Invented by Laurence Fayadat-Dilman, Veronica Juan, Shireen Khan, Shaopeng Huang, Hua Ying, Eric Escobar Cabrera, Genevieve Desjardins, ZYMEWORKS Inc, Zymeworks BC Inc, Merck Sharp and Dohme LLC
The ZYMEWORKS Inc, Zymeworks BC Inc, Merck Sharp and Dohme LLC invention works as followsThe present invention provides anti-PD-1 and antigen binding fragments. These antibodies and antigen binding fragments can be used to treat cancer and infectious diseases.
Background for Anti-PD-1/LAG3 bispecific antibodies
PD-1 has been recognized as a key molecule for immune regulation and peripheral tolerance maintenance. PD-1 expression is low on naive T and B cells, and increased by TB cell signaling in lymphocytes, myeloid and monocyte cells.
Two known ligands of PD-1 (B7H1) and PDL2 (B7DC) are expressed in cancers arising from various human tissues. In large samples of e.g. In large sample sets of melanoma and melanoma (e.g. PD-1 on tumor-infiltrating lymphocytes marked dysfunctional T cells for breast cancer, melanoma and renal cancer (14-15). It has been suggested that PD-L1 expressing tumour cells interact with PD-1 expressing cells of T cells in order to reduce T cell activation, evade immune surveillance and contribute to an impaired immune reaction against the tumor.
Several monoclonal antibody (mAb) inhibiting the interaction between PD-1, and either one or both of its ligands PD-L1 or PD-L2, are currently in clinical development to treat cancer. The efficacy of these antibodies may be increased if they are administered with other cancer treatments, such as radiation, surgery or chemotherapeutics.
LAG3 is a cell-surface molecule that is expressed on activated T lymphocytes (Huard et. Immunogenetics 39:213-217, 1994), NK cells (Triebel et al. J Exp Med 171:1393-1405, 1990), B cells (Kisielow et al. Eur J Immunol, 35:2081-2088 (2005), and plasmacytoid Dendritic Cells (Workman et.al. J Immunol (182:1885-1891 2009) plays an important part in the function these lymphocyte subsets. The interaction between LAG3’s major ligand Class II MHC and LAG3 is also thought to modulate dendritic cells function (Andreae, et al. J Immunol 168:3874-3880, 2002). Recent preclinical research has documented that LAG-3 plays a role in CD8 T cell exhaustion. (Blackburn, et.al. Nat Immunol 10:29-37, 2009). Like chronic viral infections, tumor-specific CD4+/CD8+ T-cells display an impaired effector function as well as an exhausted phenotype that is characterized by decreased pro-inflammatory production and hyporesponsiveness when antigenic stimulation is re-stimulated. Cell extrinsic and cell intrinsic mechanisms are responsible for this, including regulatory T-cells. These inhibitory mechanisms are a formidable barrier for effective antitumor immune response.
LAG3 expression on tolerized TILs suggests that they contribute tumor-mediated immunity suppression. Inhibition LAG3 could lead to increased activation of antigen specific T cells, which may have therapeutic benefits. “There is a requirement in the field for high-efficacy therapeutic antibodies that antagonize LAG3 and PD-1 activity, which can be used as a tool to generate a robust immunological response against tumors.
The anti-PD-1/LAG-3 monospecific antibody is provided herein as a bispecific antigen-binding antibody that comprises: a humanized antigen-binding Fab98, 99 or 100 affinity-matured heavy and/or light chain CDRs with or without a G56A deamidation-site correction (sequential numbersing) and/or S61N glycosylation-site correction in the CDRH2 regions, and a anti-LAG3 antigen
The invention also provides a bispecific anti-PD-1/LAG-3 antibody that comprises: an antigen-binding Fab98, 99 or 100 humanized affinity-matured heavy and variable regions, with or without a S61N site correction, or a G56A site correction in the CDRH2 area, and a antigen binding fragment of anti-LAG3 humanized 22D2 Ab6 heavy and variable regions.
Also provided herein is an anti PD-1/LAG-3 monospecific antibody, comprising: a PD-1 antigen binding fragment including humanized 08A heavy chain and light-chain CDR regions and a PD-3 antigen binding fragment comprising heavy and lighter chain CDRs of the anti LAG3 antibody 22D2 ab6. This invention also provides an anti PD-1/LAG-3 monospecific antibody that comprises: an antigen-binding fraction comprising humanized heavy and lighter chain variable region with or without a G56A deamidation and/or S61N glycosylation correction in the CDRH2 area and an antigen binding fragment consisting of the heavy and lightweight chain variable regions from the anti-LAG3 22D2 antibody.
Also provided herein is a bispecific anti-PD-1/LAG-3 antibody that comprises: an antigen-binding PD-1 fragment comprising humanized 08A affinity-matured Fab128, 133 or 138 heavy and lighter chain CDRs with or without G56A deamidation sites corrections in the CDRH2 regions and an antigen binding anti-LAG3 fragment comprising heavy and lightweight chain CDRs of anti-LAG3 22D2 ab6. This invention also provides an anti PD-1/LAG-3 monospecific antibody that comprises: an anti PD-1 antigen binding fragment consisting of humanized 08A-affinity matured Fab128, 133 or 138 heavy and 139 light chain variable region with or without G56A deamidation sites corrections in the CDRH2 regions and an anti PD-1 antigen binding fragment consisting of the heavy and the light chain variable areas from anti-LAG3 22D2 ab6.
In certain embodiments, an antigen-binding anti-PD-1 fragment consists of a heavy-chain kappa-constant region containing mutations in Q124R and T178R. The antigen binding anti-LAG3 fragment consists of an antigen-binding anti-LAG3-heavy-chain kappa-constant region containing mutations in S181K.
In other embodiments, the IgG1 heavy chain constant regions of the anti-PD-1 and anti-LAG3 antigen-binding fragments further comprise pairs of CH3 mutations selected from the group consisting of: L351Y/F405A/Y407V and T366I/K392M/T394W; T350V/L351Y/F405A/Y407V and T350V/T366L/K392L/T394W; and T350V/L351Y/F405A/Y407V and T350V/T366L/K392M/T394W, wherein the mutations are in EU numbering.
The anti-LAG3 heavy chain and anti-PD-1 heavy chain each contain one or more mutations of the following: L234A, L234D, D265S, D265A, G237A, and N297A.
Also, herein are isolated RNAs encoding any of the anti PD-1/LAG3 monospecific antibodies or antigen binding fragments that we provide. Expression vectors containing such nucleic acids are also provided (where said polypeptides may optionally contain a leader sequence). These isolated nucleic acid and expression vectors can be used to express antibodies or antigen binding fragments of them in recombinant hosts cells. Host cells containing such isolated nucleic acid are also provided. In one embodiment the host cell is a Chinese hamster cell. In one embodiment the host cell can be a yeast, such as a Pichia or Pichia pastoris cell.
To make the invention easier to understand, we have defined certain technical and scientific words below. All other technical and science terms are used in this document unless they are defined in another part of this document. They have the same meaning as a person with ordinary knowledge in the field to which the invention belongs.
The singular forms of words like?a?,?,?,?,?,?,?,?,?,?,?,?,?,?,?,? ?an,? The plural forms of?an,? Include their plurals unless the context is clear.
?Administration? “?Administration? As it relates to an animal or human subject, cell or tissue, organ or biological liquid, the term refers to the contact of an exogenous composition, pharmaceutical, therapeutic or diagnostic agent. The treatment of a single cell includes contact of the reagent with the cell as well as the contact of the reagent with a fluid that is in contact the cell. ?Administration? Treatment? Also means ex vivo or in vitro treatments. For example, of a single cell by a reagent.
?Treat? “?Treat? Administering a therapeutic agent (such as a composition that contains any of the antibodies, antigen-binding fractions, or other components provided herein) to a patient or subject who has one or more symptoms of disease or is suspected to have a disease for which the agent exhibits therapeutic activity. The agent is typically administered at a dose that will alleviate the symptoms of a disease in the subject or population being treated, either by causing the regression or inhibiting progression of the symptoms by any clinically measureable degree. The amount of a drug that can be used to relieve a particular disease symptom will vary depending on factors like the severity of the disease, the age and weight of the subject, as well as its ability to produce a desired reaction in the subject. The clinical measurements that are used by doctors or other healthcare professionals to determine the severity of a symptom or its progression can be used to determine whether a symptom is alleviated.
The term “antibody” is used in this context. Any form of antibody that exhibits a desired biological activity is considered an antibody. It is used in a broad sense to include all types of antibodies that exhibit the desired biological activity.
The basic structural unit of an antibody is a tetramer.” Each tetramer contains two pairs of identical polypeptide chains. Each pair has one “light” Each tetramer contains two identical pairs of polypeptide chains, each pair having one ‘light? The amino-terminal portion of each chain includes a variable region of about 100 to 110 or more amino acids that is primarily responsible for antigen recognition. The amino-terminal part of each chain contains a variable region of 100 to 110 amino acids or more, which is primarily responsible for antigen detection. The carboxy-terminal part of the heavy chains can define a constant area primarily responsible for effects function.
The term “light chain” is used to describe a polypeptide chain of about 25 kDa, where the amino-terminal portion includes a variable region of approximately 100 to 110 or more amino acids and whereas the carboxy-terminal portion includes a constant region. When used to refer to an antigen, the term “light chain” refers to a 25 kDa polypeptide, with a variable amino-terminal region of 100 to 110 amino acids or more, and a carboxyterminal region that is constant. A light chain’s approximate length is between 211 and 217 amino acid. Human light chains are usually classified into kappa Typically, human light chains are classified as kappa (?) Light chains are based on the sequence of amino acids in the constant domains. The amino acid sequences of light chains are well-known in the art. “A light chain can also be a human-light chain.
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