Patent Grant
10919966
This application is a U.S. national phase under 35 U.S.C. § 371 of International Patent Application No. PCT/KR17/08495 filed Aug. 7, 2017, which in turn claims the priorities under 35 U.S.C. § 119 of Korean Patent Application No. 10-2016-0100211 filed Aug. 5, 2016 and Korean Patent Application 10-2017-0099673 filed Aug. 7, 2017. The disclosures of such International Patent Application No. PCT/KR17/08495, Korean Patent Application No. 10-2016-0100211, and Korean Patent Application 10-2017-0099673 are hereby incorporated herein by reference in their respective entireties, for all purposes.
The present disclosure relates to an antibody to human programmed cell death-ligand 1 (PD-L1) or an antigen-binding fragment thereof, a nucleic acid encoding the same, a vector including the nucleic acid, a cell transformed with the vector, a method for producing the antibody or an antigen-binding fragment thereof, and a composition for preventing or treating cancer or infectious diseases containing the same.
The immune response of antigen-specific T-lymphocyte cells is a process that is very complicated and regulated delicately. First of all, activation of T-lymphocytes begins when the T-cell antigen receptor (TCR) present on the surface of T-lymphocytes recognizes the major histocompatibility complex (MHC) of the antigen-presenting cells (APCs), and, in humans, antigens bound to class II molecules of HLA (human leucocyte antigen). In this case, for sufficient activation of T-lymphocytes, in addition to the recognition of antigens, co-stimulatory signals are required, which are obtained, when CD80, CD40 and the like expressed in antigen presenting cells simultaneously bind to CD28, CD40L and the like, which are ligands present on the surface of T-lymphocyte cells. As a result, the secretion of cytokines is activated. Activation of T-lymphocytes is not achieved in the absence of transfer of co-stimulatory signals, although the antigen is recognized by the binding of TCR-MHC/epitope.
However, co-inhibitory signals are also activated so that activated T-lymphocytes become inactive after a period of time. This can prevent tissue damage and the like due to excessive immune stimulation. There are a variety of co-inhibitory signals and representatively, cytotoxic T lymphocyte antigen (CTLA)-4 and programmed death-1 (PD-1) of T lymphocytes and antigen-presenting cell ligands corresponding thereto are involved in CD80 and CD86, and PD-L1 (programmed death-ligand 1). CTLA-4 functions to inactivate naive or memory T-lymphocytes by binding to the ligands, CD80 and CD86. PD-1 functions to regulate functions of T-lymphocytes in peripheral tissues through PD-L1 and PD-L2.
The immune function of the human body is to recognize antigens and, at the same time, to regulate the overall T lymphocyte functions through regulation of these co-stimulatory and co-inhibitory signals. This regulatory mechanism is called “immune checkpoint”. The human immune function is to detect tumor-specific neo-antigens expressed by variations such as mutations occurring in tumor cells and thereby to eliminate tumor cells or virus infection sources.
On the other hand, in order to avoid such immune attacks, some tumor cells inhibit immune functions by altering the tumor microenvironments or perform immune escape by T-cell immunity tolerance or immuno-editing.
One of immune escape strategies is to inhibit the functions of tumor-specific T lymphocytes through changes in immune checkpoint functions. That is, the attack of tumor-specific T-lymphocyte cells is avoided by activating such an inhibitory immune checkpoint in tumor cells. In this regard, activities and effects of inhibited tumor-specific T-lymphocyte cells are improved by inhibiting functions thereof using monoclonal antibodies against PD-1 or a ligand thereof, PD-L1, so that antitumor effects can be obtained.
Under these technical backgrounds, the present inventors have made efforts to develop antibodies specifically binding to PD-L1. As a result, the present inventors have developed anti-PD-L1 antibodies that bind with a high affinity to PD-L1, and have found that the anti-PD-L1 antibody can serve as the desired immune anticancer agent or therapeutic agent for infectious diseases by inhibiting the formation of the PD-1/PD-L1 complex, thus completing the present disclosure.
Therefore, it is one object of the present disclosure to provide a novel antibody to PD-L1 or an antigen-binding fragment thereof.
It is another object of the present disclosure to provide a nucleic acid encoding the antibody or an antigen-binding fragment thereof.
It is another object of the present disclosure to provide a vector including the nucleic acid, a cell transformed with the vector, and a method for preparing the same.
It is another object of the present disclosure to provide a composition for preventing or treating cancer or infectious diseases containing the antibody or antigen-binding fragment thereof.
In accordance with the present disclosure, the above and other objects can be accomplished by the provision of an antibody binding to PD-L1 or an antigen-binding fragment thereof including a heavy chain variable region including a heavy chain CDR1 including a sequence having a sequence identity of 90% or higher with a sequence selected from the group consisting of sequences as set forth in synthetic construct: 1 to 7, a heavy chain CDR2 including a sequence having a sequence identity of 90% or higher with a sequence selected from the group consisting of sequences as set forth in SEQ ID NOS: 8 to 15, and a heavy chain CDR3 including a sequence having a sequence identity of 90% or higher with a sequence selected from the group consisting of sequences as set forth in SEQ ID NOS: 16 to 25, and a light chain variable region including a light chain CDR1 including a sequence having a sequence identity of 90% or higher with a sequence selected from the group consisting of sequences as set forth in SEQ ID NOS: 88 to 102, a light chain CDR2 including a sequence having a sequence identity of 90% or higher with a sequence selected from the group consisting of sequences as set forth in SEQ ID NOS: 103 to 119, and a light chain CDR3 including a sequence having a sequence identity of 90% or higher with a sequence selected from the group consisting of sequences as set forth in SEQ ID NOS: 120 to 144.
In accordance with another aspect of the present disclosure, provided is a nucleic acid encoding the antibody or an antigen-binding fragment.
In accordance with another aspect of the present disclosure, provided is an expression vector including the nucleic acid.
In accordance with another aspect of the present disclosure, provided is a cell transformed with the expression vector.
In accordance with another aspect of the present disclosure, provided is a method for producing the antibody or an antigen-binding fragment thereof, including (a) culturing the cell, and (b) recovering the antibody or an antigen-binding fragment thereof from the cultured cell.
In accordance with another aspect of the present disclosure, provided is a composition for preventing or treating cancer or infectious diseases containing, as an active ingredient, the antibody or an antigen-binding fragment thereof.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
The above and other objects, features and other advantages of the present disclosure will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which:
hPD-L1 has the sequence FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQ HSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNA (SEQ ID NO: 252);
M1 has the sequence FSITASKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQH SSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNA (SEQ ID NO: 253);
M2 has the sequence FTVTVPKDLYVVEYGSNVTLECRFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQ HSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNA (SEQ ID NO: 254);
M3 has the sequence FTVTVPKDLYVVEYGSNMTIECKFPVERELNLLVLIVYWEMEDKNIIQFVHGEEDLKVQ HSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNA (SEQ ID NO: 255);
M4 has the sequence FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWGKEDEQVIQFVHGEEDLKVQ HSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNA (SEQ ID NO: 256);
M5 has the sequence FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVNGKEDPNPQ HSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNA (SEQ ID NO: 257);
M6 has the sequence FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQ HSNFHGRAQLPKDQLSLGNAALQITDVKLQDAGVYRCMISYGGADYKRITVKVNA (SEQ ID NO: 258);
M7 has the sequence FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQ HSSYRQRARLLKDQLLKGKAVLQITDVKLQDAGVYRCMISYGGADYKRITVKVNA (SEQ ID NO: 259); and
M8 has the sequence FTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWEMEDKNIIQFVHGEEDLKVQ HSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYCCIISYGGADYKRITVKVNA (SEQ ID NO: 260);
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those appreciated by those skilled in the field to which the present disclosure pertains. In general, nomenclature used herein is well-known in the art and is ordinarily used.
In one aspect, the present disclosure is directed to an antibody binding to PD-L1 or an antigen-binding fragment thereof including: a heavy chain variable region including a heavy chain CDR1 including a sequence having a sequence identity of 90% or higher with a sequence selected from the group consisting of sequences as set forth in SEQ ID NOS: 1 to 7, a heavy chain CDR2 including a sequence having a sequence identity of 90% or higher with a sequence selected from the group consisting of sequences as set forth in SEQ ID NOS: 8 to 15, and a heavy chain CDR3 including a sequence having a sequence identity of 90% or higher with a sequence selected from the group consisting of sequences as set forth in SEQ ID NOS: 16 to 25; and a light chain variable region including a light chain CDR1 including a sequence having a sequence identity of 90% or higher with a sequence selected from the group consisting of sequences as set forth in SEQ ID NOS: 88 to 102, a light chain CDR2 including a sequence having a sequence identity of 90% or higher with a sequence selected from the group consisting of sequences as set forth in SEQ ID NOS: 103 to 119, and a light chain CDR3 including a sequence having a sequence identity of 90% or higher with a sequence selected from the group consisting of sequences as set forth in SEQ ID NOS: 120 to 144.
As used herein, the term “PD-L1” is a ligand for an immunosuppressive receptor “programmed death receptor 1 (PD-1)” that is predominantly expressed in activated T and B cells, which can negatively regulate antigen receptor signaling. The ligands (PD-L1 and PD-L2) for PD-1 may be constitutively expressed or may be derived into a number of cell types, including non-hematopoietic cell tissues and various tumor types. PD-L1 is expressed in B cells, T cells, bone marrow cells and dendritic cells (DCs), but also on non-lymphatic organs such as peripheral cells, pseudo-vascular endothelial cells and heart, lungs and the like. In contrast, PD-L2 is found only in macrophages and dendritic cells. The expression pattern of the PD-1 ligand suggests the role of PD-1 in maintaining peripheral tolerance and may contribute to the regulation of autoreactive T-cell and B-cell responses in the periphery. Both ligands are type I transmembrane receptors that contain both IgV- and IgC-like domains in the extracellular domain. Both ligands include a short cytoplasmic region having an unknown signaling motif.
A number of studies have shown that the interaction between PD-1 and ligands thereof inhibits lymphocyte proliferation in vitro and in vivo. Disruption of the PD-1/PD-L1 interaction is known to improve proliferation of T cells and production of cytokine and to block the progression of cell cycle. Blocking of the PD-1/PD-L1 interaction can lead to improved tumor-specific T-cell immunity, thus contributing to cleaning of tumor cells with the immune system. In addition, in chronic HIV infection, HIV-specific CD8+ T cells are functionally impaired, exhibiting a reduced ability to produce cytokine and effector molecules and a reduced ability to proliferate the same, and PD-1 is highly expressed in HIV-specific CD8+ T cells, which can improve T cell activity or anti-viral immune reactions by enhancing the ability to proliferate HIV-specific T cells and the ability to produce cytokines in response to HIV peptide stimuli through blocking the PD-1/PD-L1 interaction.
As used herein, the term “antibody” refers to an anti-PD-L1 antibody that specifically binds to PD-L1. The scope of the present disclosure includes not only a complete antibody specifically binding to PD-L1, but also an antigen-binding fragment of the antibody molecule.
The complete antibody refers to a structure having two full-length light chains and two full-length heavy chains, wherein each light chain is linked to the corresponding heavy chain by a disulfide bond. The heavy chain constant region has gamma (γ), mu (μ), alpha (α), delta (δ) and epsilon (ε) types and is subclassed into gamma 1 (γ1), gamma 2 (γ2), gamma (γ3), gamma 4 (γ4), alpha 1 (α1) and alpha 2 (α2). The constant region of the light chain has kappa (κ) and lambda (λ) types.
The antigen-binding fragment of an antibody or the antibody fragment refers to a fragment that at least has an antigen-binding capacity and includes Fab, F(ab′), F(ab′)2, and Fv. Among the antibody fragments, Fab refers to a structure including a variable region of each of the heavy chain and the light chain, the constant domain of the light chain, and the first constant domain (CH1) of the heavy chain, each having one antigen-binding site. Fab′ is different from Fab in that it further includes a hinge region including at least one cysteine residue at a C-terminus of the CH1 domain of the heavy chain. F(ab′)2 is created by a disulfide bond between cysteine residues in the hinge region of Fab′. Fv is the minimal antibody fragment having only a heavy chain variable region and a light chain variable region, and recombinant technology for producing Fv, is disclosed in PCT International Publications such as WO88/01649, WO88/06630, WO88/07085, WO88/07086 and WO 88/09344. Two-chain Fv is a fragment wherein the variable region of the heavy chain and the variable region of the light chain are linked by a non-covalent bond, and single-chain Fv is a fragment wherein the variable region of the heavy chain and the variable region of the light chain are generally linked by a covalent bond via a peptide linker between, or are directly linked at the C-terminal, forming a dimer-like structure, like the two-chain Fv. Such antibody fragments may be obtained using proteases (e.g., Fabs can be obtained by restriction-cleaving the whole antibody with papain, and the F(ab′) fragment can be obtained by restriction-cleaving the whole antibody with pepsin), and may be prepared by genetic recombination techniques.
In one embodiment, the antibody of the present disclosure is an Fv form (for example, scFv), Fab or a complete antibody form. In addition, the heavy chain constant region may be selected from the isotypes consisting of gamma (γ), mu (u), alpha (α), delta (δ) or epsilon (c). For example, the constant region may be gamma 1 (IgG1), gamma 3 (IgG3) or gamma 4 (IgG4). The light chain constant region may be kappa or lambda.
As used herein, the term “heavy chain” encompasses both a full-length heavy chain, which includes a variable domain (VH) containing an amino acid sequence having a sufficient variable region sequence for imparting a specificity to an antigen and three constant domains (CH1, CH2 and CH3), and a fragment thereof. As used herein, the term “light chain” encompasses both a full-length light chain, which includes a variable domain (VL) containing an amino acid sequence having a sufficient variable region sequence for imparting specificity to an antigen and a constant domain (CL), and a fragment thereof.
The antibody of the present disclosure includes, but is limited to, monoclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, short chain Fvs (scFVs), short chain antibodies, Fab fragments, F(ab′) fragments, disulfide-bond Fvs (sdFVs), anti-idiotypic (anti-Id) antibodies, or epitope-binding fragments of such antibodies, or the like.
The monoclonal antibody refers to the same antibody, excluding possible naturally occurring mutations where an antibody obtained from a population of substantially homogeneous antibodies, that is, each antibody constituting the population, may be present in a minor amount. Monoclonal antibodies are highly specific and are induced against a single antigenic site. In contrast to conventional (polyclonal) antibody preparations that typically contain different antibodies directed by different determinants (epitopes), each monoclonal antibody is directed by a single determinant on the antigen.
The term “epitope” means a protein determinant to which an antibody can specifically bind. An epitope is usually composed of chemically active surface molecule groups, for example, amino acids or sugar side chains, and generally has specific three dimensional structural characteristics as well as specific charge characteristics. The steric and non-steric epitopes are distinguished from each other in that binding to steric epitopes is lost in the presence of a denaturing solvent, but binding to non-steric epitopes is not lost.
The non-human (e.g., murine) antibody of the “humanized” form is a chimeric antibody containing a minimal sequence derived from non-human immunoglobulin. In most cases, the humanized antibody is a human immunoglobulin (receptor antibody) wherein a residue from the hypervariable region of a receptor is replaced with a residue from the hypervariable region of non-human species (donor antibody), such as a mouse, rat, rabbit or non-human primate having the desired specificity, affinity and ability.
The term “human antibody” means a molecule derived from human immunoglobulin, wherein all the amino acid sequences constituting the antibody including a complementarity-determining region and a structural region are composed of human immunoglobulin.
Some of the heavy chain and/or light chain is identical to or homologous with the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining chain(s) include “chimeric” antibodies (immunoglobulins) which are identical to or homologous with corresponding sequences in an antibody derived from another species or belonging to another antibody class or subclass as well as fragments of such antibody exhibiting desired biological activity.
As used herein, the term “antibody variable domain” refers to the light and heavy chain regions of an antibody molecule including the amino acid sequences of a complementarity determining region (CDR; i.e., CDR1, CDR2, and CDR3) and a framework region (FR). VH refers to a variable domain of the heavy chain. VL refers to a variable domain of the light chain.
The term “complementarity determining region” (CDR; i.e., CDR1, CDR2, and CDR3) refers to an amino acid residue of the antibody variable domain, which is necessary for antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3.
In the present disclosure, the antibody binding to PD-L1 or an antigen-binding fragment thereof includes: a heavy chain variable region including a heavy chain CDR1 selected from the group consisting of SEQ ID NOS: 1 to 7, a heavy chain CDR2 selected from the group consisting of SEQ ID NOS: 8 to 15, and a heavy chain CDR3 selected from the group consisting of SEQ ID NOS: 16 to 25; and a light chain variable region including a light chain CDR1 selected from the group consisting of SEQ ID NOS: 88 to 102, a light chain CDR2 selected from the group consisting of SEQ ID NOS: 103 to 119, and a light chain CDR3 selected from the group consisting of SEQ ID NOS: 120 to 144.
Specifically, the antibody binding to PD-1 or an antigen-binding fragment thereof according to the present disclosure includes:
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 8 and the heavy chain CDR3 of SEQ ID NO: 16;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 18;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 3, the heavy chain CDR2 of SEQ ID NO: 10 and the heavy chain CDR3 of SEQ ID NO: 19;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 4, the heavy chain CDR2 of SEQ ID NO: 11 and the heavy chain CDR3 of SEQ ID NO: 20;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 5, the heavy chain CDR2 of SEQ ID NO: 12 and the heavy chain CDR3 of SEQ ID NO: 21;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 6, the heavy chain CDR2 of SEQ ID NO: 13 and the heavy chain CDR3 of SEQ ID NO: 22;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 23;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 7, the heavy chain CDR2 of SEQ ID NO: 14 and the heavy chain CDR3 of SEQ ID NO: 24;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 15 and the heavy chain CDR3 of SEQ ID NO: 25; or
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17.
In addition, the antibody binding to PD-1 or an antigen-binding fragment thereof includes:
a light chain variable region including the light chain CDR1 of SEQ ID NO: 88, the light chain CDR2 of SEQ ID NO: 103 and the light chain CDR3 of SEQ ID NO: 120;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 121;
a light chain variable region including a light chain CDR1 of SEQ ID NO: 90, the light chain CDR2 of SEQ ID NO: 105 and the light chain CDR3 of SEQ ID NO: 122;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 91, the light chain CDR2 of SEQ ID NO: 106 and the light chain CDR3 of SEQ ID NO: 123;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 107 and the light chain CDR3 of SEQ ID NO: 124;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 92, the light chain CDR2 of SEQ ID NO: 108 and the light chain CDR3 of SEQ ID NO: 122;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 93, the light chain CDR2 of SEQ ID NO: 109 and the light chain CDR3 of SEQ ID NO: 125;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 94, the light chain CDR2 of SEQ ID NO: 110 and the light chain CDR3 of SEQ ID NO: 126;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 95, the light chain CDR2 of SEQ ID NO: 111 and the light chain CDR3 of SEQ ID NO: 127;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 96, the light chain CDR2 of SEQ ID NO: 112 and the light chain CDR3 of SEQ ID NO: 128;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 108 and the light chain CDR3 of SEQ ID NO: 129;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 105 and the light chain CDR3 of SEQ ID NO: 130;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 113 and the light chain CDR3 of SEQ ID NO: 131;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 97, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 132;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 133;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 97, the light chain CDR2 of SEQ ID NO: 114 and the light chain CDR3 of SEQ ID NO: 134;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 92, the light chain CDR2 of SEQ ID NO: 115 and the light chain CDR3 of SEQ ID NO: 135;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 98, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 130;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 116 and the light chain CDR3 of SEQ ID NO: 121;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 108 and the light chain CDR3 of SEQ ID NO: 136;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 99, the light chain CDR2 of SEQ ID NO: 105 and the light chain CDR3 of SEQ ID NO: 137;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 117 and the light chain CDR3 of SEQ ID NO: 138;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 118 and the light chain CDR3 of SEQ ID NO: 133;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 119 and the light chain CDR3 of SEQ ID NO: 139;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 100, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 140;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 108 and the light chain CDR3 of SEQ ID NO: 141;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 105 and the light chain CDR3 of SEQ ID NO: 139;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 142;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 105 and the light chain CDR3 of SEQ ID NO: 143;
a light chain variable region including the light chain CDR1 of SEQ ID NO: 101, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 141; or
a light chain variable region including the light chain CDR1 of SEQ ID NO: 102, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 144.
In one embodiment of the present disclosure, the antibody or an antigen-binding fragment thereof according to the present disclosure may include the following heavy chain variable regions and light chain variable regions:
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 1, the heavy chain CDR2 of SEQ ID NO: 8 and the heavy chain CDR3 of SEQ ID NO: 16, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 88, the light chain CDR2 of SEQ ID NO: 103 and the light chain CDR3 of SEQ ID NO: 120;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 121;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 18, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 90, the light chain CDR2 of SEQ ID NO: 105 and the light chain CDR3 of SEQ ID NO: 122;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 3, the heavy chain CDR2 of SEQ ID NO: 10 and the heavy chain CDR3 of SEQ ID NO: 19, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 91, the light chain CDR2 of SEQ ID NO: 106 and the light chain CDR3 of SEQ ID NO: 123;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 4, the heavy chain CDR2 of SEQ ID NO: 11 and the heavy chain CDR3 of SEQ ID NO: 20, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 107 and the light chain CDR3 of SEQ ID NO: 124;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 5, the heavy chain CDR2 of SEQ ID NO: 12 and the heavy chain CDR3 of SEQ ID NO: 21, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 92, the light chain CDR2 of SEQ ID NO: 108 and the light chain CDR3 of SEQ ID NO: 122;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 6, the heavy chain CDR2 of SEQ ID NO: 13 and the heavy chain CDR3 of SEQ ID NO: 22, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 93, the light chain CDR2 of SEQ ID NO: 109 and the light chain CDR3 of SEQ ID NO: 125;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 23, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 94, the light chain CDR2 of SEQ ID NO: 110 and the light chain CDR3 of SEQ ID NO: 126;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 7, the heavy chain CDR2 of SEQ ID NO: 14 and the heavy chain CDR3 of SEQ ID NO: 24, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 95, the light chain CDR2 of SEQ ID NO: 111 and the light chain CDR3 of SEQ ID NO: 127; or
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 15 and the heavy chain CDR3 of SEQ ID NO: 25, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 96, the light chain CDR2 of SEQ ID NO: 112 and the light chain CDR3 of SEQ ID NO: 128.
According to one embodiment of the present disclosure, the antibody is further screened through an optimization procedure, and the antibody or an antigen-binding fragment thereof according to the invention may include the following heavy chain variable regions and light chain variable regions:
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 108 and the light chain CDR3 of SEQ ID NO: 129;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 105 and the light chain CDR3 of SEQ ID NO: 130;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 113 and the light chain CDR3 of SEQ ID NO: 131;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 97, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 132;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 133;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 97, the light chain CDR2 of SEQ ID NO: 114 and the light chain CDR3 of SEQ ID NO: 134;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 92, the light chain CDR2 of SEQ ID NO: 115 and the light chain CDR3 of SEQ ID NO: 135;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 98, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 130;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 116 and the light chain CDR3 of SEQ ID NO: 121;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 108 and the light chain CDR3 of SEQ ID NO: 136;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 99, the light chain CDR2 of SEQ ID NO: 105 and the light chain CDR3 of SEQ ID NO: 137;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 117 and the light chain CDR3 of SEQ ID NO: 138;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including and the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 118 and the light chain CDR3 of SEQ ID NO: 133;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 119 and the light chain CDR3 of SEQ ID NO: 139;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 100, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 140;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 108 and the light chain CDR3 of SEQ ID NO: 141;
a heavy chain variable region including heavy chain CDR1 of SEQ ID NO: 2, heavy chain CDR2 of SEQ ID NO: 9 and heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 105 and the light chain CDR3 of SEQ ID NO: 139;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 142;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 105 and the light chain CDR3 of SEQ ID NO: 143;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 101, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 141; or
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 102, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 144.
Specifically, the antibody or an antigen-binding fragment thereof according to the invention may include the following heavy chain variable regions and light chain variable regions:
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 121;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 105 and the light chain CDR3 of SEQ ID NO: 130;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 104 and the light chain CDR3 of SEQ ID NO: 133;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 108 and the light chain CDR3 of SEQ ID NO: 136;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 119 and the light chain CDR3 of SEQ ID NO: 139;
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 105 and the light chain CDR3 of SEQ ID NO: 139; or
a heavy chain variable region including the heavy chain CDR1 of SEQ ID NO: 2, the heavy chain CDR2 of SEQ ID NO: 9 and the heavy chain CDR3 of SEQ ID NO: 17, and a light chain variable region including the light chain CDR1 of SEQ ID NO: 89, the light chain CDR2 of SEQ ID NO: 105 and the light chain CDR3 of SEQ ID NO: 143.
The “framework region” (FR) refers to a variable domain residue other than a CDR residue. Each variable domain typically has four FRs identified as FR1, FR2, FR3, and FR4.
According to one embodiment of the present disclosure, the antibody or an antigen-binding fragment thereof may include:
a heavy chain variable region FR1 selected from the group consisting of SEQ ID NOS: 26 to 34;
a heavy chain variable region FR2 selected from the group consisting of SEQ ID NOS: 35 to 41;
a heavy chain variable region FR3 selected from the group consisting of SEQ ID NOS: 42 to 49; or
a heavy chain variable region FR4 selected from the group consisting of SEQ ID NOS: 50 to 54.
In addition, the antibody or an antigen-binding fragment thereof may include:
a light chain variable region FR1 selected from the group consisting of SEQ ID NOS: 145 to 163;
a light chain variable region FR2 selected from the group consisting of SEQ ID NOS: 164 to 184;
a light chain variable region FR3 selected from the group consisting of SEQ ID NOS: 185 to 210; or
a light chain variable region FR4 selected from the group consisting of SEQ ID NOS: 211 to 216.
The “Fv” fragment is an antibody fragment containing complete antibody recognition and binding sites. Such region includes a dimmer, for example, scFv, that consists of one heavy chain variable domain and one light chain variable domain substantially tightly covalently connected to each other.
A “Fab” fragment contains the variable and constant domains of the light chain, and a variable and first constant domain (CH1) of the heavy chain. A F(ab′)2 antibody fragment generally includes a pair of Fab fragments covalently linked via a hinge cysteine located therebetween near the carboxyl end thereof.
The “single chain Fv” or “scFv” antibody fragment includes VH and VL domains of the antibody, wherein these domains are present in a single polypeptide chain. The Fv polypeptide may further include a polypeptide linker between the VH domain and the VL domain in order for the scFv to form a desired structure for antigen binding.
The PD-L1 antibody is monovalent or divalent, and includes short or double chains. Functionally, the binding affinity of PD-L1 antibody ranges from 10−5 M to 10−12 M. For example, the binding affinity of the PD-L1 antibody is 10−6 M to 10−12 M, 10−7 M to 10−12 M, 10−8 M to 10−12 M, 10−9 M to 10−12 M, 10−5 M to 10−11 M, 10−6 M to 10−11 M, 10−7 M to 10−11 M, 10−8 M to 10−11 M, 10−9 M to 10−11 M, 10−10 M to 10−11 M, 10−5 M to 10−10 M, 10−6 M to 10−10 M, 10−7 M to 10−10 M, 10−8 M to 10−10 M, 10−9 M to 10−10 M, 10−5 M to 10−9 M, 10−6 M to 10−9 M, 10−7 M to 10−9 M, 10−8 M to 10−9 M, 10−5 M to 10−8 M, 10−6 M to 10−8 M, 10−7 M to 10−8 M, 10−5 M to 10−7 M, 10−6 M to 10−7 M, or 10−5 M to 10−6 M.
The antibody binding to PD-L1 or an antigen-binding fragment thereof may include a heavy chain variable region including a sequence having a sequence identity of 90% or higher with a sequence selected from the group consisting of sequences as set forth in SEQ ID NOS: 57 to 87. The antibody binding to PD-L1 or an antigen-binding fragment thereof may include a heavy chain variable region selected from the group consisting of sequences as set forth in SEQ ID NOS: 57 to 87. In one embodiment of the present disclosure, the antibody binding to PD-L1 or an antigen-binding fragment thereof may include a heavy chain variable region of SEQ ID NO: 58, 68, 71, 76, 80, 83 or 85.
In addition, the antibody binding to PD-L1 or an antigen-binding fragment thereof may include a light chain variable region including a sequence having a sequence identity of 90% or higher with a sequence selected from the group consisting of sequences as set forth in SEQ ID NOS: 217 to 247. The antibody binding to PD-L1 or an antigen-binding fragment thereof may include a light chain variable region selected from the group consisting of sequences as set forth in SEQ ID NOS: 217 to 247. In one embodiment of the present disclosure, the antibody binding to PD-L1 or an antigen-binding fragment thereof may include a light chain variable region of SEQ ID NO: 218, 228, 231, 236, 240, 243 or 245.
In a specific embodiment according to the present disclosure, the antibody binding to PD-L1 or an antigen-binding fragment thereof may include: ???
a heavy chain variable region of SEQ ID NO: 58 and a light chain variable region of SEQ ID NO: 218;
a heavy chain variable region of SEQ ID NO: 68 and a light chain variable region of SEQ ID NO: 228;
a heavy chain variable region of SEQ ID NO: 71 and a light chain variable region of SEQ ID NO: 231;
a heavy chain variable region of SEQ ID NO: 76 and a light chain variable region of SEQ ID NO: 236;
a heavy chain variable region of SEQ ID NO: 80 and a light chain variable region of SEQ ID NO: 240;
a heavy chain variable region of SEQ ID NO: 83 and a light chain variable region of SEQ ID NO: 243; or
a heavy chain variable region of SEQ ID NO: 85 and a light chain variable region of SEQ ID NO: 245.
“Phage display” is a technique for displaying a mutant polypeptide as a fusion protein with at least a part of a coat protein, for example, on the surface of the particle of a phage, for example, a fibrous phage. The usefulness of phage display is to rapidly and efficiently classify sequences that bind to target antigens with high affinity in large libraries of randomized protein mutants. Displaying peptides and protein libraries on phages has been used to screen millions of polypeptides in order to identify polypeptides with specific binding properties.
Phage display technology has offered a powerful tool for generating and screening novel proteins that bind to specific ligands (e.g., antigens). Using the phage display technology, large libraries of protein mutants can be generated, and sequences binding with high affinity to target antigens can be rapidly classified. The nucleic acid encoding mutant polypeptides is fused with the sequence of nucleic acid encoding viral coat proteins, e.g., gene III proteins or gene VIII proteins. A monophasic phage display system, in which a nucleic acid sequence encoding protein or polypeptide is fused with a nucleic acid sequence encoding a part of the gene III protein, has been developed. In the monophasic display system, a fused gene is expressed at a low level, a wild-type gene III protein is also expressed, and thus particle infectivity is maintained.
It is important to demonstrate the expression of peptides on the fibrous phage surface and the expression of functional antibody fragments in the peripheral cytoplasm of E. coli for the development of antibody phage display libraries. Libraries of antibody- or antigen-binding polypeptides are prepared by a number of methods, for example, of modifying a single gene by inserting a random DNA sequence, or cloning a related gene sequence. The libraries can be screened for the expression of antibody- or antigen-binding proteins with desired characteristics.
Phage display technology has several advantages over conventional hybridomas and recombinant methods for producing antibodies with desired characteristics. This technique provides the generation of large antibody libraries with a variety of sequences within a short time without using animals. The production of hybridomas and the production of humanized antibodies may require a production time of several months. In addition, since no immunity is required, the phage antibody libraries can generate antibodies against antigens that are toxic or have low antigenicity. The phage antibody libraries can also be used to produce and identify novel therapeutic antibodies.
Techniques for generating human antibodies from non-immunized humans, germline sequences, or naive B cell Ig repertoires that have been immunized using phage display libraries can be used. Various lymphatic tissues can be used to prepare native or non-immunogenic antigen-binding libraries.
Techniques for identifying and separating high-affinity antibodies from phage display libraries are important for the separation of new therapeutic antibodies. The separation of high-affinity antibodies from the libraries can depend on the size of the libraries, the production efficiency in bacterial cells and the variety of libraries. The size of the libraries is reduced by inefficient folding of the antibody- or antigen-binding protein and inefficient production due to the presence of the stop codon. Expression in bacterial cells can be inhibited when the antibody- or antigen-binding domain is not properly folded. The expression can be improved by alternately mutating residues on the surface of the variable/constant interfaces or the selected CDR residues. The sequence of the framework region is an element to provide appropriate folding when generating antibody phage libraries in bacterial cells.
It is important to generate various libraries of antibody- or antigen-binding proteins in the separation of high-affinity antibodies. CDR3 regions have been found to often participate in antigen binding. Since the CDR3 region on the heavy chain varies considerably in terms of size, sequence and structurally dimensional morphology, various libraries can be prepared using the same.
Also, diversity can be created by randomizing the CDR regions of variable heavy and light chains using all 20 amino acids at each position. The use of all 20 amino acids results in antibody sequences with an increased diversity and an increased chance of identifying new antibodies.
The antibody or antibody fragment according to the present disclosure may include sequences of the anti-PD-L1 antibody of the present disclosure described herein as well as biological equivalents thereto so long as the antibody or antibody fragment can specifically recognize PD-L1. For example, an additional variation can be made to the amino acid sequence of the antibody in order to further improve the binding affinity and/or other biological properties of the antibody. Such a variation include, for example, deletion, insertion and/or substitution of amino acid sequence residues of the antibody. Such an amino acid variation are made, based on the relative similarity (identity) of amino acid side chain substituent, such as hydrophobicity, hydrophilicity, charge or size. Analysis of the size, shape and type of amino acid side chain substituent, demonstrates that all of arginine, lysine and histidine are positively charged residues, alanine, glycine and serine have similar sizes, and phenylalanine, tryptophan and tyrosine have similar shapes. Thus, based on these considerations, arginine, lysine and histidine; and alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine are considered to be biologically functional equivalents.
When considering variations having the biologically equivalent activity, the antibody or nucleic acid encoding the same according to the present disclosure is also interpreted to include a sequence showing a substantial identity with the sequence set forth in the corresponding SEQ ID NO. The term “sequence showing a substantial identity” means a sequence that shows an identity of at least 90%, most preferably, at least 95%, 96% or more, 97% or more, 98% or more, or 99% or more, when aligning the sequence of the present disclosure so as to correspond to any other sequence as much as possible and analyzing the aligned sequence using an algorithm commonly used in the art. Alignment methods for sequence comparison are well-known in the art. The NCBI basic local alignment search tool (BLAST) is accessible from NBCI and can be used in conjunction with sequence analysis programs such as blastp, blasm, blastx, tblastn and tblastx on the Internet. BLSAT is available at www.ncbi.nlm.nih.gov/BLAST/. A method for comparing a sequence identity using this program can be found at ncbi.nlm.nih.gov/BLAST/blast_help.html.
Based on this, the antibody or an antigen-binding fragment thereof according to the present disclosure can have a sequence identity (homology) of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more. Such an identity can be determined by the comparison and/or alignment of sequences by methods known in the art. For example, the percent sequence identity of the nucleic acid or protein according to the present disclosure can be determined using a sequence comparison algorithm (i.e., BLAST or BLAST 2.0), manual alignment or visual inspection.
In another aspect, the present disclosure is directed to a nucleic acid encoding the antibody or an antigen-binding fragment thereof.
The antibody or antigen-binding fragment thereof can be recombinantly produced by isolating the nucleic acid encoding the antibody or antigen-binding fragment thereof according to the present disclosure. The nucleic acid is isolated and inserted into a replicable vector to conduct further cloning (amplification of DNA) or further expression. Based on this, in another aspect, the present disclosure is directed to a vector containing the nucleic acid.
The term “nucleic acid” is intended to encompass both DNA (gDNA and cDNA) and RNA molecules, and nucleotides, which are basic constituent units of the nucleic acid, include naturally derived nucleotides as well as analogues wherein sugar or base moieties are modified. The sequence of the nucleic acid encoding heavy and light chain variable regions of the present disclosure can be varied. Such a variation include addition, deletion, or non-conservative substitution or conservative substitution of nucleotides.
The DNA encoding the antibody can be easily separated or synthesized using conventional procedures (for example, using an oligonucleotide probe specifically binding to DNA encoding heavy and light chains of the antibody). A variety of vectors are obtainable. Vector components generally include, but are not limited to, one or more of the following components: signal sequences, replication origins, one or more marker genes, enhancer elements, promoters and transcription termination sequences.
As used herein, the term “vector” refers to a means for expressing target genes in host cells and includes: plasmid vectors; cosmid vectors; and viral vectors such as bacteriophage vectors, adenovirus vectors, retroviral vectors and adeno-associated viral vectors. The nucleic acid encoding the antibody in the vector is operatively linked to a promoter.
The term “operatively linked” means a functional linkage between a nucleic acid expression regulation sequence (e.g., promoter, signal sequence or array of transcription regulator binding site) and another nucleic acid sequence, and is regulated by transcription and/or translation of the nucleic acid sequence.
When a prokaryotic cell is used as a host, the vector generally includes a potent promoter capable of conducting transcription (such as tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, or T7 promoter), a ribosome binding site to initiate translation, and a transcription/translation termination sequence. In addition, for example, when a eukaryotic cell is used as a host, the vector includes a promoter (e.g., a metallothionein promoter, a β-actin promoter, a human hemoglobin promoter and a human muscle creatine promoter) derived from the genome of mammalian cells, or a promoter derived from animal virus such as adenovirus late promoter, vaccinia virus 7.5K promoter, SV40 promoter, cytomegalovirus (CMV) promoter, HSV tk promoter, mouse breast tumor virus (MMTV) promoter, HIV LTR promoter, Moloney virus promoter, Epstein Barr virus (EBV) promoter, and Rous sarcoma virus (RSV) promoter), and generally has a polyadenylation sequence as a transcription termination sequence.
Optionally, the vector may be fused with another sequence to facilitate purification of the antibody expressed therefrom. The sequence to be fused includes, for example, glutathione S-transferase (Pharmacia, USA), maltose-binding protein (NEB, USA), FLAG (IBI, USA), 6×His (hexahistidine; Quiagen, USA) and the like.
The vector includes antibiotic-resistant genes commonly used in the art as selectable markers and examples thereof include genes resistant to ampicillin, gentamycin, carbenicillin, chloramphenicol, streptomycin, kanamycin, geneticin, neomycin and tetracycline.
In another aspect, the present disclosure is directed to a cell transformed with the above-mentioned vector. The cell used to produce the antibody of the present disclosure may be a prokaryote, yeast or higher eukaryotic cell, but is not limited thereto.
Strains of the genus Bacillus such as Escherichia coli, Bacillus subtilis and Bacillus tuligensis, Streptomyces, Pseudomonas (for example, Pseudomonas putida), and prokaryotic host cells such as Proteus mirabilis and Staphylococcus (for example, Staphylococcus carnosus) can be used.
The interest in animal cells is the largest and examples of useful host cell lines include, but are not limited to, COS-7, BHK, CHO, CHOK1, DXB-11, DG-44, CHO/−DHFR, CV1, COS-7, HEK293, BHK, TM4, VERO, HELA, MDCK, BRL 3A, W138, Hep G2, SK-Hep, MMT, TRI, MRC 5, FS4, 3T3, RIN, A549, PC12, K562, PER.C6, SP2/0, NS-0, U20S, or HT1080.
In another aspect, the present disclosure is directed to a method for producing the antibody or antigen-binding fragment thereof including: (a) culturing the cells; and (b) recovering the antibody or an antigen-binding fragment thereof from the cultured cells.
The cells can be cultured in various media. Any commercially available medium can be used as a culture medium without limitation. All other essential supplements well-known to those skilled in the art may be included in appropriate concentrations. Culture conditions such as temperature and pH have already been used with selected host cells for expression, which will be apparent to those skilled in the art.
The recovery of the antibody or antigen-binding fragment thereof can be carried out, for example, by centrifugation or ultrafiltration to remove impurities, and purification of the resulting product, for example, using affinity chromatography. Additional other purification techniques such as anion or cation exchange chromatography, hydrophobic interaction chromatography, hydroxyl apatite chromatography and the like may be used.
In another aspect, the present disclosure is directed to a composition for preventing or treating cancer containing the antibody as an active ingredient.
The present disclosure provides, for example, a composition for preventing or treating cancer or infectious disease containing: (a) a pharmaceutically effective amount of the antibody to PD-L1 or antigen-binding fragment thereof according to the invention; and (b) a pharmaceutically acceptable carrier. The present disclosure also relates to a method for preventing or treating cancer or infectious disease including administering the antibody to PD-L1 or antigen-binding fragment thereof according to the present disclosure in an effective amount required for a patient.
Since the composition uses, as an active ingredient, the anti-PD-L1 antibody or antigen-binding fragment thereof according to the present disclosure described above, repeated description thereof is omitted.
The binding of PD-L1 to PD-1 negatively regulates T cell antigen-specific responses important for tolerance and prevention of autoimmunity and immunopathology. However, excessive PD-L1/PD-1 interaction, which may be induced by chronic antigen stimulation, may cause inhibition of T cell antigen-specific responses and loss of T cells, which are characteristics of T cell depletion. T cell depletion is a condition of T cell dysfunction that may occur in chronic infections and cancers. T cell depletion is defined as a poor effector function, continuous expression of inhibitory receptors, or a transcriptional state different from functional effectors or memory T cells. Depletion interferes with the progression of infections and tumors.
As demonstrated in the following examples, the antibody or an antigen-binding fragment thereof according to the invention binds with high affinity to PD-L1 to inhibit formation of the PD-1 and PD-L1 complex, thereby being useful for the treatment of cancer inducing T cell depletion that evades anti-tumor T cell activity.
In some cases, an anti-cancer therapeutic agent other than the aforementioned antibody may be used in combination to effectively target tumor cells overexpressing PD-L1, to enhance the anti-tumor T cell activity and thereby to improve the immune response targeting tumor cells. The aforementioned antibody may be used in combination with other anti-neoplastic or immunogenic agents [for example, weaken cancer cells, tumor antigens (including recombinant proteins, peptides and carbohydrate molecules)], antigen-presenting cells such as dendritic cells pulsed with tumor-derived antigens or nucleic acid, cells transfected with immunostimulatory cytokine (e.g., IL-2, IFNα2, GM-CSF), and genes encoding immunostimulatory cytokine (including, but not limited to, GM-CSF); standard cancer therapy (e.g., chemotherapy, radiation therapy or surgery), or other antibodies (including, but not limited to, VEGF, EGFR, Her2/neu, VEGF receptors, other growth factor receptors, CD20, CD40, CTLA-4, OX-40, 4-IBB and ICOS).
Anti-PD-L1 antibodies can induce apoptosis (cell death). Apoptosis is induced by direct or indirect mechanisms. For example, binding of anti-PD-L1 antibodies to PD-L1 can cause complement dependent cytotoxicity (CDC). In some cases, the anti-PD-L1 antibody binds to PD-L1 and causes the mobilization of secondary cell types to kill PD-L1-expressing target cells. Representative mechanisms, by which anti-PD-L1 antibodies mediate apoptosis by the mobilization of secondary cell types, include, but are not limited to, antibody-dependent cytotoxicity (ADCC) and antibody-dependent cellular cytotoxicity (ADCP). Target PD-L1-expressing cell types include tumors and T cells such as activated T cells.
In addition, the antibody or an antibody fragment thereof according to the present disclosure can be used to prevent or treat infections and infectious diseases.
As used herein, the term “prevention” means any action that inhibits cancer or infectious diseases or delays the progress of the same by administration of a composition and, as used herein, the term “treatment” means inhibition of the development of cancer, or alleviation or elimination of cancer, or inhibition, alleviation or elimination of infectious diseases.
Cancer, the disease to which the composition is applied, typically includes cancer that responds to immunotherapy, and cancer that has been not involved in immunotherapy to date. Non-limiting examples of preferred cancer in need of treatment include, but are not limited to, melanoma (e.g., metastatic malignant melanoma), renal cancer (e.g., clear cell carcinoma), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), pancreatic adenocarcinoma, breast cancer, colon cancer, lung cancer (e.g., non-small cell lung cancer), esophageal cancer, head and neck squamous cell carcinoma, liver cancer, ovarian cancer, cervical cancer, thyroid cancer, glioblastoma, glioma, leukemia, lymphoma, and other neoplastic carcinomas. In addition, the present disclosure includes refractory or recurrent cancers growth of which can be inhibited using the antibodies of the invention.
The antibody or antibody fragment may be used alone or in combination with a vaccine to stimulate an immune response to pathogens, toxins and auto-antigens. The antibody or an antigen-binding fragment thereof can be used to stimulate immune responses to human-infecting viruses, including, but not limited to, human immunodeficiency virus, hepatitis viruses A, B and C, Epstein-Barr virus, human cytomegalovirus, human papilloma and Herpes virus. The antibody or an antigen-binding fragment thereof can be used to stimulate immune responses to infection with bacterial or fungal parasites and other pathogens.
The pharmaceutically acceptable carriers, which are contained in the composition of the present disclosure, include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil and the like, which are conventionally used for drug preparation. In addition to the above components, the composition of the present disclosure may further contain a lubricant, a wetting agent, a sweetener, a flavor, an emulsifier, a suspending agent, a preservative or the like.
The pharmaceutical composition of the present disclosure can be administered orally or parenterally. In the case of parenteral administration, the pharmaceutical composition can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, endothelial administration, topical administration, intranasal administration, intrapulmonary administration, rectal administration and the like.
When administered orally, the protein or peptide may be digested. For this reason, the oral composition should be formulated to coat the active agent or protect the protein or peptide from digestion in the stomach. In addition, the pharmaceutical composition may be administered by any device enabling the active agent to be transferred to the target cell.
The appropriate dosage of the composition according to the present disclosure may vary depending on factors such as formulation method, administration method, age, body weight, gender, pathological condition and food of a patient, administration time, administration route, excretion rate and responsiveness. A skilled physician can readily determine and prescribe a dosage effective for desired treatment or prevention. For example, the daily dosage of the pharmaceutical composition of the present disclosure is 0.0001 to 100 mg/kg. As used herein, the term “pharmaceutically effective amount” means an amount sufficient to prevent or treat cancer.
The pharmaceutical composition of the present disclosure may be prepared into a unit dose form or incorporated into a multi-dose vial by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method which can be easily carried out by a person having ordinary skill in the technical field to which the present disclosure pertains. The formulation may be in the form of a solution, suspension or emulsion in oil or aqueous media, or in the form of an excipient, powder, suppository, powder, granule, tablet or capsule, and may further contain a dispersant or a stabilizing agent.
The composition of the present disclosure may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents.
In another aspect, the present disclosure is directed to a composition for diagnosing cancer containing the antibody to PD-L1 or an antigen-binding fragment thereof according to the present disclosure. Also, the present disclosure is directed to a method for diagnosing cancer by treatment with the antibody to PD-L1 or an antigen-binding fragment thereof according to the present disclosure.
Cancer can be diagnosed by measuring the level of PD-L1 expression in a sample through the antibody to PD-L1 according to the present disclosure. The level of expression can be measured by a conventional immunoassay method that includes, but is not limited to, radioimmunoassay, radioimmunoprecipitation, immunoprecipitation, immunohistochemical staining, enzyme-linked immunosorbent assay (ELISA), captured-ELISA, inhibition or competition analysis, sandwich analysis, flow cytometry, immunofluorescent staining and immunoaffinity purification using the antibody to PD-L1.
Cancer can be diagnosed by analyzing the intensity of the final signal by the immunoassay process. That is, when protein of a marker according to the present disclosure is highly expressed in a biological sample and thus the signal of biological sample is stronger than that of a normal biological sample (for example, normal stomach tissue, blood, plasma or serum), cancer is diagnosed.
In another aspect, the present disclosure is directed to a kit for diagnosing cancer containing the composition for diagnosing cancer. The kit according to the present disclosure includes the antibody to PD-L1 according to the present disclosure and can diagnose cancer by analyzing a signal generated upon reaction between a sample and the antibody. The signal may include, but is not limited to, an enzyme coupled to an antibody such as alkaline phosphatase, β-galactosidase, horseradish peroxidase, luciferase or cytochrome P450. In this case, when alkaline phosphatase is used as an enzyme, as a substrate for the enzyme, a chromogenic reaction substrate such as bromochloroindole phosphate (BCIP), nitroblue tetrazolium (NBT), naphthol-AS-B1-phosphate and ECF (enhanced chemifluorescence) are used, and when horseradish peroxidase is used, a substrate such as chloronaphthol, aminoethylcarbazole, diaminobenzidine, D-luciferin, lucigenin (Bis-N-methyl acridinium nitrate), resorufin benzyl ether, luminol, Amplex Red reagent (10-acetyl-3,7-dihydroxy phenoxazine), HYR (p-phenylenediamine-HCl and pyrocatechol), TMB (tetramethylbenzidine), ABTS (2,2′-Azine-di[3-ethylbenzthiazoline sulfonate]), o-phenylenediamine (OPD) and naphthol/pyronin, glucose oxidase, t-NBT (nitroblue tetrazolium) or m-PMS (phenzaine methosulfate) is used, but the present disclosure is not limited thereto.
In addition, the kit according to the present disclosure may also include a label for generating a detectable signal and the label may include a chemical (e.g., biotin), an enzyme (alkaline phosphatase, β-galactosidase, horseradish peroxidase and cytochrome P450), a radioactive substance (such as C14, I125, P32 and S35), a fluorescent substance (such as fluorescein), a luminescent substance, a chemiluminescent substance and FRET (fluorescence resonance energy transfer), but is not limited thereto.
Measurement of the activity of the enzyme used for cancer diagnosis or measurement of the signal can be carried out by a variety of methods known in the art. Thus, PD-L1 expression can be qualitatively or quantitatively analyzed.
Hereinafter, the present disclosure will be described in more detail with reference to examples. However, it is obvious to those skilled in the art that these examples are provided only for illustration of the present disclosure and should not be construed as limiting the scope of the present disclosure.
1. Production of PD-1 Protein Expression Vectors
For cloning of PD-L1, amplification was conducted through polymerase chain reaction (PCR) using primers for PD-L1 containing restriction enzyme SfiI sites at 5′ and 3′ (Table 1) in order to obtain only an extracellular domain using Jurkat cell cDNA libraries (Stratagene, USA). The amplified PCR product was prepared by fusing human Fc (SEQ ID NO: 248) and mouse Fc (SEQ ID NO: 249) to a carboxyl terminal using N293F vector (
2. Expression and Purification of PD-L1 Antigen
In order to express an antigen in animal cells, HEK-293F cells were transfected with plasmid DNA. The polyplex reaction solution for transfection was prepared by mixing 25 μg of plasmid DNA with 3 ml of a Freestyle 293 expression medium and further mixing 2 mg/ml of PET (polyethylenimine, polyplusA-transfection, USA) with the resulting mixture again. The polyplex reaction solution was reacted at room temperature for 15 minutes and then cultured in 40 ml of the culture medium (1×106 cells/ml) for 24 hours at 37° C. and 8% CO2 at 120 rpm. After 24 hours of transfection, Soytone (BD, USA), as a supplement, is added to a final concentration of 10 g/L. Antibodies were produced using a transient expression system using HEK-293F for 7 days. Affinity chromatography was performed to obtain the antigen from the culture medium. The supernatant was obtained by centrifugation at 5,000 rpm for 10 minutes to remove cells and cell debris from the culture medium recovered on the 7th day. The supernatant was reacted with a recombinant protein A agarose resin washed with DPBS at 4° C. for 16 hours.
When the recombinant protein A agarose resin was used, the protein was eluted with 0.1M glycine and neutralized with 500 μl of 1M Tris-HCl to perform primary purification. The primarily purified protein was secondarily purified using Superdex 200 (1.5 cm*100 cm) gel filtration chromatography.
The purity of the purified protein was identified by SDS-PAGE gel and size exclusion chromatography [TSK-GEL G-3000 SWXL size-exclusion chromatography (SEC) (Tosoh)].
As a result, it was confirmed that the purified PD-L1 protein had a purity of 95% or more, as shown in
1. Antigen Preparation
PD-L1-hFc and PD-L1-mFc prepared in Example 1 and PD-L1-his (Catalog Number, 10084-H08H) purchased from Sino Biological Inc. as protein antigens were coated in a dose of 50 ug on an immunosorbent tube and then blocked.
2. Bio-Panning
A human antibody library phage was obtained by infecting a human scFv library with a variety of 2.7×1010 with bacteria and then culturing at 30° C. for 16 hours. After culturing, the culture solution was centrifuged, and the supernatant was concentrated with PEG, and then dissolved in PBS buffer to prepare a human antibody library. The human antibody library phage was charged into an immune tube, followed by reaction at room temperature for 2 hours. After washing with 1×PBS/T and 1×PBS, only the scFv-phages specifically bound to the antigen were eluted. The eluted phages were infected with E. coli again and amplified (panning process) to obtain a pool of positive phages. The second and third round panning processes were conducted using the phages amplified in the first round of panning in the same manner as above, except that only the number of times of the PBST washing step was increased. As a result, as shown in Table 2, it was seen that the number of phages bound to the antigen (output) during the third round panning was slightly increased, as compared to the input phages.
3. Polyphage ELISA
The cell stock frozen after the first to third panning processes was added to a medium containing 5 ml of 2×YTCM, 2% glucose and 5 mM MgCl2 to OD600 of 0.1 and then cultured at 37° C. for 2 to 3 hours (OD600=0.5 to 0.7). M1 helper phages were infected and cultured in a medium containing 2×YTCMK, 5 mM MgCl2, and 1 mM IPTG at 30° C. for 16 hours. The cultured cells were centrifuged (4,500 rpm, 15 min, 4° C.), and the supernatant was transferred to a new tube (first to third-panned poly scFv-phages). Two kinds of antigens were each coated at a density of 100 ng/well on 96-well immuno-plates (NUNC 439454) with coating buffer at 4° C. for 16 hours, and each well was blocked using 4% skim milk dissolved in PBS.
Each well was washed with 0.2 ml of PBS/T, and 100 μl of the first to third-panned poly scFv-phage was added to each well, followed by reaction at room temperature for 2 hours. Again, each well was washed 4 times with 0.2 ml of PBS/T, and the secondary antibody, anti-M13-HRP (Amersham 27-9421-01) was diluted at 1:2000 and reacted at room temperature for 1 hour. After washing with PBS/T, OPD tablets (Sigma. 8787-TAB) were dissolved in PC buffer, and the resulting solution was added at a concentration of 100 μl/well to induce color development for 10 minutes. Then, absorbance was measured at 490 nm with a spectrophotometer (Molecular Device).
The results are shown in
4. Screening of Positive Phages
Colonies obtained from the polyclonal phage antibody group (third panning) with high binding capacity were cultured in a 1 ml 96-deep well plate (Bioneer 90030) at 37° C. for 16 hours. 100 to 200 μl of the cells grown thus were added to a medium containing 2×YTCM, 2% glucose and 5 mM MgCl2, to OD600 of 0.1, and were added to a medium containing 1 ml of 2×YTCM, 2% glucose and 5 mM MgCl2, and then cultured in a 96-deep well plate at 37° C. for 2 to 3 hours to OD600 of 0.5 to 0.7. M1 helper phages were infected at an MOI of 1:20 and cultured in a medium containing 2×YTCMK, 5 mM MgCl2, 1 mM IPTG at 30° C. for 16 hours.
The antigen PD-L1 was coated at a density of 100 ng/well on a 96-well immunoplate at 4° C. for 16 hours and each well was blocked using 4% skim milk dissolved in PBS. Each monoclonal scFv-phage (100 scFv-phage) washed with 0.2 ml PBS/T and cultured for 16 hours was added in a dose of 100 μl to each well and reacted at room temperature for 2 hours. Again, each well was washed 4 times with 0.2 ml of PBS/T, and the secondary antibody, anti-M13-HRP, was diluted to 1/2000 and reacted at room temperature for 1 hour. After washing with 0.2 ml of PBS/T, color development was performed and absorbance was measured at 490 nm.
As a result, as shown in
5. Base Sequence Analysis of Positive Phage Antibodies
The selected single clones were subjected to DNA-prep using a DNA purification kit (Qiagen, Germany) to obtain DNAs, and sequence analysis for DNAs was requested (Solgent). The CDR regions of VH and VL of the selected antibodies were identified, based on results of sequence analysis and the similarity (identity) between these antibodies and germ line antibody groups was investigated using an Ig BLAST program on the NCBI website at ncbi.nlm.nih.gov/igblast/. As a result, 10 species of phage antibodies specific to PD-L1 were obtained and are summarized in Table 3 below.
Antibodies including the heavy and light-chain CDRs and FR sequences of the selected antibodies, and heavy chain variable regions and light chain variable regions including the same are shown in Tables 4 and 5 below.
1. Conversion of scFv Form to IgG Form
PCR (iCycler iQ, BIO-RAD) was performed on the heavy and light chains to convert the selected 10 species of monoclonal phage antibodies to PD-L1 from phages to IgG whole vector. As a result, heavy and light chains were obtained, and the vectors and the heavy and light chains of each of the clones were cut (digested) with restriction enzymes. DNAs were eluted from each of the vector and heavy chain with a DNA-gel extraction kit (Qiagen). Ligation was performed by mixing 1 μl (10 ng) of the vector, 15 μl (100-200 ng) of the heavy chain, 2 μl of 10× buffer, 1 μl of ligase (1 U/μl) and distilled water, allowing the mixture to stand at room temperature for 1 to 2 hours, injecting the resulting mixture into transformed cells (competent cells, XL1-blue), placing the cells on ice for 5 minutes and subjecting the cells to heat-shock at 42° C. for 90 seconds.
After the heat shock, 1 ml of the medium was added to the cells, and then the cells were grown at 37° C. for 1 hour, spread on an LB Amp plate and incubated at 37° C. for 16 hours. The colony thus obtained was inoculated with 5 ml of LB Amp medium, cultured at 37° C. for 16 hours and subjected to DNA-prep using a DNA-prep kit (Nuclogen). Sequence analysis of the obtained DNAs was requested (Solgent).
As a result, it was confirmed that the sequences of heavy chains and light chains of 11 clones for PD-L1 converted into the whole IgG corresponded to the sequences of phage antibodies. In order to transfect into HEK 293F cells, the heavy and light chains of respective clones converted into whole IgG were grown in 100 ml of LB Amp medium, and DNAs were obtained using a Midi-prep kit (QIAgen).
2. Human Antibody Production
The cloned pNATVH and pNATVL vectors were co-transfected at a ratio of 6:4 into HEK293F cells and the supernatant was collected on the 7th day, the cells and debris were removed through centrifugation and a 0.22 μm top filter, and the supernatant was collected and subjected to protein A affinity chromatography to purify the IgG antibody. After purification, the antibody was separated through a glycine buffer, and buffer was changed such that the final resuspension buffer was PBS. Purified antibodies were quantitated by BCA and nano drop, and each of 15 species of antibodies was loaded in a dose of 5 ug under reducing and non-reducing conditions, and analyzed by SDS-PAGE to determine purity and mobility of the purified protein (
As a result, as shown in
1. Evaluation of Antibody Activity
Testing for activity of the selected antibodies was carried out using a PD1/PD-L1 blockade bioassay kit (promega, J1250). A CHO cell line highly expressing PD-L1 was spread on a 96-well plate, cultured for 16 hours or longer, treated with each antibody serially diluted at a constant concentration and then cultured together with a Jurkat cell line highly expressing human PD-1, for 6 hours. The degree of recovery of the inhibition of the antibody was determined with a spectrophotometer (SpectraMax M5 spectrophotometer, Molecular Devices, USA), which was determined from a luminescent intensity resulting from degradation of the substrate by luciferase. The activity of 10 species of PD-L1 antibodies was found based on the value to recover a reduced signal by formation of a PD-1/PD-L1 complex, and 16E12 exhibited similar activity to the control antibody (
In order to measure activity of the PD-L1 antibody, 16E12 in a concentration-dependent manner, serial dilution and PD-1/PD-L1 blockade bioassay were performed again to recover the reduced signal in a concentration gradient dependent manner. The degree of recovery can be expressed as EC50 (effective concentration of mAb at 50% level of recovery signal), analyzed using Graphpad Prism6, and in vitro efficacy inhibition recovery ability of EC50 is shown in
2. Affinity of PD-L1 Antibody to Overexpressed Cells
Regarding transformation cell pools highly expressing PD-L1, HEK293E was transformed with a plasmid pcDNA3.1 containing human PD-L1 and screened in a selective medium containing 150 ug/ml Zeocin (#R25001, Thermo Fisher). Each cell pool was identified and selected by fluorescence activated cell sorting (FACS) analysis using anti-PD-L1 and used for functional assays such as FACS binding assays or FACS competition assays.
0.5 to 1×106 cells per sample were each prepared from the transformation cell pools highly expressing human PD-L1, and antibodies were serially diluted at a constant dilution rate and reacted with the prepared cells at 4° C. for 20 minutes. Then, the cells were washed three times with PBS (#LB001-02, Welgene) containing 2% fetal bovine serum and reacted at 4° C. for 20 minutes with an anti-human IgG antibody (#FI-3000, Vectorlabs) conjugated with a FITC (fluorescein isothiocyanate) fluorescent substance. Then, the cells were subjected to the same washing process as above and then suspended in 0.5 ml of PBS containing 2% FBS (#26140-079, Thermo Fisher) with an FACSCanto II flow cytometer (BD Biosciences, USA) as a flow cytometer. As a result, the PD-L1 antibody, 16E12, was specifically bound and the binding capacity thereof was determined from an equilibrium dissociation constant (Kd) obtained through an analysis function of Graphpad Prism6.
As a result, as can be seen from
3. Affinity of PD-L1 Antibody Using ProteOn XPR36
A ProteOn XPR36 (BioRad) instrument was used. The GLC sensor chip (BioRad) was mounted on the instrument and washed with PBST buffer, and the carboxymethyldextran surface was activated with an EDC/sulfo-NHS mixed solution. PD-L1-hFc dissolved at a concentration of 5 ug/ml in a 10 mM sodium acetate buffer solution (pH 5.0) was injected and immobilized on the GLC sensor chip.
In order to deactivate the activated carboxyl groups that remain unreacted with the PD-L1 protein, 1 M ethanolamine was flowed and 10 mM glycine (pH 2.0) was injected in order to wash proteins that remain unbound to the sensor chip. Then, sensogram data were collected during binding and dissociation over time while allowing the antibodies to flow at a flow rate of 30 μL/min (30 nM to 0.123 nM) for 10 min using PBST buffer.
The equilibrium dissociation constant (KD) was calculated by plotting and fitting the sensogram data in the equilibrium state depending on concentration. As a result, 16E12 exhibited KD of 0.045 nM, indicating high affinity to the PD-L1 antigen (
1. Production of Libraries for Optimization of PD-L1-16E12 Antibody
For antibody optimization, new LC shuffling libraries were produced by immobilizing the heavy chain and injecting a 105-106 light chain (LC) pool owned by Ybiologics, Inc. Also, antibody optimization was conducted by the following three methods: LC shuffling; core packing+LC shuffling including comparatively analyzing the residues of structurally important sites such as hydrophobic cores of heavy chains, exposed residues, charge clusters, salt bridges, mutating the same into conserved residues and then conducting LC shuffling; and CDR hotspot+LC shuffling, in the case of DNAs in antibody variable regions, including randomly mutating mutational hot spots that can be mutated frequently in the process of in vivo affinity maturation and then conducting LC shuffling.
In order to produce LC shuffling libraries, LC genes of the 16E12 antibody were cut (digested) with BstX I and then used as vectors and the library pools owned by Ybiologics, Inc. were cut (digested) into BstX I and used as inserts. After ligation with a ligase, transformation was carried out using cells for electroporation transformation. The antibody libraries were produced by collecting the transformed cells on a square plate. As a result, about 1.5×107 various libraries were obtained. The result of sequence analysis showed that all HC sequences were identical and LC sequences were different from each other.
In order to produce the core packing+LC shuffling libraries, the framework (FR) sites of the 16E12 antibody were replaced with conserved amino acid sequences, the LC genes were cut with BstX I and then used as vectors, and the library pools owned by Ybiologics, Inc. were cut with BstX I and then used as inserts. After ligation with a ligase, transformation was carried out using cells for electroporation transformation. The antibody libraries were produced by collecting the transformed cells on a square plate. As a result, about 8.4×106 various libraries were obtained. The result of sequence analysis showed that the FR sites of HC were replaced with conserved amino acid sequences and LC sequences were different from each other.
In order to produce the core hot spot+LC shuffling libraries, the framework (FR) sites of the 16E12 antibody were replaced with conserved amino acid sequences, the hot spot libraries of CDR1 were cut with Sfi I and used as inserts, and the library pools owned by Ybiologics, Inc. were cut with Sfi I and then used as vectors. After ligation with a ligase, transformation was carried out using cells for electroporation transformation. The antibody libraries were produced by collecting the transformed cells on a square plate. As a result, about 5.6×106 various libraries were obtained. The result of sequence analysis showed that the FR sites of HC were replaced with conserved amino acid sequences, the hot spot sequences of CDR1 were randomly mutated and LC sequences were different from each other.
1. Antigen Preparation
PD-L1-hFc and PD-L1-mFc produced by Ybiologics, Inc, and PD-L1-his (Catalog Number, 10377-H08H) purchased from Sino Biological Inc. as protein antigens were coated in a dose of 50 ug on an immunosorbent tube and then blocked.
2. Bio-Panning
A human antibody library phage was obtained by infecting a human scFv library with a variety of 2.7×1010 with bacteria and then culturing at 30° C. for 16 hours. After culturing, the culture solution was centrifuged, and the supernatant was concentrated with PEG, and then dissolved in PBS buffer to prepare a human antibody library. The human antibody library phage was charged into an immune tube, followed by reaction at room temperature for 2 hours. After washing with 1×PBS/T and 1×PBS, only the scFv-phages specifically bound to the antigen were eluted.
The eluted phages were infected with E. coli again and amplified (panning process) to obtain a pool of positive phages. For antibody optimization, only the first round of panning was conducted. As a result, as shown in Table 6, it was seen that the number of phages bound to the antigen (output) during the first round of panning was slightly increased, as compared to the input phages.
3. Screening of Positive Phages
Colonies obtained from panning were cultured in a 1 ml 96-deep well plate (Bioneer 90030) at 37° C. for 16 hours. 100 to 200 μl of the cells grown thus were added to a medium containing 2×YTCM, 2% glucose and 5 mM MgCl2, to OD600 of 0.1, and were added to a medium containing 1 ml of 2×YTCM, 2% glucose and 5 mM MgCl2, and then cultured in a 96-deep well plate at 37° C. for 2 to 3 hours to OD600 of 0.5 to 0.7. M1 helper phages were infected at an MOI of 1:20 and cultured in a medium containing 2×YTCMK, 5 mM MgCl2, and 1 mM IPTG at 30° C. for 16 hours.
The antigen PD-L1 was coated at a density of 100 ng/well on a 96-well immunoplate at 4° C. for 16 hours and each well was blocked using 4% skim milk dissolved in PBS. Each monoclonal scFv-phage (100 scFv-phage) washed with 0.2 ml of PBS/T and cultured for 16 hours was added in a dose of 1 μl to each well and reacted at room temperature for 2 hours. Again, each well was washed 4 times with 0.2 ml of PBS/T, and the secondary antibody, anti-M13-HRP, was diluted to 1/2000 and reacted at room temperature for 1 hour. After washing with 0.2 ml of PBS/T, color development was performed and absorbance was measured at 490 nm.
As a result, single-phage clones having higher binding capacity to each antigen than the parent antibody (16E12, red-marked, 6D) were obtained and results are shown in
4. Base Sequence Analysis of Positive Phage Antibodies
The selected single clones were subjected to DNA-prep using a DNA purification kit (Qiagen, Germany) to obtain DNA, and sequence analysis for DNA was requested (Solgent). The CDR regions of VH and VL of the selected antibodies were identified, based on results of sequence analysis and the similarity (identity) between these antibodies and germ line antibody groups was investigated using an Ig BLAST program on the NCBI website at ncbi.nlm.nih.gov/igblast/. As a result, 21 species of phage antibodies having higher binding capability than the parent antibody were obtained and are summarized in Table 7 below.
Antibodies including the heavy and light-chain CDRs and FR sequences of the selected antibodies, and heavy chain variable regions and light chain variable regions including the same are shown in Tables 8 and 9 below.
1. Conversion of scFv Form to IgG Form
PCR (iCycler iQ, BIO-RAD) was performed on the heavy and light chains to convert the selected 21 species of monoclonal phage antibodies to PD-L1 from phages to IgG whole vector. As a result, heavy and light chains were obtained, and the vectors and the heavy and light chains of each of the clones were cut (digested) with restriction enzymes. DNAs were eluted from each of the vector and heavy chain with a DNA-gel extraction kit (Qiagen). Ligation was performed by mixing 1 μl (10 ng) of the vector, 15 μl (100-200 ng) of the heavy chain, 2 μl of 10× buffer, 1 μl of ligase (1 U/μl) and distilled water, allowing the mixture to stand at room temperature for 1 to 2 hours, injecting the resulting mixture into transformed cells (competent cells, XL1-blue), placing the cells on ice for 5 minutes and subjecting the cells to heat-shock at 42° C. for 90 seconds.
After the heat shock, 1 ml of the medium was added to the cells, and then the cells were grown at 37° C. for 1 hour, spread on an LB Amp plate and incubated at 37° C. for 16 hours. The colony thus obtained was inoculated with 5 ml of LB Amp medium, cultured at 37° C. for 16 hours and subjected to DNA-prep using a DNA-prep kit (Nuclogen). Sequence analysis of the obtained DNAs was requested (Solgent).
As a result, it was confirmed that the sequences of heavy chains and light chains of 21 clones for PD-L1 converted into the whole IgG corresponded to the sequences of the phage antibodies. In order to transfect into HEK 293F cells, the heavy and light chains of respective clones converted into whole IgG were grown in 100 ml of LB Amp medium, and DNAs were obtained using a Midi-prep kit (QIAgen).
2. Human Antibody Production
The cloned pNATVH and pNATVL vectors were co-transfected at a ratio of 6:4 into HEK293F cells and the supernatant was collected on the 7th day, the cells and debris were removed through centrifugation and a 0.22 μm top filter, and the supernatant was collected and subjected to protein A affinity chromatography to purify the IgG antibody. After purification, the antibody was separated through a glycine buffer, and buffer was changed such that the final resuspension buffer was PBS. Purified antibodies were quantitated by BCA and nano drop, and each of 21 species of antibodies was loaded in a dose of 5 ug under reducing and non-reducing conditions, and analyzed by SDS-PAGE to determine purity and mobility of the purified protein. In addition, some of the supernatants were loaded on SDS-PAGE to compare the expression rates with the parent antibody, the majority of the antibodies were more expressed than the parent antibody.
1. Evaluation of Antibody Activity
Testing for activity of the selected antibodies was carried out using a PD-1/PD-L1 blockade bioassay kit (promega, J1250). A CHO cell line highly expressing PD-L1 was spread on a 96-well plate, cultured for 16 hours or longer, treated with each antibody serially diluted at a constant concentration, and then cultured together with a Jurkat cell line highly expressing human PD-1, for 6 hours. The degree of recovery of the inhibition of the antibody was determined with a spectrophotometer (SpectraMax M5 spectrophotometer, Molecular Devices, USA), which was determined from a luminescent intensity resulting from degradation of the substrate by luciferase. The activity of 21 species of PD-L1 antibodies was found based on the value to recover a reduced signal by formation of a PD-1/PD-L1 complex, and 4A7, 4A11, 4C9, 4F5, 4H5 and 4H8 exhibited higher activity than the parent antibody and similar activity to the control antibody (
In order to measure activity of 6 species of PD-L1 antibodies (4A7, 4A11, 4C9, 4F5, 4H5, 4H8) in a concentration-dependent manner, serial dilution and PD-1/PD-L1 blockade bioassay were performed again to recover the reduced signal in a concentration gradient dependent manner. The degree of recovery can be expressed as EC50 (effective concentration of mAb at 50% level of recovery signal), analyzed using Graphpad Prism6, and 4F5 exhibited the highest in vitro efficacy inhibition recovery ability of EC50 (
2. Affinity of PD-L1 Antibody to Overexpressed Cells
Regarding transformation cell pools highly expressing human PD-1, HEK293E was transformed with a pcDNA3.1 plasmid containing human PD-1 (NM_005018.2) or human PD-L1 (NM_014143.2) and screened in a selective medium containing 400 ug/ml Zeocin (#R25001, Thermo Fisher). Each cell pool was identified and selected by fluorescence activated cell sorting (FACS) analysis using anti-PD-1 (#557860, BD) and used for functional assays such as FACS binding assays or FACS competition assays. 0.5 to 1×106 cells per sample were prepared from the transformation cell pools highly expressing human PD-L1, and antibodies were serially diluted at a constant dilution rate and reacted with the prepared cells at 4° C. for 20 minutes. Then, the cells were washed three times with PBS (#LB001-02, Welgene) containing 2% fetal bovine serum and reacted at 4° C. for 20 minutes with an anti-human IgG antibody (#FI-3000, Vectorlabs) conjugated with a FITC (fluorescein isothiocyanate) fluorescent substance. Then, the cells were subjected to the same washing process as above and then suspended in 0.5 ml of PBS containing 2% FBS (#26140-079, Thermo Fisher) with an FACSCanto II flow cytometer (BD Biosciences, USA) as a flow cytometer (Table 11).
0.5 to 1×106 cells per sample were each prepared from the transformation cell pools highly expressing human PD-L1, and antibodies were serially diluted at a constant dilution rate and reacted with the prepared cells at 4° C. for 20 minutes. Then, the cells were washed three times with PBS (#LB001-02, Welgene) containing 2% fetal bovine serum and reacted at 4° C. for 20 minutes with an anti-human IgG antibody (#FI-3000, Vectorlabs) conjugated with a FITC (fluorescein isothiocyanate) fluorescent substance. Then, the cells were subjected to the same washing process as above and then suspended in 0.5 ml of PBS containing 2% FBS (#26140-079, Thermo Fisher) with an FACSCanto II flow cytometer (BD Biosciences, USA) as a flow cytometer (
3. Inhibitory Ability of Antibody Against Formation of PD-1/PD-L1 Complex by Enzyme Immunoadsorption
Human PD-1-Fc (S1420, Y-Biologics) was added to wells of a 96-well immuno microplate (#439454, Thermo) and then washed three times with PBS containing 0.05% tween-20 (#P9416, Sigma-Aldrich), followed by washing with 4% skim milk (#232120, Becton, Dickinson and Company) and allowing to stand at room temperature for 1 hour to block non-specific binding. At the same time, human PD-L1-His (S1479, Y-Biologics) was reacted with antibodies serially diluted at a constant dilution rate at room temperature for 1 hour, followed by allowing to stand in the prepared microplate at room temperature for 1 hour. After the resulting product was subjected to the same washing method as above, the anti-biotin-His antibody (#MA1-21315-BTIN, Thermo) diluted to 1:2000 was added to the well of microplate, allowed to react at room temperature for 1 hour, and Streptavidin poly-HRP antibody (#21140, Pierce) diluted to 1:5000 was added to the well of microplate, reacted at room temperature for 1 hour and then washed in the same manner. 100 ul of a TMB substrate solution (#T0440, Sigma-Aldrich) was added to the reaction product, light was shielded, and the reaction product was allowed to stand at room temperature for 3 minutes, 50 μL of 2.5 M sulfuric acid (#S1478, Samchun) was added to stop the reaction, and absorbance was measured at 450 nm using a spectrophotometer (#GM3000, Glomax® Discover System Promega). The results are shown in
4. Affinity of PD-L1 Antibody Using ProteOn XPR36
A ProteOn XPR36 (BioRad) instrument was used. The GLC sensor chip (BioRad) was mounted on the instrument and washed with PBST buffer, and the carboxymethyldextran surface was activated with an EDC/sulfo-NHS mixed solution. PD-L1-hFc dissolved at a concentration of 5 ug/ml in a 10 mM sodium acetate buffer solution (pH 5.0) was injected and immobilized on the GLC sensor chip.
In order to deactivate the activated carboxyl groups that remain unreacted with the PD-L1 protein, 1 M ethanolamine was flowed and 10 mM glycine (pH 2.0) was injected in order to wash proteins that remain unbound to the sensor chip. Then, sensogram data were collected during binding and dissociation over time while allowing the antibodies to flow at a flow rate of 30 μL/min (30 nM to 0.123 nM) for 10 min using PBST buffer.
The equilibrium dissociation constant (KD) was calculated by plotting and fitting the sensogram data in the equilibrium state depending on concentration. As a result, 16E12(4F5) exhibited KD of 0.001 nM, indicating high affinity to the PD-L1 antigen (
Comparison in affinity of PDL1-16E12, LS and 4F5 to human, monkey and mouse PD-L1 proteins is as shown in Table 12.
An antigen PD-L1 wild type (WT) or several mutants were coated at a density of 100 ng/well on a 96-well immunoplate at 4° C. for 16 hours and the wells were blocked with 4% skim milk dissolved in PBS. Each well was washed with 0.2 ml of PBS/T, and then a single clone scFv-phage (each 100 scFv-phage) cultured for 16 hours was added in a dose of 100 μl to each well and reacted at room temperature for 2 hours. Again, each well was washed 4 times with 0.2 ml of PBS/T, and then the second antibody, anti-Fab, was diluted to 1/2000 and reacted at room temperature for 1 hour. After washing with 0.2 ml of PBS/T, color development was performed and absorbance was measured at 490 nm.
As a result, it was confirmed that the control antibody and PD-L1 mutants had different binding behaviors and thus different epitopes (
T cells were mixed with monocyte-derived dendritic cells separated from different humans at a ratio of 1:10 and cultured for 5 days, and the amount of interferon gamma in the culture medium was measured. As a result, culture media containing the parent antibody of 16E12 exhibited a concentration-dependent increase in amount of interferon gamma (
In order to identify the in vivo efficacy of 16E12-2B9 PD-L1 monoclonal antibody, 8×106 CT-26 cells as colon cancer cells were subcutaneously injected into the flank of BALb/C mice, and tumor growth was observed, while administering the monoclonal antibody at a dose of 5 mg/kg twice a week over 3 weeks, from the time when the size of tumor was similar to that of a millet. As a result, a significant decrease in tumor size was observed in the PD-L1 monoclonal antibody-administered group (
The antibody protein was diluted in DPBS to 3 uM, 45 ul, mixed with 5 ul of 200×Sypro orange dye (#S6650, Thermo) and then aliquoted in a dose of 50 ul into a qPCR Tube (#B77009, B57651, bioplastics). QPCR was performed using a Biorad CFX96 real time PCR system. The qPCR conditions were given as follows: reaction at 25° C. for 30 seconds, elevation of the temperature by 1° C. up to 99° C. and at the same time, reaction at each temperature for 1 min, and final reaction at 25° C. for 10 seconds. Tm (melting temperature) was used as a rate constant at which the antibody structure was un-bound. The results are shown in Table 13 below.
In order to identify binding of the anti-PD-L1 antibody to PD-L2, human PD-L2-Fc (#10292-H02H, Sino) was immobilized on wells of a 96-well immuno microplate (#439454, Thermo) at 4° C. for 16 hours, and then washed three times with PBS containing 0.05% tween-20 (#P9416, Sigma-Aldrich), followed by allowing to stand in a cleaning solution containing 4% skim milk (#232120, Becton, Dickinson and Company) at room temperature for 1 hour to block non-specific binding. At the same time, each antibody serially diluted at a constant dilution rate or human PD-1-His (S1352, Y-Biologics) used as a positive control was reacted with at room temperature for 1 hour, followed by allowing to stand in the prepared microplate at room temperature for 1 hour. After the resulting product was subjected to the same washing method as above, the anti-biotin-His antibody (#MA1-21315-BTIN, Thermo) diluted to 1:2000 was added to the well of microplate, allowed to react at room temperature for 1 hour, and the streptavidin poly-HRP antibody (#21140, Pierce) diluted to 1:5000 was added to the well of microplate, reacted at room temperature for 1 hour and then washed in the same manner. 100 ul of a TMB substrate solution (#T0440, Sigma-Aldrich) was added to the reaction product, light was shielded, and the reaction product was allowed to stand at room temperature for 3 minutes, 50 μL of 2.5 M sulfuric acid (#S1478, Samchun) was added to stop the reaction, and absorbance was measured at 450 nm using a spectrophotometer (#GM3000, Glomax® Discover System Promega). The results are shown in
The novel antibodies binding to PD-L1 or antigen-binding fragments thereof according to the present disclosure can bind to PD-1 with high affinity, and inhibit the formation of the PD-1/PD-L1 complex, thereby inhibiting T cell depletion that evades PD-1/PD-L1-mediated T cell activity. Accordingly, the antibodies binding to PD-L1 or antigen-binding fragments thereof according to the present disclosure are useful for the prevention or treatment of target cancer or infectious diseases.
Although specific configurations of the present disclosure have been described in detail, those skilled in the art will appreciate that this description is provided as preferred embodiments for illustrative purposes and should not be construed as limiting the scope of the present disclosure. Therefore, the substantial scope of the present disclosure is defined by the accompanying claims and equivalents thereto.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10-2016-0100211 | Aug 2016 | KR | national |
| 10-2017-0099673 | Aug 2017 | KR | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/KR2017/008495 | 8/7/2017 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/026249 | 2/8/2018 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 20090055944 | Korman et al. | Feb 2009 | A1 |
| 20110209230 | Korman et al. | Aug 2011 | A1 |
| Number | Date | Country |
|---|---|---|
| 2920173 | Feb 2015 | CA |
| 2008544755 | Dec 2008 | JP |
| 8801649 | Mar 1988 | WO |
| 8806630 | Sep 1988 | WO |
| 8807085 | Sep 1988 | WO |
| 8807086 | Sep 1988 | WO |
| 8809344 | Dec 1988 | WO |
| WO2010077634 | Jul 2010 | WO |
| WO2015061668 | Apr 2015 | WO |
| 2015097536 | Jul 2015 | WO |
| 2016000619 | Jan 2016 | WO |
| 2016007235 | Jan 2016 | WO |
| WO2016061142 | Apr 2016 | WO |
| 2016090312 | Jun 2016 | WO |
| Entry |
|---|
| Brown et al J. Immunol. May 1996; 156(9):3285-3291 (Year: 1996). |
| Vajdos et al (J. Mol. Biol. Jul. 5, 2002;320(2); 415-428) (Year: 2002). |
| Paul, Fundamental Immunology, 3rd Edition, 1993, pp. 292-295 (Year: 1993). |
| Strome et al., The Oncologist, 2007; 12:1084-95 (Year: 2007). |
| Brand et al., Anticancer Res. 2006; 26:463-70 (Year: 2006). |
| Kataja et al., Ann Oncol 2009; 20(sup 4): iv10-14 (Year: 2009). |
| Nelson et al., Ann. Intern Med. 2009; 151:727-737 (Year: 2009). |
| Balmana et al. Ann Oncol 2009; 20(supp 4):iv19-20 (Year: 2009). |
| Dyck et al Eur. J. Immununol. 47:765 (2017) (Year: 2017). |
| Jager, V., et al., “High level transient production of recombinant antibodies and antibody fusion proteins in HEK293 cells”, “BMC biotechnology”, 2013, Page(s) http://www.bomedcentral.com/1472-6750/13/52, vol. 13, No. 52, Publisher: BioMed Central. |
| Kuhn, P., et al., “Recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by phage display”, “Proteomics Clin. Appl.”, 2016, pp. 922-948, vol. 10, Publisher: Wiley-VCH. |
| Rudikoff, S., et al., “Single amino acid substitutions altering antigen-binding specificity”, “Proc. Natl. Acad. Sci. USA: Immunology”, 1982, pp. 1979-1983, vol. 79. |
| Tamura, M., et al., “Structural Correlates of an Anticarcinoma Antibody: Identification of Specificity-Determining Residues (SDRs) and Development of a Minimally Immunogenic Antibody Variant by Retention of SDRs Only”, “The Journal of Immunology”, Feb. 2000, pp. 1432-1441, vol. 164, No. 3, Publisher: The American Association of Immunologists. |
| Panka, D., et al., “Variable region framework differences result in decreased or increased affinity of variant anti-digoxin antibodies”, “Immunology”, May 1988, pp. 3080-3084, vol. 85. |
| Xiang, J., et al., “Modificiation in Framework Region I Results in a Decreased Affinity of Chimeric Anti-Tag72 Antibody”, “Molecular Immunology”, 1991, pp. 141-148, vol. 28, No. ½. |
| Caldas, C., et al., “Humanization of the anti-CD18 antibody 6.7: an unexpected effect of a framework residue in binding to antigen, Molecular Immunology”, 2003, pp. 941-952, vol. 39, No. 15, Publisher: Pergamon. |
| Du, J., et al., “Molecular Basis of Recognition of Human Osteopontin by 23C3, a Potential Therapeutic Antibody for Treatment of Rheumatoid Arthritis”, “Journal of Molecular Biology”, 2008, pp. 835-842, vol. 382, No. 4, Publisher: Elsevier. |
| Number | Date | Country | |
|---|---|---|---|
| 20190322750 A1 | Oct 2019 | US |
