRL2 is selected from the group consisting of: CDRL2 as shown in SEQ ID NO: 2 and by adding or deleting or The CDRL2 which is different from the CDRL2 shown in SEQ ID NO: 2 by substituting 1, 2 or 3 amino acids, and the CDRL3 is selected from the group consisting of: CDRL3 shown in SEQ ID NO: 2 and by Add or delete or replace 1, 2 or 3 amino acids and CDRL3 different from CDRL3 shown in SEQ ID NO: 2.

Embodiment 2. The binding protein according to embodiment 1, which includes: CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: CDRH1 as shown in SEQ ID NO: 1 and by adding or deleting or Substitute 1, 2, or 3 amino acids for CDRH1 different from CDRH1 shown in SEQ ID NO: 1, where CDRH2 is selected from the group consisting of: CDRH2 shown in SEQ ID NO: 1 and by adding Or delete or replace 1, 2 or 3 amino acids and CDRH2 different from CDRH2 shown in SEQ ID NO: 1, and CDRH3 is selected from the group consisting of: CDRH3 shown in SEQ ID NO: 1 And CDRH3 different from CDRH3 shown in SEQ ID NO: 1 by adding or deleting or replacing 1, 2 or 3 amino acids; and the anti-body system specific to human LAG-3 includes CDRL1 and CDRL2, CDRL1 CDRL1 is selected from the group consisting of: CDRL1 as shown in SEQ ID NO: 2 and different from CDRL1 as shown in SEQ ID NO: 2 by adding or deleting or replacing 1, 2 or 3 amino acids CDRL1 and CDRL2 are selected from the group consisting of: CDRL2 as shown in SEQ ID NO: 2 and by adding or In addition, two or three or a substituted amino acids with the SEQ ID NO: 2 shown in the different CDRL2 CDRL2.

Embodiment 3. The binding protein according to embodiment 1 or paragraph 2, which is an antibody.

Embodiment 4. The binding protein according to embodiment 3, wherein the antibody specific for human LAG-3 is an IgA or IgG antibody.

Embodiment 5. The binding protein according to embodiment 4, wherein the antibody specific for human LAG-3 is an IgG antibody.

Example 6. The binding protein according to any of the preceding examples, which has> 50% inhibition of LAG3-MHCII interaction in competitive flow cytometry analysis.

Embodiment 7. The binding protein as defined in any one of embodiments 3 to 6, wherein one or more regions specific for human checkpoint inhibitors other than LAG-3 are linked to humans by a linker The LAG-3 specific antibody is linked to the C-terminus of the heavy chain.

Example 8. The binding protein according to Example 7, wherein there are two regions specific for human checkpoint inhibitors other than LAG-3, one region and two each of the antibody specific for LAG-3 The C end of the chain is connected.

Example 9. The binding protein according to Example 7 or Example 8, which can neutralize human checkpoint inhibitors other than the LAG-3.

Example 10. A binding protein having the general formula (III): Wherein: H (LAG-3) is an IgG antibody heavy chain including CDRH1, CDRH2 and CDRH3, wherein the CDRH1 is selected from: CDRH1 shown in SEQ ID NO: 1, and by adding or deleting or replacing 1, 2 or 3 amino acids and CDRH1 different from CDRH1 shown in SEQ ID NO: 1; wherein CDRH2 is selected from: CDRH2 shown in SEQ ID NO: 1 and by adding or deleting or replacing 1, 2 or 3 Amino acids and CDRH2 different from CDRH2 shown in SEQ ID NO: 1; and CDRH3 is selected from: CDRH3 shown in SEQ ID NO: 1 and by adding, deleting or replacing 1, 2 or 3 Amino acid and CDRH3 different from CDRH3 shown in SEQ ID NO: 1; L (LAG-3) is an IgG antibody light chain including CDRL1 and CDRL2, wherein the CDRL1 is selected from: SEQ ID NO: 2 CDRL1 shown, and CDRL1 different from CDRL1 shown in SEQ ID NO: 2 by adding or deleting or replacing 1, 2 or 3 amino acids; and CDRL2 is selected from: SEQ ID NO: 2 CDRL2 and CDRL2 different from CDRL2 shown in SEQ ID NO: 2 by adding or deleting or substituting 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 capable Areas that bind to human checkpoint inhibitors other than LAG-3.

Embodiment 11. The binding protein according to any of the preceding embodiments, which includes CDRH1, CDRH2 and CDRH3 as shown in SEQ ID NO.1.

Embodiment 12. The binding protein according to any of the preceding embodiments, which includes CDRL1 and CDRL2 as shown in SEQ ID NO.2.

Example 13. The binding protein according to paragraph 12, which comprises CDRL1, CDRL2 and CDRL3 as shown in SEQ ID NO.2.

Embodiment 14. The binding protein according to paragraph 11, wherein CDRH1 has the sequence defined by SEQ ID NO: 4, CDRH2 has the sequence defined by SEQ ID NO: 5, and CDRH3 has the sequence defined by SEQ ID NO: 6 The defined sequence.

Embodiment 15. The binding protein according to embodiment 13, wherein CDRL1 has the sequence defined by SEQ ID NO: 7, CDRL2 has the sequence defined by SEQ ID NO: 8 and CDRL3 has the sequence defined by SEQ ID NO: 9 The defined sequence.

Embodiment 16. The binding protein according to any one of embodiments 3 to 15, wherein the heavy chain or H line of the antibody specific for human LAG-3 includes the sequence as defined in SEQ ID NO. 1, or has the highest 10 amino acid additions, deletions, or substitutions of variants of SEQ ID NO. 1 that differ.

Embodiment 17. The binding protein according to embodiment 16, wherein up to 10 amino acid additions, deletions or substitutions are not within the CDR region.

Embodiment 18. The binding protein according to any one of embodiments 3 to 17, wherein the light chain or L line of the antibody specific for human LAG-3 includes the sequence as defined in SEQ ID NO. 2 or has up to 10 Amino acid additions, deletions, or substitutions of variants of SEQ ID NO. 2 that differ.

Embodiment 19. The binding protein according to embodiment 18, wherein up to 10 amino acid additions, deletions or substitutions are not within the CDR region.

Embodiment 20. The binding protein according to any one of embodiments 3 to 15, wherein the heavy chain or H line of the antibody specific for human LAG-3 includes the sequence as defined in SEQ ID NO. 1 or the SEQ ID NO.1 sequence has 90% sequence identity.

Embodiment 21. The binding protein according to embodiment 20, wherein the variation occurs outside the CDR region.

Embodiment 22. The binding protein according to any one of embodiments 3 to 15 or 20 to 21, wherein the light chain or L of the antibody specific for human LAG-3 includes the sequence as defined in SEQ ID NO. 2. Or a sequence having 90% sequence identity with the sequence of SEQ ID NO.2.

Embodiment 23. The binding protein according to embodiment 22, wherein the variation occurs outside the CDR region.

Embodiment 24. The binding protein according to any one of paragraphs 3 to 23, wherein the heavy chain or H line of the antibody specific for human LAG-3 includes the sequence as defined in SEQ ID NO.1.

Embodiment 25. The binding protein according to any one of embodiments 3 to 24, wherein the light chain or L line of the antibody specific for human LAG-3 includes the sequence as defined in SEQ ID NO.2.

Embodiment 26. The binding protein according to any one of embodiments 7 to 25, wherein the linker or A is a peptide linker.

Embodiment 27. The binding protein according to embodiment 26, wherein the linker or A has the sequence of SEQ ID NO.30.

Embodiment 28. The binding protein according to any one of embodiments 10 to 27, which exhibits> 50% inhibition of LAG3-MHCII interaction in competitive flow cytometry analysis, and neutralization of human examinations other than LAG-3 Point inhibitor.

Embodiment 29. An isolated nucleic acid encoding a binding protein as defined in Embodiment 1 or Embodiment 2.

Embodiment 30. An isolated nucleic acid encoding the heavy chain of the binding protein as defined in any one of embodiments 3 to 9.

Embodiment 31. An isolated nucleic acid that encodes the light chain of the binding protein as defined in any one of embodiments 3 to 9.

Embodiment 32. An isolated nucleic acid encoding HA-CPI, wherein H, A, and V _H (PD-1) 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 the nucleic acid as defined in any one of embodiments 29 to 33.

Embodiment 35. The carrier according to embodiment 34, which is a performance carrier.

Embodiment 36. A host cell comprising the vector according to embodiment 35.

Embodiment 37. A method of manufacturing a binding protein as defined in Embodiment 1 or Embodiment 2, which includes culturing the host cell according to Embodiment 36 under conditions suitable for protein expression, and isolating the binding protein, wherein the host cell The nucleic acid defined in Example 29 is contained in one or more expression vectors.

Embodiment 38. A method of manufacturing a binding protein as defined in any one of Embodiments 3 to 9, which comprises culturing the host cell according to Embodiment 36 under conditions suitable for protein expression, and isolating the binding protein, wherein the host The cell contains the nucleic acids defined in Example 30 and Example 31 in one or more expression vectors.

Embodiment 39. A method of manufacturing a binding protein as defined in any one of Embodiments 10 to 28, comprising culturing the host cell according to Embodiment 36 under conditions suitable for protein expression, and isolating the binding protein, wherein the host The cell contains the nucleic acids defined in paragraphs 32 and 33 in one or more expression vectors.

Embodiment 40. A pharmaceutical composition comprising the binding protein according to any one of embodiments 1 to 28 and a pharmaceutically acceptable excipient.

Embodiment 41. A method of treating cancer comprising 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. The method of treating infectious diseases according to embodiment 42, wherein the infectious diseases are bacterial infections, parasitic infections, viral infections, 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. The method of treating HIV according to Embodiment 44, further comprising 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. The method of curing HIV according to Embodiment 46, further comprising administering one or more antiretroviral agents.

Embodiment 48. The binding protein as defined in any one of Embodiments 1 to 28 is used as a medicine.

Embodiment 49. The binding protein as defined in any one of Embodiments 1 to 28 is used to treat cancer.

Embodiment 50. The binding protein as defined in any one of Embodiments 1 to 28 is used for the treatment of infectious diseases.

Embodiment 51. The binding protein used according to embodiment 50, wherein the infectious disease is bacterial infection, parasitic infection, viral infection, or sepsis.

Embodiment 52. The binding protein as defined in any one of embodiments 1 to 28 is for the treatment of HIV.

Embodiment 53. The binding protein as defined in any one of Embodiments 1 to 28 is used to cure HIV.

Embodiment 54. A binding protein as defined in any one of Embodiments 1 to 28 and one or more antiretroviral agents for individual treatment of HIV simultaneously or sequentially.

Embodiment 55. A binding protein as defined in any one of Embodiments 1 to 28 and one or more antiretroviral agents for individual or simultaneous use to cure HIV.

Embodiment 56. The use of a binding protein as defined in any one of Embodiments 1 to 28 for the manufacture of medicines for the treatment of cancer.

Embodiment 57. The use of a binding protein as defined in any one of Embodiments 1 to 28 for the manufacture of pharmaceutical products for the treatment of infectious diseases.

Embodiment 58. Use of the binding protein according to embodiment 57, wherein the infectious disease is bacterial infection, parasitic infection, viral infection or sepsis.

Embodiment 59. The use of a binding protein as defined in any one of Embodiments 1 to 28 for the manufacture of pharmaceutical products for the treatment of HIV.

Embodiment 60. The use of a binding protein as defined in any one of Embodiments 1 to 28 for the manufacture of medicines for the cure of HIV.

Embodiment 61. The use of a binding protein as defined in any one of Embodiments 1 to 28 and one or more antiretroviral agents for the manufacture of pharmaceutical products for the treatment of HIV simultaneously or sequentially.

Embodiment 62. The use of a binding protein as defined in any one of Embodiments 1 to 28 and one or more antiretroviral agents for the manufacture of pharmaceutical products for individual or simultaneous use to cure HIV.

Examples Example 1: Identify a simple variable region that binds to PD-1

According to method 1, the epitope binding region (also called regional antibody or dAb) specific for PD-1 is identified by selecting a strain from a natural bacteriophage library, and the bacteriophage library presents individual VH The variable region (dAb) is fused with the bacteriophage coat protein III of the extracellular domain of anti-human PD-1 (hPD-1) via a c-myc tag as an antigen adsorbed on the immune test tube.

    Method 1: Passive bacteriophage selection    

Three rounds of selection were used to identify dAbs present in phage bound to hPD-1. Discard the non-specifically bound bacteriophage to display the dAb, pre-culture the bacteriophage bank with an empty immunotube in the first round, and coat the bacteriophage bank with glycated human serum albumin (Sigma A8301 in the second round ) Of the pre-cultivation of the immune test tube, no pre-cultivation in the third round. In all 3 rounds, the bacteriophage pool was then added to an immunoassay tube coated with hPD-1 antigen and incubated to allow the bacteriophage to bind to the antigen. After binding, unbound (and weakly bound) bacteriophages are washed away by washing several times. Then, trypsin (c-myc tag cleavage) is used to dissociate the remaining bacteriophage, thereby dissolving the bacteriophage. The dissociated bacteriophage is then used to infect E. coli and the infected cells are implanted on a selective medium, where the resulting culture of the selected strain is used to amplify the dissociated bacteriophage for selection in the next round.

After 3 rounds of selection, individual E.coli-infected E. coli selection strains were selected and grown overnight, and the supernatant containing the generated bacteriophage was screened by ELISA according to Method 2.

    Method 2: ELISA screening    

Add 50 μl hPD-1 (1 μg / ml) dissolved in PBS to the well of a 96-well Maxisorp ^TM immunoassay disk (Nunc, Denmark) and incubate the disk at 4 ° C overnight (using 2 mg / ml albumin glycation) HSA (Sigma A83Q1) as a control). The wells were washed with PBS and then blocked with 2% Marvel in PBS. A 1: 1 supernatant mixture containing a single strain of bacteriophage-dAb group and 2% Marvel in PBS was added to each well. The bound bacteriophage-dAb particles were detected with anti-M13 (bacteriophage coat protein) specific monoclonal antibody-HRP conjugate. A color matrix (SureBlue 1-active component TMB Microwell peroxidase solution) was added and the optical density (OD) was measured at 450 nm. If the OD450 (hPD-1 antigen) / OD450 (HSA) ratio is greater than 3, dAb-bacteriophage strains from 3 rounds of passive selection are considered as positive binders in ELISA analysis.

Sequence the positive clones. By splicing with overlap extension (SOE) PCR side fragments (5 'fragment-start-signal-anti-RSV VH-CH1-3-3' side fragment-stop codon-polyA), the unique selection The plants were recombined into anti-RSV (IgG1 Fc failed heavy chain) mAb-dAb fusions. The SOE PCR products were used for linear DNA transfection of mammalian HEK293 cells. Using hPD-1 / hPDL-1 ligand binding inhibition assay (Method 3), the supernatant of transfected cells was screened at a single concentration for the ability to inhibit the interaction between hPD1 and hPDL1 and SPR (surface plasmon resonance analysis) The selected plants were ranked by dissociation constant for hPD-1 binding.

Screened more than 2000 selected plants and 18 anti-RSV-PD1-mAb-dAb selected plants in hPD-1 / hPDL-1 ligand binding inhibition analysis to verify the combination of inhibition and SPR will be slowly dissociated to colonize Anti-RSV mAb heavy chain (IgG1 Fc failure) in pTT5 mammalian expression vector. Display anti-RSV-PD1-mAb-dAb molecule and purify mAb-dAb. The purified mAbdAb was used for hPD1-hPDL-1 ligand binding inhibition and steady-state kinetics SPR.

    Method 3: PD-1 / PDL-1 ligand binding inhibition analysis         Human PD-1 / PDL-1 ligand binding inhibition analysis    

This analysis includes serial dilutions (1: 3) of each tested mAb-dAb or positive control GRITS50150 (all maximum concentrations are 34 μg / ml). A test sample was prepared by mixing 30 μl of antibody (diluted as described above) with 30 μl 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. The positive control is prepared by replacing the test antibody only with buffer.

A Mosquito liquid dispensing console was used to coat a 96-well dish with 1 μl / well of 20 μg / ml human PD1.his in PBS. The analysis disk was sealed and incubated at 4 ° C overnight. The analysis dish was then cleaned using an automatic dish washer, then 200 μl of blocking buffer was added and incubated at room temperature with gentle shaking for 1 hour. Then use an automatic dishwashing machine to clean the analysis disk again, blot it dry and immediately add 25 μl of the test sample. The analysis disk was incubated for 2 hours under gentle shaking, and then washed with an automatic disk washing machine. Add 150 μl / well of 2X interpretation buffer T containing surfactant to each well and immediately interpret on MSD Sector Imager 6000.

The background signal is subtracted by subtracting the negative control average of each analysis disk. The% inhibition of the positive control was then determined using the following equation: 100-((data value / positive control) x 100). Graph Pad Prism is used to plot the data as the average of two repetitive wells, where the error line shows the SEM, using X = log [x] conversion and nonlinear regression "log (inhibitor) vs. reaction-variable slope ( Four-parameter fitting is used to determine IC ₅₀ (50% inhibitory concentration that causes the difference between the highest and lowest dose response curves). Please note that the MW of mAb-dAb is assumed to be 170,000 kDa. The MW of GRITS50150 is assumed to be 150,000 kDa.

    Rhesus monkey PD-1 / Crab-eating macaque PDL-1 ligand binding inhibition analysis    

This analysis includes serial dilutions (1: 3) of each tested mAb-dAb or positive control GRITS50150 (all maximum concentrations are 34 μg / ml). A test sample was prepared by mixing 30 μl of antibody (diluted as described above) and 30 μl of 2 μg / ml cynomolgus cynomolgus monkey PDL-1 Fc.flag-ST. Negative controls were prepared by substituting buffer for the test antibody and cynomolgus monkey PDL-1 Fc.flag-ST. The positive control is prepared by replacing the test antibody only with buffer.

A Mosquito liquid dispensing console was used to coat a 96-well dish with 1 μl / well of 20 μg / ml human PD1.his in PBS. The analysis disk was sealed and incubated at 4 ° C overnight. The analysis dish was then cleaned using an automatic dish washer, then 200 μl of blocking buffer was added and incubated at room temperature with gentle shaking for 1 hour. Then use an automatic dishwashing machine to clean the analysis disk again, blot it dry and immediately add 25 μl of the test sample. The analysis disk was incubated for 2 hours under gentle shaking, and then washed with an automatic disk washing machine. Add 150 μl / well of 2X interpretation buffer T containing surfactant to each well and immediately interpret on MSD Sector Imager 6000.

The background signal is subtracted by subtracting the negative control average of each analysis disk. The% inhibition of the positive control was then determined using the following equation: 100-((data value / positive control) x 100). Graph Pad Prism is used to plot the data as the average of two repetitive wells, where the error line shows the SEM, using X = log [x] conversion and nonlinear regression "log (inhibitor) vs. reaction-variable slope ( "Four-parameter" fitting is used to determine IC ₅₀ (the 50% inhibitory concentration that causes the difference between the highest and lowest dose response curves). Please note that the MW of mAb-dAb is assumed to be 170,000 kDa. The MW of GRITS50150 is assumed to be 150,000 kDa.

    Method 4: Surface Plasma Resonance (SPR)    

Using BIACORE ^™ 3000 or 4000, the binding kinetics of mAbdAb and the affinity for binding to PD-1 were evaluated. 2050 resonance units (RU) of biotinylated human PD-1 and 835RU biotinylated rhesus monkey PD1 were loaded onto streptavidin-coated Biacore wafers. A single concentration of the tested mAbdAb dissolved in the buffer was passed through the wafer and the binding curve was recorded (where a series of concentrations were used). This item is operated at 25 ° C to repeat the same Biacore operation. The curve is a double-reference curve using a buffer injection curve and then fitted with a 1: 1 combination model included in the Biacore Evaluation software.

    Method 5: Determination of mAb-dAb concentration in supernatant and analysis of mAb concentration after small-scale purification    

Use Octet Red 384 instrument and 8.0.2.5 version software, use basic wizard template 'Quantitation with regeneration to quantify samples. First, the protein A sensor tip was immersed in PBSF for 10 minutes before use. Then, ten-fold dilutions (15 μl of each sample and 135 μl of PBSF) were incubated with the sensor tip. Standard curve: It was generated by using a continuous double PBSF dilution of mAb-dAb standard from 100 μg / ml to 0.78 ug / ml. The sensor was reset with 10 mM glycine pH 1.5 between each sample. Then use version 8.0 data analysis software to analyze the data.

    Method 6: Determination of the concentration of purified mAb and mAb-dAb    

The concentration of the purified mAbdAb in the buffer was determined spectrophotometrically by measuring the absorbance of UV light at 280 nm using a Nanodrop 1000 instrument (Thermo Scientific).

51A09 strains that exhibited inhibitory effects in hPD1-hPDL-1 ligand binding inhibition analysis and exhibited slow dissociation with SPR were affinity matured to increase the efficacy of dAbs.

Example 2: Affinity is mature

Mutazyme ^™ DNA polymerase (Stratagene model 200550) was used to change the mutation rate of 3.5 amino acids per dAb (both framework and CDR regions) to diversify the dAb sequence of the selected strain 51A09. The diverse dAb gene pool was then cloned into pre-cut bacteriophage vector DNA (pDOM4), and then transformed into TG1 electroporated competent cells (C2987). Select diversified colonies as described in Method 7.

    Method 7: Soluble bacteriophage selection    

The bacteriophage library based on the diversified 51A09 selection strains and sufficient streptavidin-coated (or neutravidin) -coated magnetic beads are blocked separately to reduce the non-specific binding of bacteriophages during subsequent selection steps . Before each round of selection, the bacteriophage library was pre-cultured with unsupported microbeads as an additional step to reduce the amount of non-specific bacteriophage. In the first round, a diverse bacteriophage library based on 51A09 selection strains was incubated with 50 nM biotinylated hPD-1-His at room temperature for 1 hour. After incubation, the antigen-bacteriophage complex is captured onto the microbeads via the interaction of biotin and streptavidin (or neutravidin). Remove the microbeads from the solution by using a magnetic stand and repeat washing to remove non-specific bacteriophage and weak conjugates. The remaining bacteriophage was trypsinized and recovered. After transfection with E. coli, the selected bacteriophage will be selected for other rounds under the following conditions:

Round 2: 5nM hPD1 at room temperature for 15 minutes (+ 100nM glycated HSA)

Round 3: 0.5nM hPD1 at room temperature for 30 minutes

Round 4: 2 hours at 60 ° C, followed by 0.5 nM hPD1 at room temperature for 15 minutes (cultivation of this bacteriophage library at 60 ° C, followed by blocking and antigen binding steps to select colonies with good physiological properties ).

The bacteriophage DNA was isolated from the selection results in the fourth round. The dAb encoding gene library from the pDOM4 bacteriophage presentation vector was amplified with primers (pre-primer 5'-cccggaaagggatccacaggactggactccccgacagaggtgcagctgttggagtct-3 '(SEQ ID NO: 46); inverted primer 3'-ggggatctagaattcatcagctcgagacggtgaccagggtt 47)) to generate a fragment suitable for In-Fusion colonization to the mammalian expression vector pTT5 containing the anti-RSV mAb heavy chain (IgG1 Fc failure). To help colonization, a BamH1 position 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 to the PD-1 dAb. The pTT5 anti-RSV vector was linearized with BamH1 and EcoR1 and an In Fusion reaction was performed using the In-Fusion HD selection kit (Clontech) according to the manufacturer's instructions with purified PCR products from the selected selection strain. The In Fusion reaction was transformed into NEB 5-α competent E. coli (high efficiency; model C2987) cells. The DNA isolated from the transformed individual clones was sequenced to identify unique clones and provide materials for small-scale transfection and performance in HEK293 cells. The supernatant of transfected cells was screened using hPD1-hPDL-1 ligand binding inhibition assay (Method 3). Then select a group of selected strains based on their potency and sequence for repeated transfection and purification, and use Jurkat PD1 + analysis (Method 8) for utility screening.

    Method 8: PD1 + Jurkat analysis    

The PDL1 + CHO cells containing the NFAT driver reporter gene and PD1 + Jurkat cells were mixed in the presence or absence of the tested mAbdAb. The mAbdAb was tested for its ability to block gene expression from reporters using the following method: basically according to the method disclosed by Promega Corporation (CS187109), using Promega Jurkat NFAT-luc2 / PD-1 effector stimulated with CHO PD-L1 + cells T cells undergo functional PD-1 analysis. Briefly, 100 μl of 4x105 cells / m1 (40,000 cells / well) was dissolved in each well of a 96-well plate in 10% FBS, 0.25 mg / ml G418 and 0.2 mg / ml hygromycin B ( CHO PDL1 + cells of F-12 nutrient mixture (HAM) of hygromycin B). Incubate at 37 ° C and 5% CO2 for 16h. Remove 95 μl of medium from each well, then add 40 μl of test antibody or control dissolved in RPMI medium containing HEPES plus 2% FBS and 40 μl of Jurkat NFAT-luc2 in RPMI medium containing HEPES plus 2% FBS / PD1 cells are 1.25x106 cells / ml (50,000 cells / well). The general anti-system is prepared as a 9-point dose curve with a 3-fold dilution step starting from 200 μg / ml. The analysis disk was incubated at 37 ° C and 5% CO2 for 6 hours, and then incubated at room temperature for 5 minutes. Then 80 μl of BioGlo ^™ reagent (prepared as described in the Bio-Glo Luciferase Assay System, Promega model G7940) was added to each well. After 5-10 minutes of incubation, the luminescence was measured on a disc reader. The original data value is divided by the control well without antibody, and the value obtained as "fold over no treatment" is called "fold induction". The nonlinear regression curve fitting software was used to determine the EC50 with the smallest asymptotic line approaching the value 1 (fitting method: least square (normal) fitting).

    Method 9: PD1 + / LAG3 + Jurkat cell binding analysis    

The binding of anti-LAG3 / PD-1 antibodies to cell surface expression LAG3 and PD-1 was evaluated using LAG3 expressing Jurkat cells purchased from Promega and PD-1 expressing Jurkat cells (CS194801 and CS187102 respectively). Promega cell lines were supplemented with antibodies supplemented with 1% FBS in PBS at concentrations ranging from 100 nM to 0.001 nM for 1 hour at 4 ° C, including the “antibody-free” control group. Wild-type Jurkat cells were incubated with 100 nM antibody at 4 ° C for 1 hour as a negative control. After incubation, the cells were washed three times with 1% FBS in PBS. Multiple strains of PE-linked anti-human IgG1 Fc F (ab ') 2 (Invitrogen, # H10104) were added to dilute 1/250 in 1% FBS in PBS to treat the cells. The cells were incubated in the dark at 4 ° C for 30 minutes. Wash once with 1% FBS in PBS and again with PBS. NIR dead cell stain (Invitrogen # L10119) diluted 1 to 1000 in PBS was added to the cells to select viable cells. The cells were incubated in the dark at 4 ° C for 30 minutes. The cells were washed once with PBS and suspended in 100 μl of PBS for measurement using MACSQuant flow cytometer (Miltenyi). Select 50μl absorption, fast mode and high flow rate to operate the flow cytometer. In addition to the forward and side scattered light, adjust the instrument settings for the appropriate voltage for PE fluorescence and NIR fluorescence. The valve is set to the NIR fluorescence scattering pattern (non-fluorescent cells) and 10,000 pieces are collected from the valve. The data is exported from MACSQuant and analyzed using FlowJo version 10 software. Collect the cells in the front scatter area (FSC) and the side scatter area (SSC), the FSC to NIR fluorescence selected by the live cells and the FSC height to the FSC area to guard the single cell population. The median PE fluorescence density (MFI) of these live single cells was measured with FlowJo and exported to Windows Excel. In Excel, the MFI value of each antibody treatment is divided by the MFI value of cells treated only with PE-linked anti-human IgG1 Fc F (ab ') 2 ("no antibody" control group). This MFI data was copied to GraphPad prism V 5.0.4 where the pM concentration was converted to a logarithmic value (log) and the data was plotted as an inverse curve dose response (variable slope) curve. The EC50 value is generated from a curve representing the half-maximal effective concentration (the concentration that triggers a half-response between baseline and maximum after a specific exposure).

After dividing by MFI from "PE-linked anti-human IgG1 Fc F (ab ') 2 Ab", all MFI data generated from Jurkat WT cells treated at the highest concentration of 100 nM are very close, which proves that these antibodies Cannot combine with wild-type cells.

    The result    

The series of specific selected strains showing IC50 <5nM in hPD1-hPDL-1 ligand binding inhibition analysis and showing cross-reactivity of rhesus monkeys (rhesus) on Biacore are as follows (Table 4):

The CDR sequence series of these strains are listed in Table 5.

Table 5 shows one amino acid substitution in CDRH1 (one position in CDRH1), two amino acid substitutions in CDRH2 (two positions in CDRH2), and four amino acids in CDRH3 (four positions in CDRH3) Position) dAbs with 2 amino acid substitutions showed IC50 <5nM in hPD1-hPDL-1 ligand inhibition analysis and rhesus monkey cross-reactivity.

Further comparison of these sequences identified the following common CDR sequences: CDRH1 of V _H (PD-1): THYMX ₄ (SEQ ID NO: 57), where X ₄ is V or A; CDRH2 of V _H (PD-1): FIGPAGDX ₅ TYYADSVX ₆ G (SEQ ID NO: 58), where X ₅ is T, F or S and X ₆ is K or E; CDRH3 of V _H (PD-1): YTX ₇ TSX ₈ X ₉ DX ₁₀ YDV ( SEQ ID NO: 59), wherein X ₇ is A or E, X ₈ is G, S or D, X ₉ is V, F or Y, and X ₁₀ is T or S.

Example 3: Further mutation

51A09-188 is an example of dAb identified after affinity maturation, which binds to human and rhesus monkey PD1. In order to reduce the impact of any pre-existing anti-drug antibodies, alanine residues were added to the C-terminus of the regional antibody, and the C-terminal amino acid sequence LVTVSSA (SEQ ID NO: 81), in which the reverse primer line was mutated. The mutated version was cloned into pTT5 anti-RSV heavy chain vector by In Fusion homologous recombination as described in Example 2. This selected strain is called 51A09-188001.

The purified mAbdAb was detected by PD1 + Jurkat function analysis to determine whether the C-terminal modification had any effect on dAb function.

In the hope of further enhancing affinity and efficacy in bioanalysis, mutations from other affinity enhancing selection strains in the 51A09 lineage were introduced into the 51A09-188001 sequence. Introduce mutations by targeting mutations 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 mutant version was cloned into pTT5 anti-RSV heavy chain vector by In Fusion homologous recombination as described in Example 2. The mAbdAb protein was purified and analyzed by PD1 + Jurkat functional analysis.

    The result    

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

A yeast-based platform was used for three rounds of natural antibody selection to generate anti-hLAG3 binding molecules. 100 nM human LAG3-His (hLAG3-His) was cultured in a natural yeast antibody library and the primary initial enrichment was performed by magnetic bead separation to select the results. This is followed by a round of FACS selection, whereby gating and cell sorting are used to isolate the yeast cell population that can bind to 100 nM hLAG3-His. Then, the antibody heavy chains from the separated binding group are reconfigured in different light chain libraries and selected for several rounds to optimize the pairing of the heavy chain and light chain of the selected antibody. Then reconfigure the heavy chain and light chain to enrich the group. 50nM hLAG3-His was used for one round of Zengfeng magnetic bead selection, followed by 50nM cynomolgus monkey LAG3-His for FACS selection. This produces a pure binding group that binds to both human and rhesus monkey LAG3-His. The binding group is then mixed with a non-specific reagent and an unrelated antigen with a 6-His tag for negative FACS selection, and then the generated result is subjected to affinity pressurization by binding to 0.5 nM hLAG3-His. The final selection results are produced by gated and sorted cells in FACS selection, and individual selections are isolated by implantation of selective media. The isolated colonies were sequenced and expressed unique mAbs and tested for cells that bound to soluble antibodies and expressed hLAG3.

With more than 350 selected plants, the following methods were tested and 82 selected plants met the following criteria:

˙KD <1nM of SPR (surface plasmon resonance; method 9) of human and macaque LAG-3

˙Inhibition of LAG3-MHCII interaction (Method 10)> 50% in competitive flow cytometry analysis

˙Combined with LAG3 positive cells> 2 times over background in cell-bound flow cytometry analysis (Method 11)

˙Combined with LAG3 negative cells in cell-bound flow cytometry analysis (Method 11) exceeds background by less than 2 times

˙SEC analysis 65% monolayer has <16.5 residence time or> 80% monolayer and residence time> 16.5 (Method 12)

˙Functional LAG-3 analysis (Method 13) EC50 <5μg / ml

˙ (DNA) sequencing did not identify CDR cysteine

Method 10: Surface plasmon resonance

Using BIACORE ^™ 4000, the binding kinetics of the antibody and the affinity for binding to LAG-3 were evaluated. The LAG-3 antibody was captured to 1 to 5 of each flow cytometer of protein A coated with CM5 series S sensor wafer. The antibodies of 50 nM, 12.5 nM, 3.125 nM and 0.78 nM (blank = 0 nM) were flowed through LAG-3 of human or cynomolgus monkey at 37 ° C. Use Biacore 4000 data analysis software version 1.0, apply 1: 1 combined model to analyze data. With some curves, kinetics may not be determined, but only steady-state models may be used to determine affinity. For some antibodies, a longer dissociation time is required in order to accurately determine the kd (detachment rate), because a 10% decrease in reaction is not observed during the 10-minute dissociation time. For some strains (typically those with an affinity of about 100 pM or less), a lower concentration range should be required for accurate determination of kinetics and affinity, as higher concentrations may be used in this experiment Can't get a good fit.

Method 11: Competitive flow cytometry analysis

1. Dissolve 100 μl of Daudi cell suspension (Biocat, model 117942) at 1x106 cells / ml in analysis buffer (prepared by mixing Dulbecco's PBS 10: 1 with fetal bovine serum) in 96-well V -In the hole of the chassis.

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

3. Resuspend the cells in 25 μl of analysis buffer containing soluble biotinylated human LAG-3.Fc.

4. Add 25μl containing control antibody 11E3 (Caltag / Abioscience, model AG-20B-0011-C100), control antibody 17B4 (GSK) or control antibody 6D511 (US Biological), or test antibody (all antibody concentration ranges) From 60 pM to 1000 nM) analysis buffer.

5. Add 150 μl of analysis buffer to the well. Centrifuge at 250-350g for 3 min at 4 ° C, then remove the supernatant.

6. Rinse the cells twice with 200 μl analysis buffer.

7. Resuspend the cells in 50 μl of streptavidin PE (Biolegend, model 45204) diluted 1: 200 in assay buffer, or 50 μl containing 0.5 μl I3-RD1 PE conjugated anti-MHCII (Beckman Coulter Inc., Model 6604366) in the analysis buffer.

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

9. Add 150 μl of PBS to the analysis well and centrifuge the analysis plate at 250-350 g at 4 ° C for 3 min, and then remove the supernatant. The cells were washed twice with Dulbecco's PBS (200 µl / well).

10. Resuspend the cell pellet in 50-100 µl Dulbecco's PBS.

11. Add equal volume (50-100 μl) of 2 μM Sytox Blue (Molecular Probes, model S11348) to each analysis well, and then use FACS Canto II (BD Biosciences) and FACSDiva 8.01 software to obtain the data.

12. Use unstained sample wells to determine appropriate flow cytometer parameters, and then apply the settings to all data samples. Sytox blue dead cell exclusion staining was used to identify live and dead cells.

Method 12: Analysis of cell combined with flow cytometry

1. Dissolve 100 μl at 1x106 cells / ml in analysis buffer (prepared by mixing Dulbecco's PBS 10: 1 with fetal bovine serum) to overexpress human LAG3 (LAG3 positive cells) or not express LAG3 (LAG3 negative cells ) The EL4 cell suspension was transferred to the well of a 96-well V-chassis.

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

3. Resuspend the cells in 50 μl concentration between 1-100 nM with or without control antibody 11E3 (Catag / Abioscience, model AG-20B-0011-C100), control antibody 17B4 (Enzo Life Sciences, model ALX-804 -806PF-C100) or test antibody in the analysis buffer. The analysis plate was incubated on ice for 30 minutes.

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

5. Wash the cells twice with 200 μl analysis buffer.

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

7. Resuspend cells in 50 μl of detection antibody or separate analysis buffer.

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

9. Add 150 μl of PBS to the analysis well and centrifuge the analysis plate at 250-350 g at 4 ° C for 3 min, and then remove the supernatant. The cells were washed twice with Dulbecco's PBS (200 µl / well).

10. Resuspend the cell pellet in 50 µl Dulbecco's PBS.

11. Add 50 μl of 2 μM Sytox Blue (Molecular Probes, model S11348) to each analysis well, and then use FACS Canto II (BD Biosciences) and FACSDiva 8.01 software to obtain the data.

12. Use unstained sample wells to determine appropriate flow cytometer parameters, and then apply the settings to all data samples. Sytox blue dead cell exclusion staining was used to identify live and dead cells.

Method 13: Open access to analytical size exclusion chromatography (aSEC)

Open access analytical size exclusion chromatography was performed on the Agilent 1100 (including degasser, Quat pump, autosampler ALS, column chamber, and DAD detector) using TSK G300SWXL columns at ambient temperature. The column was equilibrated with buffer (200 mM sodium dihydrogen phosphate, 250 mM NaCl, pH 6.0), and then 20 μg of test antibody was loaded. Samples were operated with standard SEC method using equilibration buffer at a flow rate of 0.5 ml / min. The dissolved protein was detected by monitoring the absorbance at 214nm. The percentage of monomers and aggregates was determined with Chemstation using peak integration.

Method 14: Functional LAG-3 analysis

Basically, according to the method disclosed by Promega Corporation (CS194801), functional LAG-3 analysis was performed using Promega LAG-3 + Jurkat / NFAT-luc effector T cells stimulated with Raji cells and SEE. Briefly, add to each well of a 96-well plate: 25μl of anti-LAG-3 antibody ten-point dose curve starting from 25μg / ml (or control antibody dose curve) 25μl dissolved in analysis vehicle (containing 1% FBS RMPI 1640 vehicle) LAG 3-effector cells (4x10 ⁶ ; CS194801) 25 μl of 1: 1 Raji cells / SEE (Raji cells (2 x 10 ⁶ / ml; ATCC model CCL-86) dissolved in the analysis vehicle) Mixture and 300ng / ml Staphylococcal enterotoxin D, reducing endotoxin (Toxin Technology, Inc., model DT303red).

The analysis disk was incubated in a CO ₂ incubator at 37 ° C for 6 hours. It was then allowed to reach room temperature and 75 μl of BioGlo ^™ reagent (prepared as described by the Bio-Glo Luciferase Assay System, Promega model G7940) was added to each well. After 5-10 minutes of incubation, the luminescence was measured with a disc reader. The EC50 of antibody response was determined using a nonlinear regression curve fitting software (fitting method: least squares (normal) fitting).

The selected strains meeting these criteria are subjected to epitope binding. This method works on Octet Red 384 using version 8.0.2.5 software at 25 ° C. Each selected strain (diluted to 15 μg / ml) was sequentially loaded into the ProA sensor (180 s, immersed in PBSF for at least 10 minutes before use). The sensor was then blocked using EpoFix IgG1 W T (50 μg / ml, 300 s), then washed with PBSF and immersed in each selected strain (15 μg / ml, 60 s). This item is a self-binding check to confirm that no binding occurs in the absence of LAG3. After the baseline in PBSF at 60 seconds, the sensor was then immersed in huLAG3 (50 nM, 180 s) and then into each other antibody (10ug / ml dissolved in PBSF) for 180 seconds. Use ForteBio data analysis software version 8.0 for dissociation analysis. When the secondary antibody is seen to bind to LAG3 in the presence of the primary antibody, the two antibodies are not competitive. When no secondary antibody binding is seen, the two anti-systems compete with LAG3 for binding and are assigned to the same group.

    The result    

Selected strains 57B02, 57E02, 57C06 and 57H08 competed with LAG3 and were assigned to the same epitope group. It has the following properties: The CDR series of these strains are listed in Table 8.

Table 8 shows that the selected strains showed variations in their CDRL1 and CDRL3 sequences. The mAb with three amino acid substitutions in CDRL1 (compared to the CDRL1 sequence of 57E02 in 3 positions in CDRL1) met the criteria stated in Example 4. This table also shows that a large degree of variation is allowed in CDRL3, which means that the precise sequence of this CDR may not be necessary.

By comparing the CDRL1 sequences, the following common sequence can be identified: RASQX ₁ ISSX ₂ LX ₃ (SEQ ID NO: 56), wherein X ₁ is G or S, X ₂ is W, F or Y, and X ₃ is A or N.

Example 5: Selection of LAG-3 mAb

Use the auto-optimized version of the VH and VL sequences of the selected strain 57E02 for codon optimization (which is the baseline sequence compared to the pre-optimized embryonic sequence. "Frequency" codon will be mutated at the difference). The position of the Apal restriction enzyme identified in the optimized VH sequence was manually removed. The In-fusion oligo sequence was added to the 5 'and 3' ends of the light chain sequence (5 '= GGCCACCGCCACCGGTGTGCACAGC (SEQ ID NO: 48); 3' = CGTACGGTGGCCGCCC (SEQ ID NO: 49)).

The amino acid sequence of the light chain is shown in SEQ ID NO: 50

The DNA sequence of the light chain is shown in SEQ ID NO: 51

The amino acid sequence of the heavy chain is shown in SEQ ID NO: 52

The DNA sequence of the heavy chain is shown in SEQ ID NO: 53.

The heavy and light chain DNA sequences were ordered as gBlock from IDT and screened using the In-Fusion HD colonization kit (Clontech) according to the manufacturer's instructions to contain IgG1 LAGA invalid heavy chains digested with AgeI-HF and ApaI-HF. PTT5 expression vector in the light chain constant region.

Example 6: Selection and performance of PD-1 / LAG-3 mAbdAb

Initially, the mAb-dAb was generated by amplifying the mAb and dAb sequences, and then the PCR products were combined using SOE PCR, and then using AgeI and EcoRI to restrict the endonuclease recognition position colonization into the pTT5 vector for In Fusion colonization. Quotient instructions.

To generate DNA encoding 57E02x51A09-188001 mAb-dAb, the VH region of the mAb-dAb containing 51A09-188001 dAb (produced in Example 3) was used as the gBlock containing the 57E02 VH region to clone 57E02 mAb as described above (see 7 ) The same colonization strategy replacement.

The In Fusion reaction was transformed into NEB 5-α competent E. coli (High Efficiency; model C2987) cells. The DNA isolated from the transformed individual strains was sequenced and the heavy and light chain vectors were used for transfection and expression in HEK293-6E cells. 5 days after transfection, the culture was collected by centrifugation and the clarified culture supernatant was loaded at a constant flow rate (5 ml / min) into a pre-equilibrated 5 ml HiTrap MabSelect SuRe (5 ml; GE Healthcare) connected to the Akta Xpress system (GE Healthcare). The peak portions were pooled and flowed through an anion exchange chromatography column at pH 7.5.

The light chain amino acid sequence and DNA sequence of mAbdAb are the same as the light chain amino acid sequence and DNA sequence of mAb 57E02 (produced in Example 6). The heavy chain amino acid sequence of 57E02x51A09-188001 mAbdAb is shown in SEQ ID NO: 54:

The nucleotide sequence of 57E02x51A09-188001 mAbdAb heavy chain is shown in SEQ ID NO: 55:

Example 7: Qualitative PD-1 / LAG-3 mAbdAb

The mAb-dAb produced in Example 6 was tested by the following analysis:

1. PD1 + Jurkat analysis (Method 8)

2. LAG3 + Jurkat analysis (Method 13)

3. Combine with LAG3 + Jurkat cells (Method 9)

4. Binding to human and macaque LAG3 + cells

5. Combine with PD1 + Jurkat cells (Method 9)

6. Human PD1 / PDL1 receptor binding analysis (Method 3)

7. Cynomolgus monkey PD1 / PDL1 receptor binding analysis (Method 3)

8. Mixed lymphocyte reaction analysis (Method 15)

9. Analysis of HIV hyperplasia (Method 16)

10. Intracellular cytokine analysis (Method 17)

11. Virus-induced MLR analysis (Method 18)

The results of item 1 to item are shown in Table 9:

Several LAG3 / PD1 mAbdAbs were identified and characterized by the described in vitro analysis. Three molecules with the desired functional activity and different CDR binding positions for LAG3 or PD1 were evaluated by in vitro analysis. It was determined that 57E02-51A09-188001 had the best combination of in vivo and in vitro properties, including: affinity, ability to promote T cell activation and function, in vitro target interception, pharmacokinetics, and pharmacodynamic properties.

Advantageously, please note that two tested mAbdAbs including the same PD-1 dAb component but different LAG-3 mAb exhibit different clearance in vivo (before the expected clearance of anti-drug antibody vehicle occurs). This item is an unexpected benefit of this particular combination of PD-1 dAb and LAG-3 mAb.

Method 15: Mixed lymphocyte reaction analysis

Mixed lymphocyte reaction (MLR) analysis is a correlate of T cell function in vitro. It evaluates the ability of T cells to respond to foreign objects to stimulate the production of cytokines. In this analysis, this stimulus was due to differences in major histocompatibility (MHC) antigens from dendritic cells from three different donors. This assay is used to characterize the LAG3-PD1 mAb-dAb to enhance the ability of T cells to produce cytokines.

Method: Thaw peripheral blood mononuclear cells (PBMC) from three HIV-infected donors who were stably treated with retrovirus and let stand overnight in RPMI 1640 medium containing 10% FBS. The next day CD4 + T cells were isolated from the resting PBMC (using Stemcell Technologies CD4 negative selection / enrichment kit-model 19052). The CD4 T cells at 1 x 10 ⁶ / mL were resuspended in RPMI + 10% FBS medium. CD4 T cells were ¹ × 10 ⁵ cells 100 μL / well in triplicate, and implanted into a tissue culture-treated U-shaped bottom analysis disk. Serially dilute the control or test mAb or mAbdAb in a separate analysis plate to produce a six-point, four-fold serial dilution (20X) and add 10 μL of each binding protein to each well and add the cell binding protein The mixture was incubated at room temperature for 15 mins. During the incubation, the performance from three healthy donors of the PDL-1 and MHC-II monocyte-derived dendritic cells (MDDCs) and thawed at 0.2 x 10 ⁶ / mL were resuspended in RPMI + 10% FBS medium. Add MDDC to the appropriate wells containing CD4 T cells and binding protein. A well containing only CD4 T cells was used as a negative control. The final concentration of each antibody tested was 200, 50, 12.5, 3.125, 0.781 and 0.195 nM. The analysis disk was placed in a humidified incubator at 37 ° C for 5 days with 5% CO ₂ . On Day 6, the supernatant was collected and tested for IFNγ production using the Meso Scale Diagnostics kit: V-PLEX pre-inflammatory disc 1 (human), model K151A0H-4. MLR analysis was used to compare the functional activity of the dual-specificity LAG3 / PD1 mAbdAb (57E02-51A09-188001) with the combination of the LAG3 mAb (57E02) and PD1 dAb (51A09-188001) functional arms. Antibodies evaluated in this analysis: 1) LAG3 / PD1 dual specificity 57E02-51A09-188; 2) a LAG3 monoclonal antibody 57E02; 3) control anti-respiratory fusion virus (aRSV) mAb connected to PD1 dAb to dual specificity aRSV -51A09-188001; 4) Negative control antibody (VHDUM) which contains RSV antibody linked to an unrelated dAb (no known binding activity); 5) LAG3mAb (57E02) and PD1 mAbdAb- (aRSV-51A09-188001 ) Combination. This analysis was also used to evaluate CTLA4 / PD1 mAbdAb by comparing the amount of IFNγ produced by the bispecific antibody with the same control antibody and the PD1 mAbdAb and CTLA4 mAb listed above.

Statistical analysis: Outler analysis is performed before statistical analysis. The Outlier and the statistical analysis system are carried out with log ₁₀ transform magnitude. Outlier simplified data is averaged through technical repetition and fitting a mixed model with data on antibody, dose and antibody interaction, and using dose as fixed effect and donor interaction and operating with random effect. Uncorrected p-values were obtained via pair-wise comparisons and corrected using Bonferroni correction for the 6 concentrations compared for each antibody.

Results: The results are shown in Table 10 and Figure 1. Compared with the control antibody in the MLR analysis, the antibody that blocked the PD1 inhibitory signal significantly increased IFNγ produced by CD4 T cells. In the MLR analysis, statistical significance was observed at all detected concentrations, p <0.0001 (Figure 1). Antagonizing the LAG3 signal alone did not increase CD4 IFNγ production. However, compared to blocking PD1 alone, co-culture of both LAG3 and PD1 blocking antibodies increased cytokine production. The fold difference in cytokine production between the LAG3 / PD1 mAbdAb and individual antibodies or the combination of LAG3 and PD1 antibodies was determined. On average, we observed that co-culture with dual-specific LAG3 / PD1 increased the cytokine production of CD4 T cells by 1.86 to 3.38 times relative to the combination of the two antibodies (Table 10). In addition, the bispecific antibody showed surpassing PD1 and LAG3 blocking antibodies in cytokine production, increasing by 2.08 to 4.19 and 5.95 to 17.44 times, respectively. In summary, the LAG3 / PD1 dual-specific mAbdAb significantly promotes T cells to produce more cytokines than LAG3, PD1, or a combination of the two antibodies.

Method 16: Analysis of HIV-specific hyperplasia:

When the T cell recognizes that its cognate antigen is displayed on the antigen presenting cell in vitro or in vivo, it will proliferate or expand. Blocking the inhibitory signals from LAG3 and PD1 enhances the antiviral response by helping the expansion / proliferation of virus-specific CD8 T cells. The following analysis method is used to characterize the ability of LAG3-PD1 mAb-dAb to enhance the proliferation of HIV Gag-specific CD8 T cells.

method:

1. Serially dilute the control or test mAbdAb in a 96-well U-bottom assay dish in triplicate wells to produce a four-point serial dilution of each antibody with a final concentration of 20, 2, 0.2, 0.02ug / ml and a total volume of 50μl. Make repeated analysis disks in advance and store at -20 ° C until use.

2. Melt peripheral blood mononuclear cells (PBMC) from donors infected with HIV stably treated with antiretroviral agents in AIM-V serum-free medium (Thermofisher) without Efavirenz (EFV) and DNASe (Stem cell technologies) .

3. Wash PBMC, count and resuspend cells at 5 million cells / ml in DPBS.

4. Carboxyfluorescein succinimide (CFSE) from Life Technologies Cat # C34554 is a cell-permeable fluorescent cell stain. CFSE was resuspended in dimethyl sulfoxide (DMSO) at a storage concentration of 1 mM. Each 1 ml of PBMC (0.25 μM solution) was added with 0.25 μL of diluted CFSE in a 37 ° C. incubator for CFSE calibration for 5 mins, with gentle agitation during the cultivation.

5. Stop the calibration reaction with 25ml of cold FBS and wash the calibrated cells and count.

6. Resuspend the CFSE-calibrated PBMC at 10 million cells / ml and 50 μl of cells in the triple wells of the 96-well U-bottom assay disk containing the appropriately diluted Step # 1 antibody.

7. Acquire HIV Gag overlapping peptides from JPT Inovative Peptide Solutions (PepMix Gag Ultra # PM-HIV-GAG) and resuspend in DMSO at a storage concentration of 0.5 μg / μl. Gag peptide was added to AIMV + EFV medium (4μg / ml, 2X concentration) to make Gag peptide stimulation mixture.

8. Add 100 μl of Gag peptide mixture to each well of the analysis plate containing 50 μl of calibrated PBMC and 50 μl of diluted antibody.

9. Each analysis plate has three replicate control wells, which are unstimulated (negative control), Gag stimulation lacking binding protein, or Phytohemagglutinin-M (Sigma-Aldrich) 3 μg / ml Stimulate cells (positive control).

10. Place the analysis dish in a 37 ° C humidified incubator with 5% CO _{2 for} 6 days.

11. The cells were then clumped and stained with luciferin-labeled antibody at 4 ° C for 30 minutes. Staining was performed in a 96-well V-chassis, where the pelleted cell line was resuspended in a total volume of 50 μl of the following diluted commercial antibodies of the following surface proteins: CD3 (selected strain: SP34-2), CD8 (selected) from BD Biosciences Plants: RPA-T8), CD4 (selected plant: L200), CD27 (selected plant: M-T271); PD1 from Biolegend (selected plant: EH12); CD45RO from Beckman Coulter (selected plant: UCHL1), and Live Dead Amine Aqua Dye (from Life Technologies).

12. Wash the cells twice with PBS and fix with a volume of 200 μl diluted in PBS 0.5% PFA.

13. Use a high-throughput sampler to obtain cells on BD LSRII-Fortessa.

14. Use Flowjo to analyze the sample and determine the average frequency of the HIV Gag-specific CD8 T cells that have proliferated for each antibody type and dilution.

15. The hyperplastic CD8 T cell line is defined as: CFSE dim, Live (Amine-ve), CD8 + T cells (CD3 +, CD8 +, CD4).

16. The results shown are the average of two independent analysis of operations from donors with stable treatment of HIV infection. Each operation is performed in triplicate.

Statistical analysis: The data is analyzed using a mixed effect model, which includes antibody type, concentration, antibody interaction, and concentration as fixed effects and operates as random effects. Uncorrected p-values were obtained via pairwise comparisons and Bonferroni corrections were used to correct for the 4 concentrations compared for each antibody.

Results: The dual antagonism of LAG3 and PD1 promoted the expansion of HIV Gag-specific CD8 T cells. Compared with cells cultured with control antibody, PBMC co-cultured with Gag peptide and LAG3 / PD1 mAbdAb (57E02-51A09-188001) significantly demonstrated better CD8 proliferation (% CD8 + CFSE-dim T cells) (Figure 2 ). In the dose range of 20-0.2ug / ml, statistical significance was achieved at the 0.05 Bonferroni corrected significance level.

Method 17: Intracellular cytokine analysis: This analysis is used to evaluate the ability of the LAG3 / PD1 antagonist mAbdAb (57E02-51A09-188001) to increase T cell function.

T cells are activated by MHC-mediated recognition of the antigen, and the antigen is displayed on the antigen-presenting cells. T cell receptor (TCR) binding to the MHC-peptide complex enables activation of signal pathways that trigger cytokine production and / or increased cytotoxic activity. Bacterial toxins, such as staphylococcal enterotoxin, can be used to crosslink TCR and MHC molecules in vivo, resulting in T cell activation and production of cytokines. Cytokine production and cytotoxic activity are characteristic of functional CD4 and CD8 T cells, respectively. Intracellular cytokine staining is an effective method to measure the ability of T cells to produce multiple cytokines and increase their cytotoxic potential. It is usually used in clinical trials to monitor the qualitative and quantitative nature of T cell responses.

method

1. Thaw the cryopreserved peripheral blood mononuclear cells (PBMC) from 19 HIV-infected stable donors and add RPMI 1640 (Seradigm model 1500-500) and 500nM Efavirenz (R10 + EFV) RPMI 1640 ( Invitrogen model 22400) was allowed to stand overnight and the absolute cell number was determined.

2. Dilute LAG3 / PD1 mAbdAb or control antibody to a 4x concentration of 8 μg / mL with R10 + EFV and add 50 μl to the well of a 96-well U-bottom assay dish (Falcon model 351177) for non-tissue culture treatment.

3. Resuspended the resting PBMC at 10 million pieces / mL in R10 + EFV containing protein operation inhibitor monensin (BD Biosciences model 554724), 0.7 μl / mL. 50 μl of the cell culture mixture was added to a non-tissue culture-treated 96-well U-bottom assay dish containing the corresponding 4x antibody.

4. Include CD107 also known as lysosomal associated membrane protein 1 (LAMP-1), (selected strain: H4A3, BioLegend model 328620) included in the culture medium as a measure of cytotoxic potential / degranulation.

5. Add 100 ml of 2x R10 + EFV stimulation mixture (List Biologicals) containing staphylococcal enterotoxin A & B (SEB / A), each with a final concentration of 500 ng / mL, to the corresponding well. Bacterial antigens such as SEB / A diplomatic TCR & MHC molecules in vivo, leading to T cell activation and cytokine production.

6. Set a negative control or unstimulated well for each donor, which can measure spontaneous cytokine production / degranulation.

7. Place the analysis dish in a 37 ° C humidified incubator with 5% CO _{2 for} 6 days.

8. In 96-well V-bottom tray, re-suspend the diluted commercial antibody containing the cell pellet in the total volume of 50 μl of the following surface proteins, and stain the cell surface antigen at 4 ° C for 30 minutes: CD3 (selection) from BD Biosciences Strain: SP34-2), CD8 (selective strain: RPA-T8), CD4 (selective strain: L200), CD27 (selective strain: M-T271); PD1 from Biolegend (selective strain: EH12); CD45RO (selected strain: UCHL1) from Beckman Coulter and Live / Dead Amine Aqua Dye from Life Technologies.

9. Wash the cells twice with cold Pharmingen staining buffer (BSA) (BD Pharmingen model 554657), and then fix them with cold 4% paraformaldehyde at 4 ° C for 15 minutes, followed by cold 1x BD Perm at 4 ° C / Wash the buffer through for 15 minutes.

10. Resuspend the fixed / permeabilized cells in a 50 μl total volume of cold 1x BD Perm / wash buffer containing the following intracellular cytokines in a total volume of 50 μl for 30 minutes: TNFα from BioLegend (selected strain) : Mab11), IFNγ (selected strain: 4S.B3) and IL2 (selected strain: MQ1-17H12).

11. Wash the cells twice with cold 1x BD Perm / washing buffer and then resuspend in 200 μl of cold Pharmingen staining buffer (BSA) (BD Pharmingen model 554657).

12. Use a high-throughput sampler to obtain cells on BD LSRII-Fortessa.

13. Use Flowjo software suite (Treestar, Ashland, OR) to analyze the samples.

14. Use flow cytometry gating to identify single cell populations based on area and height. Lymphocytes are gated on the basis of front scattering and side scattering properties. The amount of viable / viable cells was then determined by amine staining exclusion. T cells were guarded on the basis of CD3, CD4 and CD8 performance. Natural cells show high amounts of CD27 and lack CD45RO performance. Natural cells (CD45RO- & CD27 +) do not need to focus on memory and effector group analysis.

15. Determine the average frequency of memory & / effector CD4 / CD8 T cells that have secreted cytokines (TNFα, IFNγ, IL2) or degranulated, ie increased CD107a performance.

Statistical analysis: Analyze the data using a mixed effect model with a magnitude of ^0.2 transformation of data, which includes the interaction of stimuli, antibodies, stimuli, and the use of antibodies as fixed effects and donors as random effects. Unpaired p-values were obtained via pair-wise comparisons and Bonferroni corrections were used to correct for the two comparisons of interest.

Results: The dual antagonism of LAG3 and PD1 increased the functional capacity of T cells infected with HIV donors. Compared with cells cultured with control antibodies, T cells co-cultured with LAG3 / PD1 mAbdAb (57E02-51A09-188001) in the presence of SEB / SEA significantly produced more TNFα, IFNγ, and IL2 (Figure 3, AB) . Importantly, co-cultivation with 57E02-51A09-188001 in the absence of stimulation (unstimulated) does not trigger cytokine production. The main function of CD4 helper T cells is to produce cytokines, such as IL2 and cytotoxic CD8 T cells that enhance the function of antibody-producing B cells. Therefore, we focused on the ability of CD4 + T cells to produce IL2 and TNFα. This helper function declined significantly during chronic HIV infection. Using SEB / SEA to cross-link with MHC-TCR, we observed that LAG3 / PD1 blockade significantly increased the frequency of IL2 and TNFα-producing CD4T cells from HIV-infected donors (Figure 3, C). The function of CD8 T cells is also compromised during HIV infection. The measured value of the cytotoxic potential is the degranulation ability and the ability to produce IFNγ measured by CD107a performance. Co-culture with LAG3 / PD1 mAbdAb significantly increased the ability of CD8 T cells infected with HIV donors to express CD107a and IFNγ (Figure 3, D). In this analysis, 10 of the 19 donors tested increased the cytotoxic potential by approximately 1.5-3 times in the presence of LAG3 / PD1 relative to the control group.

Method 18: Virus-induced MLR analysis:

This analysis was used to assess the ability of the LAG3 / PD1 antagonist mAbdAb (57E02-51A09-188001) to increase HIV RNA production. CD4 T cells with persistent latent infection are the main barriers for the healing of HIV. These latently infected cells do not express or show low amounts of HIV RNA and proteins that make them undetectable by the immune system. TCR stimulation may lead to cell activation and viral resurrection. Proteins that trigger viral resurrection and express viral RNA / CD4 T cells allow the immune system to recognize and specifically target these infected cells for clearance. Delayed reversal or HIV resurrection can be assessed in vitro by the increase in HIV cell-associated RNA and protein produced by CD4 T cells. We used heterologous stimulation of dendritic cells to elicit HIV RNA performance to adapt to this mixed lymphocyte response analysis. The ability of LAG3 / PD1 to promote HIV RNA induction was then evaluated from CD4 T cells obtained from HIV-infected donors.

Methods: Peripheral blood mononuclear cells (PBMC) of 5 donors infected with HIV who were treated with retroviruses were melted and allowed to stand overnight in RPMI 1640 medium containing 10% FBS. The next day, CD4 + T cells from standing PBMC were isolated (using Stemcell Technologies CD4 negative selection / Zengfeng kit-model 19052). The CD4 T cells at 1 x 10 ⁶ / mL were resuspended in RPMI + 10% FBS medium. Twenty replicate 1 × 10 ⁵ CD4 T cells were placed in a tissue culture-treated U-shaped bottom analysis dish at 100 μL / well. Use a control of 125 nM or LAG3 / PD1 mAbdAb. Each well was added with each antibody to CD4 T cells and the mixture was incubated at room temperature for 15 minutes. During the culture, from the three healthy donors showed the PDL-1 and MHC-II monocyte-derived dendritic cells (MDDCs) and thawed at 0.2 x 10 ⁶ / mL were resuspended in RPMI + 10% FBS medium. Add MDDC to the appropriate wells containing CD4 T cells and binding protein. A well containing CD4 T cells alone was used as a negative control. The analysis disk was placed in a humidified incubator at 37 ° C for 5 days with 5% CO ₂ . On day 6, cells and supernatant were collected and gathered together. RNA was isolated from cell pellets using Qiagen model # 74104 RNeasy kit. RNA was isolated from the supernatant by using Qiaamp Model # 52904 QIAamp Viral RNA kit.

To evaluate the amount of Gag RNA expressed in cells and supernatant, qPCR was performed using the primers and probes listed in Table 11:

Use Fast Virus 1-step Master Mix reagent. A total of 5ul of separated samples was used as input. The final concentration of the primer in the master mix is 900 nM, and the probe is 250 nM. The samples were operated on QuantStudio 3 under the following PCR cycle conditions: reverse transcription at 50 degrees for 10 minutes, 95 degrees for 20 seconds until denaturation for 1 cycle, then 95 degrees for 3 seconds for denaturation for 50 cycles, followed by 60 degrees for 30 seconds Extend steps. The Gag concentration was calculated by using the number of replicas for each reaction and adjusted for the volume of DNA dissolved and normalized to the number of extracted cells.

Statistical analysis: Outler analysis is performed before statistical analysis. The Outlier and the statistical analysis system are carried out with log ₁₀ transform magnitude. The fixed effect model was fitted with antibody data as the fixed effect. Individual analysis of each donor. Unpaired p-values and comparison statements were obtained via pair-wise comparisons and corrected using Bonferroni correction for a total of 3 comparisons.

result:

CD4 + T cells with persistent latent infection are still the main barrier to cure HIV. The first step to eliminate this virus nest is to trigger HIV RNA and protein expression so that the antiviral response can identify and eliminate these cells. Because cell activation may lead to viral resurrection, in the modified MLR analysis we used heterologous stimuli from dendritic cells to trigger HIV RNA production. We verified that 4 out of 5 test donors had LAG3 / PD1 bispecific antibodies that significantly increased HIV RNA production from CD4 T cells compared to control antibodies (Figure 4).

Example 8: Selection and characterization of CTLA4 / PD-1 mAbdAb

The CTLA4 / PD-1 mAbdAb selection strain CTLA-4 x 51A09-188001 was selected, expressed and purified.

The amino acid sequence of the light chain is shown in SEQ ID NO: 79

The amino acid sequence of the heavy chain is shown in SEQ ID NO: 80

MLR analysis (Method 15) was used to assess the functional activity of CTLA4 / PD1 mAbdAb. CTLA4, PD1 antibody (aRSV-51A09-188), the combination of the two antibodies CTLA4 / PD1 mAbdAb or control antibody were co-cultured with monocyte-derived dendritic cells and the previously described CD4 T cells. After 6 days of cultivation, the amount of IFNγ produced was measured. Compared with CTLA4 or PD1 blocking or control antibody alone, co-culture with dual-specificity CTLA4 / PD1 mAbdAb increased cytokine production (1.42-12.28 times). However, 5 of the 6 tested concentrations had the CTLA4 / PD1 bispecific antibody cytokine production equivalent to the combination of CTLA4 and PD1 antibody. The following table shows the fold difference between CTLA / PD1 mAbdAb and individual antibodies or the combination of CTLA4 and PD1 antibody in the 6 concentrations evaluated by MLR analysis.

Unless indicated by ( ^a ), the CTLA4 / PD1 mAbdAb produced a cytokine system that was significantly larger than the indicated antibody after multiple corrections. Bonferroni corrected p-value is <0.05.

【Sequence Table】

SEQ ID NO: 1 (Heavy chain variable region of selected strain 37Y056-57E02-1)

SEQ ID NO: 2 (the light chain variable region of strain 37Y056-57E02-1)

SEQ ID NO: 3 (Heavy chain variable region of selected strain 22D034-51A09-188001)

SEQ ID NO: 4 (CDRH1 LAG3, Kabat definition) GYYMH

SEQ ID NO: 5 (CDRH2 LAG3, Kabat definition) WINPNSGGTNYAQKFQG

SEQ ID NO: 6 (CDRH3 LAG3, Kabat definition) EGPYDDDGFDY

SEQ ID NO: 7 (CDRL1 LAG3, Kabat definition) RASQGISSWLA

SEQ ID NO: 8 (CDRL2 LAG3, Kabat definition) AASSLQS

SEQ ID NO: 9 (CDRL3 LAG3, Kabat definition) QETNSFWT

SEQ ID NO: 10 (selected strains 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 CDRH1 (Kabat definition)) THYMV

SEQ ID NO: 11 (selected strains 22D034-51A09-188, 22D034-51A09-140, 22D034-51A09-109, 22D034-51A09-73, 22D034-51A09-30 and 22D034-51A09-13 CDRH2 (Kabat definition) ) FIGPAGDTTYYADSVKG

SEQ ID NO: 12 (selected strains 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 CDRH3 (Kabat definition)) YTATSGVDTYDV

SEQ ID NO: 13 (CDRH1 LAG-3, defined by Chothia) GYTFTGY

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

SEQ ID NO: 15 (CDRH1 PD-1, defined by Chothia) GFTFRTH

SEQ ID NO: 16 (CDRH2 PD-1, defined by Chothia) 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) WMGWINPNSGGTN

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

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

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

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

SEQ ID NO: 29 (CDRH2 PD-1, Contact definition) AAYTATSGVDTYD SEQ ID NO. 30 (linker sequence) STGLDSPT

SEQ ID NO: 31 (CDRH1 (Kabat definition) of selected strain 22D034-51A09-163) THYMA

SEQ ID NO: 32 (selected strains 22D034-51A09-183, 22D034-51A09-135, 22D034-51A09-110, 22D034-51A09-54, 22D034-51A09-10 and 22D034-51A09-4 CDRH2 (Kabat definition) ) FIGPAGDFTYYADSVKG

SEQ ID NO: 33 (selected strains 22D034-51A09-163, 22D034-51A09-126 and 22D034-51A09-16 CDRH2 (Kabat definition)) FIGPAGDSTYYADSVKG

SEQ ID NO: 34 (CDRH2 of the selected strain 22D034-51A09-55 (Kabat definition)) FIGPAGDFTYYADSVEG

SEQ ID NO: 35 (CDRH3 (selected by Kabat) of selected strain 22D034-51A09-163) YTATSSVDTYDV

SEQ ID NO: 36 (CDRH3 (Kabat definition) of selected strains 22D034-51A09-140 and 22D034-51A09-10) YTATSDVDTYDV

SEQ ID NO: 37 (CDRH3 (selected by Kabat) of selected strain 22D034-51A09-135) YTETSGVDTYDV

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

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

SEQ ID NO: 40 (CDRH3 of the selected strain 22D034-51A09-16 (Kabat definition)) YTATSGFDSYDV

SEQ ID NO: 41 (CDRL1 of the selected strain 57C06 (Kabat definition)) RASQSISSFLN

SEQ ID NO: 42 (CDRL1 of selected strain 57H08 (Kabat definition)) RASQSISSYLN

SEQ ID NO: 43 (CDRL3 of the selected strain 57B02 (Kabat definition)) QQAVDHFT

SEQ ID NO: 44 (CDRL3 of the selected strain 57C06 (Kabat definition)) QDIYSTPLT

SEQ ID NO: 45 (CDRL3 (Kabat definition) of selected strain 57H08) QQSFPAPPYT

SEQ ID NO: 46 (Pre-primer oligonucleotide) CCCGGAAAGGGATCCACAGGACTGGACTCCCCGACAGAGGTGCAGCTGTTGGAGTCT

SEQ ID NO: 47 (inverted primer oligonucleotide) GGGGATCTAGAATTCATCAGCTCGAGACGGTGACCAGGGTT

SEQ ID NO: 48 (Pre-primer oligonucleotide) GGCCACCGCCACCGGTGTGCACAGC

SEQ ID NO: 49 (reverse primer oligonucleotide) CGTACGGTGGCCGCCC

SEQ ID NO: 50 (LAG-3 mAb 57E02 and light chain amino acid sequence of PD-1 / LAG-3 mAbdAb 57E02x51A09-188001)

SEQ ID NO: 51 (LAG-3 mAb 57E02 and light chain DNA sequence of PD-1 / LAG-3 mAbdAb 57E02x51A09-188001)

SEQ ID NO: 52 (the heavy chain amino acid sequence of LAG-3 mAb 57E02) QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLE

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

SEQ ID NO: 54 (PD-1 / LAG-3 mAbdAb 57E02x51A09-188001 heavy chain amino acid sequence)

SEQ ID NO: 55 (PD-1 / LAG-3 mAbdAb 57E02x51A09-188001 heavy chain DNA sequence)

SEQ ID NO: 56 (General CDRL1 LAG3, Kabat definition) RASQX ₁ ISSX ₂ LX ₃ (where X ₁ is G or S, X ₂ is W, F or Y, and X ₃ is A or N).

SEQ ID NO: 57 (General CDRH1 PD-1, Kabat definition) THYMX ₄ (where X ₄ is V or A)

SEQ ID NO: 58 (General CDRH2 PD-1, Kabat definition) FIGPAGDX ₅ TYYADSVX ₆ G (where X ₅ is T, F or S and X ₆ is K or E)

SEQ ID NO: 59 (General CDRH3 PD-1, Kabat definition) YTX ₇ TSX ₈ X ₉ DX ₁₀ YDV (where X ₇ is A or E, X ₈ is G, S or D, X ₉ is V, F or Y , And X ₁₀ is T or S)

SEQ ID NO: 60 (DNA sequence of HIV-1 Gag pre-primer) ATCAAGCAGCTATGCAAATGTT

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

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

SEQ ID NO: 63 (when n is 0 and X ₁₁ is absent, the C-terminus extends to the epitope binding region or single variable region). VTVS

SEQ ID NO: 64 (when n is 1 and X ₁₁ is absent, the C-terminus extends to the epitope binding region or single variable region). VTVSS

SEQ ID NO: 65 (when n is 0 and X _{11 is} present, the C-terminus extends to the epitope binding region or single variable region). VTVSX ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ X ₁₈ X ₁₉ (where X ₁₂ , X ₁₃ , X ₁₄ , X ₁₅ , X ₁₆ , X ₁₇ , X ₁₈ or X ₁₉ may represent any amino acid, where the The sequence may terminate at positions 6, 7, 8, 910, 11 or 12, and the sequence between residues 5 to 12 may contain the following sequence: A, AAA or T).

SEQ ID NO: 66 (when n is 1 and X _{11 is} present, the C-terminus extends to the epitope binding region or single variable region). VTVSSX ₁₂ X ₁₃ X ₁₄ X ₁₅ X ₁₆ X ₁₇ X ₁₈ X ₁₉ (where X ₁₂ , X ₁₃ , X ₁₄ , X ₁₅ , X ₁₆ , X ₁₇ , X ₁₈ or X ₁₉ can represent any amino acid, where the The sequence may end at position 7, 8, 910, 11, 12, or 13, and the sequence between residues 6 to 13 may contain the following sequence: A, AAA, or T).

SEQ ID NO: 67 (when n is 0 and X ₁₁ is A, the C-terminus extends to the epitope binding region or single variable region). VTVSA

SEQ ID NO: 68 (when n is 1 and X ₁₁ is A, the C-terminus extends to the epitope binding region or single variable region). VTVSSA

SEQ ID NO: 69 (when n is 0 and X ₁₁ is AS, the C-terminus extends to the epitope binding region or single variable region). VTVSAS

SEQ ID NO: 70 (when n is 1 and X ₁₁ is AS, the C-terminus extends to the epitope binding region or single variable region). VTVSSAS

SEQ ID NO: 71 (when n is 0 and X ₁₁ is AST, the C-terminus extends to the epitope binding region or single variable region). VTVSAST

SEQ ID NO: 72 (when n is 1 and X ₁₁ is AST, the C-terminus extends to the epitope binding region or single variable region). VTVSSAST

SEQ ID NO: 73 (when n is 0 and X ₁₁ is ASTK, the C-terminus extends to the epitope binding region or single variable region). VTVSASTK

SEQ ID NO: 74 (when n is 1 and X ₁₁ is ASTK, the C-terminus extends to the epitope binding region or single variable region). VTVSSASTK

SEQ ID NO: 75 (when n is 0 and X ₁₁ is ASTKG, the C-terminus extends to the epitope binding region or single variable region). VTVSASTKG

SEQ ID NO: 76 (when n is 1 and X ₁₁ is ASTKG, the C-terminus extends to the epitope binding region or single variable region). VTVSSASTKG

SEQ ID NO: 77 (when n is 0 and X ₁₁ is ASTKGP, the C-terminus extends to the epitope binding region or single variable region). VTVSASTKGP

SEQ ID NO: 78 (when n is 1 and X ₁₁ is ASTKGP, the C-terminus extends to the epitope binding region or single variable region). VTVSSASTKGP

SEQ ID NO: 79 (CTLA-4 / PD-1 mAbdAb CTLA4 x51A09-18800 light chain amino acid sequence 1)

SEQ ID NO: 80 (CTLA-4 / PD-1 mAbdAb CTLA4 x51A09-188001 heavy chain amino acid sequence)

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

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<213> Artificial

<220>

<223> CDRH2 of 22D034-51A09-188, 22D034-51A09-140, 22D034-51A09-109, 22D034-51A09-73, 22D034-51A09-30 and 22D034-51A09-13

<400> 11

<210> 12

<211> 12

<212> PRT

<213> Artificial

<220>

<223> Selected strains 22D034-51A09-188, 22D034-51A09-183, 22D034-51A09-110, 22D034-51A09-109, 22D034-51A09-73, 22D034-51A09-30, 22D034-51A09-13 and 22D034- CDRH3 of 51A09-4 (Kabat definition)

<400> 12

<210> 13

<211> 7

<212> PRT

<213> Artificial

<220>

<223> CDRH1 LAG-3, Chothia definition

<400> 13

<210> 14

<211> 6

<212> PRT

<213> Artificial

<220>

<223> CDRH2 LAG-3, Chothia definition

<400> 14

<210> 15

<211> 7

<212> PRT

<213> Artificial

<220>

<223> CDRH1 PD-1, Chothia definition

<400> 15

<210> 16

<211> 6

<212> PRT

<213> Artificial

<220>

<223> CDRH2 PD-1, Chothia definition

<400> 16

<210> 17

<211> 10

<212> PRT

<213> Artificial

<220>

<223> CDRH1 LAG-3, AbM definition

<400> 17

<210> 18

<211> 10

<212> PRT

<213> Artificial

<220>

<223> CDRH2 LAG-3, AbM definition

<400> 18

<210> 19

<211> 10

<212> PRT

<213> Artificial

<220>

<223> CDRH1 PD-1, AbM definition

<400> 19

<210> 20

<211> 10

<212> PRT

<213> Artificial

<220>

<223> CDRH2 PD-1, AbM definition

<400> 20

<210> 21

<211> 6

<212> PRT

<213> Artificial

<220>

<223> CDRH1 LAG-3, Contact definition

<400> 21

<210> 22

<211> 13

<212> PRT

<213> Artificial

<220>

<223> CDRH2 LAG-3, Contact definition

<400> 22

<210> 23

<211> 12

<212> PRT

<213> Artificial

<220>

<223> CDRH3 LAG-3, Contact definition

<400> 23

<210> 24

<211> 7

<212> PRT

<213> Artificial

<220>

<223> CDRL1 LAG-3, Contact definition

<400> 24

<210> 25

<211> 10

<212> PRT

<213> Artificial

<220>

<223> CDRL2 LAG-3, Contact definition

<400> 25

<210> 26

<211> 7

<212> PRT

<213> Artificial

<220>

<223> CDRL3 LAG-3, Contact definition

<400> 26

<210> 27

<211> 6

<212> PRT

<213> Artificial

<220>

<223> CDRH1 PD-1, Contact definition

<400> 27

<210> 28

<211> 13

<212> PRT

<213> Artificial

<220>

<223> CDRH2 PD-1, Contact definition

<400> 28

<210> 29

<211> 13

<212> PRT

<213> Artificial

<220>

<223> CDRH3 PD-1, Contact definition

<400> 29

<210> 30

<211> 8

<212> PRT

<213> Artificial

<220>

<223> Linker sequence

<400> 30

<210> 31

<211> 5

<212> PRT

<213> Artificial

<220>

<223> CDRH1 (Kabat definition) of selected strain 22D034-51A09-163

<400> 31

<210> 32

<211> 17

<212> PRT

<213> Artificial

<220>

<223> CDRH2 (Kabat definition) of selected strain 22D034-51A09- / 183/135/110/54/10/4

<400> 32

<210> 33

<211> 17

<212> PRT

<213> Artificial

<220>

<223> CDRH2 of 22D034-51A09-163, 22D034-51A09-126, and 22D034-51A09-16 (Kabat definition)

<400> 33

<210> 34

<211> 17

<212> PRT

<213> Artificial

<220>

<223> CDRH2 (Kabat definition) of selected strain 22D034-51A09-55

<400> 34

<210> 35

<211> 12

<212> PRT

<213> Artificial

<220>

<223> CDRH3 (Kabat definition) of strain 22D034-51A09-163

<400> 35

<210> 36

<211> 12

<212> PRT

<213> Artificial

<220>

<223> CDRH3 (Kabat definition) of selected strains 22D034-51A09-140 and 22D034-51A09-10

<400> 36

<210> 37

<211> 12

<212> PRT

<213> Artificial

<220>

<223> CDRH3 (Kabat definition) of selected strain 22D034-51A09-135

<400> 37

<210> 38

<211> 12

<212> PRT

<213> Artificial

<220>

<223> CDRH3 (Kabat definition) of selected strains 22D034-51A09-126 and 22D034-51A09-55

<400> 38

<210> 39

<211> 12

<212> PRT

<213> Artificial

<220>

<223> CDRH3 (Kabat definition) of selected strain 22D034-51A09-54

<400> 39

<210> 40

<211> 12

<212> PRT

<213> Artificial

<220>

<223> CDRH3 (Kabat definition) of selected strain 22D034-51A09-16

<400> 40

<210> 41

<211> 11

<212> PRT

<213> Artificial

<220>

<223> CDRL1 Kabat definition of strain 57C06)

<400> 41

<210> 42

<211> 11

<212> PRT

<213> Artificial

<220>

<223> CDRL1 Kabat definition of strain 57H08)

<400> 42

<210> 43

<211> 8

<212> PRT

<213> Artificial

<220>

<223> CDRL3 Kabat definition of strain 57B02)

<400> 43

<210> 44

<211> 9

<212> PRT

<213> Artificial

<220>

<223> CDRL3 Kabat definition of strain 57C06)

<400> 44

<210> 45

<211> 10

<212> PRT

<213> Artificial

<220>

<223> CDRL3 Kabat definition of strain 57H08)

<400> 45

<210> 46

<211> 57

<212> DNA

<213> Artificial

<220>

<223> Pre-primer oligonucleotide

<400> 46

<210> 47

<211> 41

<212> DNA

<213> Artificial

<220>

<223> Reverse primer oligonucleotide

<400> 47

<210> 48

<211> 25

<212> DNA

<213> Artificial

<220>

<223> Pre-primer oligonucleotide

<400> 48

<210> 49

<211> 16

<212> DNA

<213> Artificial

<220>

<223> Reverse primer oligonucleotide

<400> 49

<210> 50

<211> 213

<212> PRT

<213> Artificial

<220>

<223> Light chain amino acid sequence of LAG-3 mAb 57E02 and PD-1 / LAG-3 mAbdAb 57E02x51A09-188001)

<400> 50

<210> 51

<211> 639

<212> DNA

<213> Artificial

<220>

<223> Light chain DNA sequence of LAG-3 mAb 57E02 and PD-1 / LAG-3 mAbdAb 57E02x51A09-188001

<400> 51

<210> 52

<211> 450

<212> PRT

<213> Artificial

<220>

<223> Heavy chain amino acid sequence of LAG-3 mAb 57E02

<400> 52

<210> 53

<211> 1350

<212> DNA

<213> Artificial

<220>

<223> LAG-3 mAb 57E02 heavy chain DNA sequence

<400> 53

<210> 54

<211> 581

<212> PRT

<213> Artificial

<220>

<223> Heavy chain amino acid sequence of PD-1 / LAG-3 mAbdAb 57E02x51A09-188001

<400> 54

<210> 55

<211> 1743

<212> DNA

<213> Artificial

<220>

<223> Heavy chain DNA sequence of PD-1 / LAG-3 mAbdAb 57E02x51A09-188001

<400> 55

<210> 56

<211> 11

<212> PRT

<213> Artificial

<220>

<223> General CDRL1 LAG3, Kabat definition

<220>

<221> variant

<222> (5) .. (5)

<223> X = "G" or "S"

<220>

<221> variant

<222> (9) .. (9)

<223> X = "F" or "W" or "Y"

<220>

<221> variant

<222> (11) .. (11)

<223> X = "A" or "N"

<400> 56

<210> 57

<211> 5

<212> PRT

<213> Artificial

<220>

<223> General CDRH1 PD-1, Kabat definition

<220>

<221> variant

<222> (5) .. (5)

<223> X = "V" or "A"

<400> 57

<210> 58

<211> 17

<212> PRT

<213> Artificial

<220>

<223> General CDRH2 PD-1, Kabat definition

<220>

<221> variant

<222> (8) .. (8)

<223> X = "T" or "F" or "S"

<220>

<221> variant

<222> (16) .. (16)

<223> X = "K" or "E"

<400> 58

<210> 59

<211> 12

<212> PRT

<213> Artificial

<220>

<223> General CDRH3 PD-1, Kabat definition

<220>

<221> variant

<222> (3) .. (3)

<223> X = "A" or "'E"

<220>

<221> variant

<222> (6) .. (6)

<223> X = "G" or "S" or "D"

<220>

<221> variant

<222> (7) .. (7)

<223> X = "V" or "F" or "Y"

<220>

<221> variant

<222> (9) .. (9)

<223> X = "T" or "S"

<400> 59

<210> 60

<211> 22

<212> DNA

<213> Artificial

<220>

<223> HIV-1 Gag pre-primer

<400> 60

<210> 61

<211> 29

<212> DNA

<213> Artificial

<220>

<223> HIV-1 Gag probe

<400> 61

<210> 62

<211> 30

<212> DNA

<213> Artificial

<220>

<223> HIV-1 Gag reverse primer

<400> 62

<210> 63

<211> 4

<212> PRT

<213> Artificial

<220>

<223> When n is 0 and X11 is not present, the C-terminus extends to the epitope binding region or a single variable region

<400> 63

<210> 64

<211> 5

<212> PRT

<213> Artificial

<220>

<223> When n is 1 and X11 is not present, the C-terminus extends to the epitope binding region or a single variable region

<400> 64

<210> 65

<211> 12

<212> PRT

<213> Artificial

<220>

<223> When n is 0 and X11 is present, the C-terminus extends to the epitope binding region or a single variable region

<220>

<221> PEPTIDE

<222> (5) .. (12)

<223> Win? It can end at positions 6, 7, 8, 910, 11, or 12. Win? From 5 to 12 can include the following sequence: A, AAA or T.

<400> 65

<210> 66

<211> 13

<212> PRT

<213> Artificial

<220>

<223> When n is 1 and X11 is present, the C-terminus extends to the epitope binding region or a single variable region

<220>

<221> PEPTIDE

<222> (6) .. (13)

<223> Win? It can end at positions 7, 8, 910, 11, 12, or 13. Win? From 6 to 13 can include the following sequence: A, AAA or T.

<400> 66

<210> 67

<211> 5

<212> PRT

<213> Artificial

<220>

<223> When n is 0 and X11 is A, the C-terminus extends to the epitope binding region or single variable region

<400> 67

<210> 68

<211> 6

<212> PRT

<213> Artificial

<220>

<223> When n is 1 and X11 is A, the C-terminus extends to the epitope binding region or single variable region

<400> 68

<210> 69

<211> 6

<212> PRT

<213> Artificial

<220>

<223> When n is 0 and X11 is AS, the C-terminus extends to the epitope binding region or single variable region

<400> 69

<210> 70

<211> 7

<212> PRT

<213> Artificial

<220>

<223> When n is 1 and X11 is A, the C-terminus extends to the epitope binding region or single variable region

<400> 70

<210> 71

<211> 7

<212> PRT

<213> Artificial

<220>

<223> When n is 0 and X11 is AST, the C-terminus extends to the epitope binding region or single variable region

<400> 71

<210> 72

<211> 8

<212> PRT

<213> Artificial

<220>

<223> When n is 1 and X11 is AST, the C-terminus extends to the epitope binding region or single variable region

<400> 72

<210> 73

<211> 7

<212> PRT

<213> Artificial

<220>

<223> When n is 0 and X11 is ASTK, the C-terminus extends to the epitope binding region or single variable region

<400> 73

<210> 74

<211> 9

<212> PRT

<213> Artificial

<220>

<223> When n is 1 and X11 is ASTK, the C-terminus extends to the epitope binding region or single variable region

<400> 74

<210> 75

<211> 9

<212> PRT

<213> Artificial

<220>

<223> When n is 0 and X11 is ASTKG, the C-terminus extends to the epitope binding region or single variable region

<400> 75

<210> 76

<211> 10

<212> PRT

<213> Artificial

<220>

<223> When n is 1 and X11 is ASTKG, the C-terminus extends to the epitope binding region or single variable region

<400> 76

<210> 77

<211> 10

<212> PRT

<213> Artificial

<220>

<223> When n is 0 and X11 is ASTKGP, the C-terminus extends to the epitope binding region or single variable region

<400> 77

<210> 78

<211> 11

<212> PRT

<213> Artificial

<220>

<223> When n is 1 and X11 is ASTKGP, the C-terminus extends to the epitope binding region or single variable region

<400> 78

<210> 79

<211> 215

<212> PRT

<213> Artificial

<220>

<223> Heavy chain amino acid sequence of CTLA-4 / PD-1 mAbdAb CTLA4x51A09-188001

<400> 79

<210> 80

<211> 579

<212> PRT

<213> Artificial

<220>

<223> Heavy chain amino acid sequence of CTLA-4 / PD-1 mAbdAb CTLA4x51A09-188001

<400> 80

Claims (60)

    A binding protein comprising an antibody specific for human LAG-3 is connected by a linker to one or more epitope binding regions specific for human PD-1, wherein the pair of human LAG-3 The specific resistance system includes one or more CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: CDRH1 as shown in SEQ ID NO: 1 and by adding or deleting or replacing 1, 2 or 3 amino acids and CDRH1 different from CDRH1 shown in SEQ ID NO: 1, wherein CDRH2 is selected from the group consisting of: CDRH2 shown in SEQ ID NO: 1 and by addition or deletion or substitution 1 , 2 or 3 amino acids and CDRH2 different from CDRH2 shown in SEQ ID NO: 1, and CDRH3 is selected from the group consisting of: CDRH3 shown in SEQ ID NO: 1 and by adding or CDRH3 that is different from CDRH3 shown in SEQ ID NO: 1 by deleting or replacing 1, 2 or 3 amino acids; wherein the anti-body system specific for human LAG-3 includes one or more CDRL1, CDRL2 and CDRL3, CDRL1 is selected from the group consisting of: CDRL1 as shown in SEQ ID NO: 2 and by adding or deleting or replacing 1 , 2 or 3 amino acids and CDRL1 different from CDRL1 shown in SEQ ID NO: 2, wherein CDRL2 is selected from the group consisting of: CDRL2 shown in SEQ ID NO: 2 and by addition or deletion Or replace 1, 2, or 3 amino acids and CDRL2 different from CDRL2 shown in SEQ ID NO: 2, and among them CDRL3 is selected from the group consisting of: CDRL3 shown in SEQ ID NO: 2 and borrow CDRL3 different from CDRL3 shown in SEQ ID NO: 2 by adding or deleting or replacing 1, 2, or 3 amino acids; wherein the one or more epitope binding regions specific for human PD-1 Including one or more CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: CDRH1 as shown in SEQ ID NO: 3 and by adding or deleting or replacing 1, 2 or 3 amino acids CDRH1 different from CDRH1 shown in SEQ ID NO: 3, wherein CDRH2 is selected from the group consisting of: CDRH2 shown in SEQ ID NO: 3 and 1, 2 or 3 amines by adding or deleting or replacing The basic acid is different from CDRH2 shown in SEQ ID NO: 3, and CDRH3 is selected from the group consisting of: CDRH3 shown in SEQ ID NO: 3 And CDRH3 different from CDRH3 shown in SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids; and wherein the linker is a group consisting of a bond or a peptide linker Selected.         The binding protein according to claim 1, wherein the anti-body system specific for human LAG-3 includes CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: CDRH1 as shown in SEQ ID NO: 1 and CDRH1 different from CDRH1 shown in SEQ ID NO: 1 by adding or deleting or substituting 1, 2 or 3 amino acids, where CDRH2 is selected from the group consisting of: as shown in SEQ ID NO: 1 CDRH2 and CDRH2 different from CDRH2 shown in SEQ ID NO: 1 by adding or deleting or replacing 1, 2 or 3 amino acids, and CDRH3 is selected from the group consisting of: SEQ ID NO : CDRH3 shown in 1 and CDRH3 different from CDRH3 shown in SEQ ID NO: 1 by adding or deleting or substituting 1, 2 or 3 amino acids; of which the specific anti-body system to human LAG-3 Including CDRL1 and CDRL2, wherein CDRL1 is selected from the group consisting of: CDRL1 as shown in SEQ ID NO: 2 and with SEQ ID NO: 2 by adding or deleting or replacing 1, 2 or 3 amino acids The CDRL1 shown is different from the CDRL1, and the CDRL2 is selected from the group consisting of: The CDRL2 shown in SEQ ID NO: 2 is CDRL2 different from CDRL2 shown in SEQ ID NO: 2 by adding or deleting or substituting 1, 2 or 3 amino acids; and one or more of the epitope binding specific to human PD-1 The block has CDRH1, CDRH2 and CDRH3, wherein CDRH1 is selected from the group consisting of: CDRH1 as shown in SEQ ID NO: 3 and by adding or deleting or replacing 1, 2 or 3 amino acids The CDRH1 shown in ID NO: 3 is different from the CDRH1, in which CDRH2 is selected from the group consisting of: CDRH2 shown in SEQ ID NO: 3 and by adding, deleting or replacing 1, 2 or 3 amino acids The CDRH2 different from the CDRH2 shown in SEQ ID NO: 3, and the CDRH3 among them are selected from the group consisting of: CDRH3 shown in SEQ ID NO: 3 and by adding or deleting or replacing 1, 2 or 3 Each amino acid is different from CDRH3 shown in SEQ ID NO: 3.         The binding protein according to claim 1 or 2, wherein the antibody specific for LAG-3 is an IgA or IgG class.         The binding protein according to claim 3, wherein the antibody specific for LAG-3 is an IgG class.         The binding protein according to any of the preceding claims, wherein the one or more epitope binding regions specific for human PD-1 are a single variable region of an antibody (wherein the single variable region is a VH region as needed).         The binding protein according to any of the preceding claims, wherein the one or more epitope binding regions are connected by a linker to the C-terminus of an antibody heavy chain specific for human LAG-3.         According to the binding protein of claim 6, there are two epitope binding regions, one is connected to the C-terminus of each heavy chain of the antibody specific for LAG-3.     A binding protein with general formula (I): Wherein: H (LAG-3) is an IgG antibody heavy chain including CDRH1, CDRH2 and CDRH3, wherein the CDRH1 is selected from: CDRH1 shown in SEQ ID NO: 1, and by adding or deleting or replacing 1, 2 or 3 amino acids and CDRH1 different from CDRH1 shown in SEQ ID NO: 1; wherein CDRH2 is selected from: CDRH2 shown in SEQ ID NO: 1 and by adding or deleting or replacing 1, 2 or 3 Amino acids and CDRH2 different from CDRH2 shown in SEQ ID NO: 1; and CDRH3 is selected from: CDRH3 shown in SEQ ID NO: 1 and by adding, deleting or replacing 1, 2 or 3 Amino acid and CDRH3 different from CDRH3 shown in SEQ ID NO: 1; L (LAG-3) is an IgG antibody light chain including CDRL1 and CDRL2, wherein the CDRL1 is selected from: SEQ ID NO: 2 The CDRL1 shown and the CDRL1 different from the CDRL1 shown in SEQ ID NO: 2 by adding or deleting or substituting 1, 2 or 3 amino acids; and the CDRL2 is selected from: SEQ ID NO: 2 CDRL2 and CDRL2 different from CDRL2 shown in SEQ ID NO: 1 by adding or deleting or substituting 1, 2 or 3 amino acids; n is an integer selected from 2, 4 and 11; A is a single bond or peptide linker; and V _H (PD-1) is An antibody heavy chain variable region having CDRH1, CDRH2 and CDRH3, wherein the CDRH1 is selected from: CDRH1 shown in SEQ ID NO: 3, and by adding or deleting or replacing 1, 2 or 3 amino acids and CDRH1 different from CDRH1 shown in SEQ ID NO: 3; wherein CDRH2 is selected from: CDRH2 shown in SEQ ID NO: 3 and is different from SEQ ID NO by adding or deleting or replacing 1, 2 or 3 amino acids : CDRH2 different from CDRH2 shown in 3; and CDRH3 is selected from: CDRH3 shown in SEQ ID NO: 3 and it is different from SEQ ID NO: 3 by adding or deleting or replacing 1, 2 or 3 amino acids The CDRH3 shown is different from the CDRH3. The binding protein according to any of the preceding claims, wherein CDRH1 of V _H (PD-1) is THYMX ₄ and X ₄ is V or A; wherein CDRH2 of V _H (PD-1) is FIGPAGDX ₅ TYYADSVX ₆ G where X ₅ Is T, F or S and X ₆ is K or E; and CDH3 of V _H (PD-1) is YTX ₇ TSX ₈ X ₉ DX ₁₀ YDV, where X ₇ is A or E and X ₈ is G and S Or D, X ₉ is V, F or Y, and X ₁₀ is T or S. The binding protein according to any of the preceding claims, wherein the CDRL1 or L of the antibody specific for LAG-3 is RASQX ₁ ISSX ₂ LX ₃ , wherein X ₁ is G or S, X ₂ is W, F or Y, and X ₃ is A or N.     The binding protein according to any of the preceding claims, wherein the anti-specific system for LAG-3 or H (LAG-3) has CDRH1, CDRH2 and CDRH3 as shown in SEQ ID NO.1.         The binding protein according to any of the preceding claims, wherein the anti-specific system for LAG or L (LAG-3) has CDRL1 and CDRL2 as shown in SEQ ID NO.2.         The binding protein according to claim 11 or 12, wherein the resistance system specific to LAG-3 or L (LAG-3) has CDRL1, CDRL2, and CDRL3 as shown in SEQ ID NO.     A binding protein according to any preceding claim, wherein one or more epitope binding regions specific for PD-1 or _VH (PD-1) have CDRH1, CDRH2 and CDRH3 as shown in SEQ ID NO.3 .     The binding protein according to claim 11, wherein in antibodies specific for LAG-3 or H (LAG-3), CDRH1 has the sequence as defined in SEQ ID NO: 4 and CDRH2 has the sequence as SEQ ID NO: The sequence defined by 5 and CDRH3 have the sequence defined by SEQ ID NO: 6.         The binding protein according to claim 13 or 15, wherein in an antibody specific for LAG-3 or L (LAG-3), CDRL1 has the sequence as defined in SEQ ID NO: 7, and CDRL2 has the sequence as SEQ ID NO : 8 defined sequence, and CDRL3 has the sequence defined as SEQ ID NO: 9.     The binding proteins according to claims 14, 15, and 16, wherein in one or more epitope binding regions specific for PD-1 or _VH (PD-1), CDRH1 has the sequence shown in SEQ ID NO: 10 For the defined sequence, CDRH2 has the sequence defined by SEQ ID NO: 11, and CDRH3 has the sequence defined by SEQ ID NO: 12.     A binding protein according to any preceding claim, wherein the heavy chain of an antibody specific for human LAG-3 or H (LAG-3) comprises the sequence as defined in SEQ ID NO. 1 or has up to 10 amino acids Add, delete or replace variants of SEQ ID NO.1 of the difference.         The binding protein according to claim 18, wherein the up to 10 amino acid additions, deletions, or substitutions are not within the CDR region.         A binding protein according to any preceding claim, wherein the light chain of an antibody specific for human LAG-3 or L (LAG-3) comprises the sequence as defined in SEQ ID NO. 2 or has up to 10 amino acids Add, delete or replace variants of SEQ ID NO.2.         The binding protein according to claim 20, wherein the up to 10 amino acid additions, deletions, or substitutions are not within the CDR region.     The binding protein according to any preceding claim, wherein one or more epitope binding regions specific for human PD1 or V _H (PD-1) include the sequence as defined in SEQ ID NO. 3 or have up to 10 Amino acid additions, deletions or substitutions of variants of SEQ ID NO. 3.     The binding protein according to claim 22, wherein the up to 10 amino acid additions, deletions, or substitutions are not within the CDR region.         The binding protein according to any one of claims 1 to 17, wherein the heavy chain of the antibody specific for human LAG-3 or H (LAG-3) includes the sequence as defined in SEQ ID NO. The ID NO.1 sequence has at least 90% sequence identity.         The binding protein according to claim 24, wherein the variation occurs outside the CDR region.         According to the binding protein of any one of claims 1 to 17 or 24 to 25, the light chain of an antibody specific for human LAG-3 or L (LAG-3) includes the sequence as defined in SEQ ID NO. 2. Or a sequence having at least 90% sequence identity with the sequence of SEQ ID NO.         The binding protein according to claim 26, wherein the variation occurs outside the CDR region.     The binding protein according to any one of claims 1 to 17 or 24 to 27, wherein one or more epitope binding regions specific for human PD1 or V _H (PD-1) include, for example, SEQ ID NO. 3 The defined sequence or a sequence having at least 90% sequence identity with the SEQ ID NO. 3 sequence.     The binding protein according to claim 28, wherein the variation occurs outside the CDR region.         The binding protein according to any preceding claim, wherein the heavy chain of the antibody specific for human LAG-3 or H (LAG-3) includes the sequence as defined in SEQ ID NO.1.         The binding protein according to any of the preceding claims, wherein the light chain of the antibody specific for human LAG-3 or L (LAG-3) includes the sequence as defined in SEQ ID NO.2.     According to the binding protein of any preceding claim, one or more of the epitope binding regions specific for human PD-1 or _VH (PD-1) includes the sequence as defined in SEQ ID NO.3.     The binding protein according to any preceding claim, wherein the linker or A is a peptide linker.         The binding protein according to claim 33, wherein the linker or A has the sequence of SEQ ID NO.30.         According to the binding protein of any of the preceding claims, the binding protein exhibits> 50% inhibition of LAG3-MHCII interaction in competitive flow cytometry analysis and has a value of less than or equal to 5 nM in the PD-1 / PDL-1 competitive analysis IC50.         An isolated nucleic acid which encodes the heavy chain of the binding protein as defined in any one of claims 1 to 7.         An isolated nucleic acid which encodes the light chain of the binding protein as defined in any one of claims 1 to 7.     An isolated nucleic acid encoding H (LAG-3) -AV _H (PD-1), wherein H (LAG-3), A and V _H (PD-1) are as described in any of claims 8 to 35 definition.     An isolated nucleic acid encoding L (LAG-3), wherein L (LAG-3) is as defined in any one of claims 8 to 35.         A vector comprising the nucleic acid as defined in any one of claims 36 to 39.         According to the carrier of claim 40, it is a performance carrier.         A host cell comprising the vector according to claim 41.         A method for producing a binding protein as defined in any one of claims 1 to 7, which comprises culturing the host cell according to claim 42 and isolating the binding protein under conditions suitable for protein expression, wherein the host cell is in one or more Each expression vector contains the nucleic acid as defined in claim 36 or 37.         A method of manufacturing a binding protein as defined in any one of claims 8 to 35, which comprises culturing the host cell according to claim 42 and isolating the binding protein under conditions suitable for protein expression, wherein the host cell is in one or more Each expression vector contains the nucleic acid as defined in claim 38 or 39.         A pharmaceutical composition comprising the binding protein according to any one of claims 1 to 35 and a pharmaceutically acceptable excipient.         The binding protein as defined in any one of claims 1 to 35 is used as medicine.         The binding protein as defined in any one of claims 1 to 35 is used to treat cancer.         The binding protein as defined in any one of claims 1 to 35 is used for the treatment of infectious diseases.         The binding protein used according to claim 48, wherein the infectious disease is bacterial infection, parasitic infection, viral infection, or sepsis.         The binding protein as defined in any one of claims 1 to 35 is used to treat HIV.         The binding protein as defined in any one of claims 1 to 35 is used to cure HIV.         The binding protein and one or more antiretroviral agents as defined in any one of claims 1 to 35 are for individual simultaneous or sequential use for the treatment of HIV.         The binding protein and one or more anti-retroviral agents as defined in any one of claims 1 to 35 are for individual simultaneous or sequential use to cure HIV.         The use of the binding protein as defined in any one of claims 1 to 35 is for the manufacture of medicines for the treatment of cancer.         The use of the binding protein as defined in any one of claims 1 to 35 is for the manufacture of pharmaceutical products for the treatment of infectious diseases.         Use of the binding protein according to claim 55, wherein the infectious disease is bacterial infection, parasitic infection, viral infection or sepsis.         The use of the binding protein as defined in any one of claims 1 to 35 is for the manufacture of pharmaceutical products for the treatment of HIV.         The use of the binding protein as defined in any one of claims 1 to 35 is for the manufacture of pharmaceutical products for the cure of HIV.         The use of the binding protein and one or more antiretroviral agents as defined in any one of claims 1 to 35 is for the manufacture of pharmaceutical products for the treatment of HIV simultaneously or sequentially.         The use of the binding protein and one or more antiretroviral agents as defined in any one of claims 1 to 35 is for the manufacture of pharmaceutical products for the treatment of HIV simultaneously or sequentially.