Patent Application
20240425593
The invention relates to the field of therapeutic monoclonal antibodies, and more particularly, to an antibody against PD-1; and to use of the antibody in preventing and/or treating a tumor, an infection, or an autoimmune disease.
Programmed cell death receptor-1 (PD-1) is a type I membrane protein having 288 amino acids, which is known as one of the main immune checkpoints (Blank et al., 2005, Cancer Immunotherapy, 54: 307-314). The PD-1/PD-L1 signaling pathway plays an important role in regulating immune tolerance, microbial infection, and tumor immune escape. Blocking the interaction between PD-1 and one of its ligands, PD-L1, improves the immunity of tumor-specific CD8+T cells, thereby attacking cancer cells, thus helping the immune system to eliminate tumor cells. Blocking PD-1/PD-L1 signaling can promote proliferation of tumor antigen-specific T cells, play a role in killing tumor cells, thereby inhibiting local tumor growth (Julie R et al., 2012, N Engl J Med. 366: 2455-2465); PD-1 and relevant research thereof were reviewed in detail in Molecular and Biochemical Aspects of the PD-1 Checkpoint Pathway, Vassiliki A. Boussiotis, NEJM n engl j med 375; 18 nejm.org Nov. 3, 2016. The expression of PD-1 (on tumor infiltrating lymphocytes) and/or PD-L1 on tumor cells has been found on a variety of human primary tumors by immunohistochemical evaluation of biopsies. Such tissues include lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, rectal cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell cancer, urethral epithelial cell cancer, pancreatic cancer and head and neck tumors, etc. It can be seen from that blocking the interaction of PD-1/PD-L1 may improve the immunocompetence of tumor-specific T cells, helping the immune system to eliminate tumor cells. Recently, studies have shown that PD-1 is highly expressed in T cells from HIV-infected individuals, additionally, an anti-PD-1 antibody can also enhance immunity to chronic viral infection. Moreover, Zhao et al. have found that depletion of PD-1-positive expressing immune cells in mice can delay the onset of a diabetic disease model and ameliorate the paralysis caused by experimental autoimmune encephalomyelitis (Zhao P. Et al. Depletion of PD-1-positive cells ameliorates autoimmune disease. Nature Biomedical Engineering 3, 292-305, 2019). Therefore, blockade of PD-1/PD-L1 may be useful in preventing and/or treating a tumor, an infection, or an autoimmune disease.
An anti-PD-1 monoclonal antibody Pembrolizumab (Keytruda, Merck) can reduce the risk of death by 31% compared to chemotherapy in PD-L1 expression positive patients with esophageal cancer, however, Pembrolizumab causes not only nausea and vomiting, but also anemia, hyperglycemia, hypoalbuminemia, hyponatremia, pain in extremity, dyspnea, motor skills disorder, and other side effects. Recurrent or refractory Hodgkin's lymphoma responds to sintilimab, an anti-PD-1 monoclonal antibody, however, 18% of patients have an adverse event of Grade 3 or 4, and 15% of patients have severe adverse events. A Phase I clinical trial of the treatment of anti-PD-1 monoclonal antibody Tislelizumab in combination with Pamiparib showed that some patients developed hepatitis or autoimmune hepatitis, and the efficacy of Tislelizumab in urethral cancer and liver cancer is still investigated in clinical trials. Therefore, the research and development of a new anti-PD-1 monoclonal antibody has become an urgent technical problem to be solved.
In the present application, firstly, the inventors have developed murine antibodies with excellent properties which is capable of specifically recognizing PD-1. Based on that, the inventors have further developed humanized antibodies of the murine antibodies by expensing creative works on deep study and modification of the murine antibodies.
The antibodies according to the invention, especially the humanized antibodies, are extremely advantageous, which not only retains (or even enhances) the functions and properties of the parental murine antibody, such as binding to PD-1 with high affinity and specificity, but also have an extremely high extent of humanization and can be safely administered to a human subject without triggering an immunogenic reaction. Therefore, the antibodies according to the invention are of great clinical value.
In one aspect, the invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to PD-1, the antibody or antigen-binding fragment thereof comprising the following complementary determining regions (CDRs):
In certain embodiments, the substitution is a conservative substitution.
Preferably, the CDR is defined by the Kabat or IMGT numbering system.
In certain embodiments, the VH and/or VL of the antibody or antigen-binding fragment thereof comprise framework regions (FRs) of an immunoglobulin derived from a human or mouse.
In certain embodiments, the antibody or antigen-binding fragment thereof binds to human PD-1.
In one aspect, the invention provides an antibody or antigen-binding fragment thereof capable of specifically binding to PD-1, the antibody or antigen-binding fragment thereof comprising a heavy chain variable region (VH) and/or a light chain variable region (VL).
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and/or a light chain variable region (VL) as follows, wherein the CDRs are defined by the IMGT numbering system:
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and/or a light chain variable region (VL) as follows, wherein the CDRs are defined by the IMGT numbering system:
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and/or a light chain variable region (VL) as follows, wherein the CDRs are defined by the Kabat numbering system:
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) and/or a light chain variable region (VL) as follows, wherein the CDRs are defined by the Kabat numbering system:
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable regions (VH) and/or a light chain variable regions (VL), wherein at least one CDR in the heavy chain variable region (VH) and/or the light chain variable region (VL) contains a mutation which is a substitution, deletion, addition of one or several amino acids or any combination thereof (for example, a substitution, deletion, addition of 1, 2 or 3 amino acids, or any combination thereof) as compared with the CDR defined by IMGT or Kabat as described above.
Preferably, the substitution of the invention is a conservative substitution.
In certain embodiments, the VH of the antibody or antigen-binding fragment thereof of the invention comprises framework regions (FRs) derived from a heavy chain variable region (VH) of a murine immunoglobulin, and/or the VL of the antibody or antigen-binding fragment thereof of the invention comprises framework regions (FRs) derived from a light chain variable region (VL) of a murine immunoglobulin. Therefore, in certain embodiments, the antibody or antigen-binding fragment thereof of the invention is murine.
In certain embodiments, the VH of the antibody or antigen-binding fragment thereof of the invention comprises framework regions (FRs) derived from a heavy chain variable region (VH) of a human immunoglobulin, and/or the VL of the antibody or antigen-binding fragment thereof of the invention comprises framework regions (FRs) derived from a light chain variable region (VL) of a human immunoglobulin. Therefore, in certain embodiments, the antibody or antigen-binding fragment thereof of the invention is humanized. In such embodiments, one or more FRs in the heavy chain variable region and/or one or more FRs in the light chain variable region of the antibody or antigen-binding fragment thereof of the invention may comprise one or more non-human (e.g. murine) amino acid residues, for example, one or more FRs in the heavy chain variable region and/or one or more FRs in the light chain variable region may comprise one or more amino acid reverse mutations in which corresponding murine amino acid residues are present.
In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:
In certain embodiments, the humanization degree of the antibody or antigen-binding fragment thereof of the invention is at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:
In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) set forth in any one of SEQ ID NOs: 1, 20, 36, 54, and 68, and/or a light chain variable region (VL) set forth in any one of SEQ ID NOs: 2, 21, 37, 55, and 69.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region (VH) set forth in any one of SEQ ID NOs: 16, 17, 34, 51, and 52, and/or a light chain variable region (VL) set forth in any one of SEQ ID NOs: 18, 19, 35, and 53.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH as set forth in SEQ ID NO: 1, and/or a VL as set forth in SEQ ID NO: 2.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH set forth in SEQ ID NO: 1, and/or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VL set forth in SEQ ID NO: 2.
In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. having a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VH set forth in SEQ ID NO: 1, and/or, a VL having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. having a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VL set forth in SEQ ID NO: 2. In a preferred embodiment, the substitution is a conservative substitution.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH set forth in SEQ ID NO: 16, and/or a VL set forth in SEQ ID NO: 18.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH set forth in SEQ ID NO: 16, and/or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 984, at least 99%, or 100% sequence identity to the VL set forth in SEQ ID NO: 18.
In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. having a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VH set forth in SEQ ID NO: 16, and/or, a VL having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. having a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VL set forth in SEQ ID NO: 18. In a preferred embodiment, the substitution is a conservative substitution.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH set forth in SEQ ID NO: 17, and/or a VL set forth in SEQ ID NO: 19.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH set forth in SEQ ID NO: 17, and/or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VL set forth in SEQ ID NO: 19.
In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. having a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VH set forth in SEQ ID NO: 17, and/or, a VL having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. having a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VL set forth in SEQ ID NO: 19. In a preferred embodiment, the substitution is a conservative substitution.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH set forth in SEQ ID NO: 20, and/or a VL set forth in SEQ ID NO: 21.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH set forth in SEQ ID NO: 20, and/or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 984, at least 99%, or 100% sequence identity to the VL set forth in SEQ ID NO: 21.
In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VH set forth in SEQ ID NO: 20, and/or, a VL having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VL set forth in SEQ ID NO: 21. In a preferred embodiment, the substitution is a conservative substitution.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH set forth in SEQ ID NO: 34, and/or a VL set forth in SEQ ID NO: 35.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH set forth in SEQ ID NO: 34, and/or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 984, at least 99%, or 100% sequence identity to the VL set forth in SEQ ID NO: 35.
In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VH set forth in SEQ ID NO: 34, and/or, a VL having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VL set forth in SEQ ID NO: 35. In a preferred embodiment, the substitution is a conservative substitution.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH set forth in SEQ ID NO: 36, and/or a VL set forth in SEQ ID NO: 37.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH set forth in SEQ ID NO: 36, and/or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 984, at least 99%, or 100% sequence identity to the VL set forth in SEQ ID NO: 37.
In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VH set forth in SEQ ID NO: 36, and/or, a VL having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VL set forth in SEQ ID NO: 37. In a preferred embodiment, the substitution is a conservative substitution.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH set forth in SEQ ID NO: 51 or 52, and/or a VL set forth in SEQ ID NO: 53.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH set forth in SEQ ID NO: 51 or 52, and/or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 984, at least 99%, or 100% sequence identity to the VL set forth in SEQ ID NO: 53.
In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VH set forth in SEQ ID NO: 51 or 52, and/or, a VL having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VL set forth in SEQ ID NO: 53. In a preferred embodiment, the substitution is a conservative substitution.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH set forth in SEQ ID NO: 54, and/or a VL set forth in SEQ ID NO: 55.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH set forth in SEQ ID NO: 54, and/or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 984, at least 99%, or 100% sequence identity to the VL set forth in SEQ ID NO: 55.
In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VH set forth in SEQ ID NO: 54, and/or, a VL having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VL set forth in SEQ ID NO: 55. In a preferred embodiment, the substitution is a conservative substitution.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH set forth in SEQ ID NO: 68, and/or a VL set forth in SEQ ID NO: 69.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the VH set forth in SEQ ID NO: 68, and/or a VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 984, at least 99%, or 100% sequence identity to the VL set forth in SEQ ID NO: 69.
In certain embodiments, the antibody or antigen-binding fragment thereof comprises a VH having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VH set forth in SEQ ID NO: 68, and/or, a VL having a substitution, deletion or addition of one or several amino acids or any combination thereof (e.g. a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) compared to the VL set forth in SEQ ID NO: 69. In a preferred embodiment, the substitution is a conservative substitution.
In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:
In any one of the above aspects, the antibody or antigen-binding fragment thereof of the invention may further comprise a constant region sequence derived from a mammal (e.g., a mouse or a human) immunoglobulin or a variant thereof. In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain constant region (CH) of a human or murine immunoglobulin or a variant thereof, wherein the variant has a substitution, deletion or addition of one or more amino acids or any combination thereof (e.g., a substitution, deletion or addition of up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids, or any combination thereof; e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) as compared to the wild-type sequence from which it is derived; and/or, the light chain of the antibody or antigen-binding fragment thereof of the invention comprises a light chain constant region (CL) of a human or murine immunoglobulin or a variant thereof, wherein the variant has a substitution, deletion or addition of one or mom amino acids or any combination thereof (e.g., a substitution, deletion or addition of up to 50, up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids, or any combination thereof; e.g., a substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids, or any combination thereof) as compared to the wild-type sequence from which it is derived.
In certain embodiments, the heavy chain of the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain constant region (CH) of a human immunoglobulin or a variant thereof, wherein the variant has conservative substitutions of up to 50 amino acids (e.g., conservative substitutions of up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) as compared to the wild-type sequence from which it is derived; and/or, the light chain of the antibody or antigen-binding fragment thereof of the invention comprises a light chain constant region (CL) of a human immunoglobulin or a variant thereof, wherein the variant has conservative substitutions of up to 50 amino acids (e.g., conservative substitutions of up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) as compared to the wild-type sequence from which it is derived.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain constant region (CH) of a murine immunoglobulin or a variant thereof, wherein the variant has conservative substitutions of up to 50 amino acids (e.g., conservative substitutions of up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) as compared to the wild-type sequence from which it is derived. In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a light chain constant region (CL) of a murine immunoglobulin or a variant thereof, wherein the variant has conservative substitutions of up to 50 amino acids (e.g., conservative substitutions of up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) as compared to the wild-type sequence from which it is derived.
In some embodiments, the constant region is altered, e.g. mutated, to modify the properties of the anti-PD-1 antibody molecule (e.g., altering one or more of the following characteristics: Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function). The functions can be altered by replacing at least one amino acid residue in the antibody constant region with a different residue, for example, to alter the affinity of the antibody for an effector ligand (e.g., FcR or complement C1q), thereby changing (e.g., decreasing) effector function. The Fc region of the antibody mediates several important effector functions, such as ADCC, phagocytosis, CDC and so on.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain constant region (Fc) selected from the group consisting of heavy chain constant regions of, for example, IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD and IgE; particularly selected from the group consisting of heavy chain constant regions of, for example, IgG1, IgG2, IgG3 and IgG4, and mole particularly selected from the group consisting of heavy chain constant regions of IgG1 and IgG4 (e.g., human IgG1 or IgG4). In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a light chain constant region selected from, for example, a kappa or lambda light chain constant region, preferably a kappa light chain constant region (e.g., a human kappa light chain constant region).
In some embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region selected from the group consisting of (1) a human IgG1 heavy chain constant region; (2) a human IgG4 heavy chain constant region; or (3) a mutant of human IgG4 heavy chain constant region (S228P).
In some embodiments, the antibody or antigen-binding fragment thereof comprises a human IgG1 constant region (uniprot ID P01857), or a human IgG4 constant region (uniprot ID P01861).
In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain constant region (CH) as set forth in SEQ ID NO: 83 or a variant thereof, wherein the variant has conservative substitutions of up to 50 amino acids (e.g., conservative substitutions of up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) as compared to SEQ ID NO: 83; and/or, a light chain constant region (CL) as set forth in SEQ ID NO: 84 or a variant thereof, wherein the variant has conservative substitutions of up to 50 amino acids (e.g., conservative substitutions of up to 45, up to 40, up to 35, up to 30, up to 25, up to 20, up to 15, up to 10, or up to 5 amino acids; e.g., conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) as compared to SEQ ID NO: 84.
In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:
In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain constant region (CH) set forth in SEQ ID NO: 82 or 83 and a light chain constant region (CL) set forth in SEQ ID NO: 84.
In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:
In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.
In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:
In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.
In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:
In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.
In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:
In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.
In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:
In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.
In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:
In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.
In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:
In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.
In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:
In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.
In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:
In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.
In some embodiments, the antibody or antigen-binding fragment thereof of the invention comprises:
In certain embodiments, the substitution in (ii) or (v) is a conservative substitution.
In certain embodiments, the antibody of the invention comprises: a heavy chain comprising a VH set forth in SEQ ID NO: 1 and a heavy chain constant region (CH) set forth in SEQ ID NO: 83, and a light chain comprising a VL set forth in SEQ ID NO: 2 and a light chain constant region (CL) set forth in SEQ ID NO: 84.
In certain embodiments, the antibody of the invention comprises: a heavy chain comprising a VH set forth in SEQ ID NO: 16 and a heavy chain constant region (CH) set forth in SEQ ID NO: 83, and a light chain comprising a VL set forth in SEQ ID NO: 18 and a light chain constant region (CL) set forth in SEQ ID NO: 84.
In certain embodiments, the antibody of the invention comprises: a heavy chain comprising a VH set forth in SEQ ID NO: 17 and a heavy chain constant region (CH) set forth in SEQ ID NO: 83, and a light chain comprising a VL set forth in SEQ ID NO: 19 and a light chain constant region (CL) set forth in SEQ ID NO: 84.
In certain embodiments, the antibody of the invention comprises: a heavy chain comprising a VH set forth in SEQ ID NO: 20 and a heavy chain constant region (CH) set forth in SEQ ID NO: 83, and a light chain comprising a VL set forth in SEQ ID NO: 21 and a light chain constant region (CL) set forth in SEQ ID NO: 84.
In certain embodiments, the antibody of the invention comprises: a heavy chain comprising a VH set forth in SEQ ID NO: 34 and a heavy chain constant region (CH) set forth in SEQ ID NO: 83, and a light chain comprising a VL set forth in SEQ ID NO: 35 and a light chain constant region (CL) set forth in SEQ ID NO: 84.
In certain embodiments, the antibody of the invention comprises: a heavy chain comprising a VH set forth in SEQ ID NO: 36 and a heavy chain constant region (CH) set forth in SEQ ID NO: 83, and a light chain comprising a VL set forth in SEQ ID NO: 37 and a light chain constant region (CL) set forth in SEQ ID NO: 84.
In certain embodiments, the antibody of the invention comprises: a heavy chain comprising a VH set forth in SEQ ID NO: 51 and a heavy chain constant region (CH) set forth in SEQ ID NO: 83, and a light chain comprising a VL set forth in SEQ ID NO: 53 and a light chain constant region (CL) set forth in SEQ ID NO: 84.
In certain embodiments, the antibody of the invention comprises: a heavy chain comprising a VH set forth in SEQ ID NO: 52 and a heavy chain constant region (CH) set forth in SEQ ID NO: 83, and a light chain comprising a VL set forth in SEQ ID NO: 53 and a light chain constant region (CL) set forth in SEQ ID NO: 84.
In certain embodiments, the antibody of the invention comprises: a heavy chain comprising a VH set forth in SEQ ID NO: 54 and a heavy chain constant region (CH) set forth in SEQ ID NO: 83, and a light chain comprising a VL set forth in SEQ ID NO: 55 and a light chain constant region (CL) set forth in SEQ ID NO: 84.
In certain embodiments, the antibody of the invention comprises: a heavy chain comprising a VH set forth in SEQ ID NO: 68 and a heavy chain constant region (CH) set forth in SEQ ID NO: 83, and a light chain comprising a VL set forth in SEQ ID NO: 69 and a light chain constant region (CL) set forth in SEQ ID NO: 84.
In certain embodiments, the antibody of the invention is a chimeric or humanized antibody. In certain embodiments, the antibody or antigen-binding fragment thereof of the invention is selected from the group consisting of scFv, Fab, Fab′, (Fab′)2, Fv fragments, disulfide linked Fv (dsFv), a diabody, a bispecific antibody, and a multispecific antibody.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention may exhibit at least one of the following properties:
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention has reduced ADCP activity.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention has reduced ADCC activity.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention has reduced CDC activity.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention does not have CDC activity.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention does not have ADCP activity.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention does not have ADCC activity.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention has reduced activity of one, two, or all of ADCP, ADCC, and CDC activities.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention does not have one, both, or all of ADCP, ADCC, and CDC activities.
The antibody or antigen-binding fragment thereof of the invention may be derivatized, for example, by linked to an additional molecule (e.g., an additional polypeptide or protein). Typically, derivatization (e.g., labeling) of an antibody or antigen-binding fragment thereof does not adversely affect its binding to PD-1 (particularly human PD-1). Thus, the antibody or antigen-binding fragment thereof of the invention also intends to include such derivative forms. For example, the antibody or antigen-binding fragment thereof of the invention may be linked to (by chemical coupling, gene fusion, non-covalently or other manners) one or more other molecular groups, e.g., another antibody (e.g. forming a bispecific antibody), a detection agent, a pharmaceutical reagent, and/or a protein or polypeptide capable of mediating the binding of the antibody or the antigen-binding fragment to another molecule (e.g. avidin or polyhistidine tag).
One type of derivative antibody (e.g., a bispecific antibody) is generated by cross-linking two or more antibodies (of same or different types). A method for obtaining a bispecific antibody is well known in the art and exemplary methods include, but not limited to, chemical cross-linking, cell engineering (hybridoma) or genetic engineering.
Another type of derivative antibody is a labeled antibody. For example, the antibody or antigen-binding fragment thereof of the invention may be linked to a detectable marker. The detectable marker of the invention may be any substance to be detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics or chemistry. Such markers are well known in the art and examples thereof include, but not limited to, enzymes (e.g. horseradish peroxidase, alkaline phosphatase, beta-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethyl rhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine ester compounds, magnetic beads (e.g., Dynabeads®), calorimetric markers such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads, and biotin for binding to avidin (e.g. streptavidin) modified by the above markers. Such markers are disclosed in patents including, but not limited to, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, all of which am incorporated herein by reference. The detectable marker described above can be detected by a method known in the art. For example, a radioactive marker may be detected using a photographic film or a scintillation calculator, and a fluorescent marker may be detected using a photodetector to detect the emitted light. An enzyme marker is generally detected by providing a substrate to the enzyme and detecting the reaction product produced by the effect of enzyme on the substrate, and a calorimetric marker is detected by a simple visual coloring marker. In certain embodiments, such markers are useful in immunoassays (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). In some embodiments, the detectable markers described above may be linked to the antibody or antigen-binding fragment thereof of the invention through linkers of different lengths to reduce potential steric hindrance.
In addition, the antibody or antigen-binding fragment thereof of the invention may also be derivatized with a chemical group, such as polyethylene glycol (PEG), methyl or ethyl, or glycosyl. These groups can be used to improve the biological characteristics of the antibody, such as increase the serum half-life.
Therefore, in one aspect, the invention provides a conjugate comprising the monoclonal antibody or antigen-binding fragment thereof of the invention and a coupling moiety which may be a detectable marker as described above, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme. The coupling moiety may also be an agent, such as a chemotherapeutics, a radionuclide or toxin, such as paclitaxel, cyclophosphamide, fluorouracil, 89Sr, duocarmycin.
As one of the derivatives of the antibody, the invention provides a multispecific antibody comprising a first antibody or fragment thereof, and an additional antibody or fragment thereof, or an antibody mimetic, wherein the first antibody or fragment thereof, the additional antibody or fragment thereof, or the antibody mimetic retains its original binding specificity. The first antibody or fragment thereof is any (monoclonal) antibody or antigen-binding fragment thereof of the invention which specifically binds to PD-1. As used herein, “antibody mimetic” refers to specifically binds to an antigen as an antibody, but without an antibody structure. They are usually artificial peptides or proteins having a molar mass of about 3 to 20 kDa, e.g. a designed ankyrin repeat protein (DARPin) and fynomer. The designed ankyrin repeat protein (DARPin) can be linked to an IgG antibody, a scFv-Fc antibody fragment or combination thereof, as described in CN104341529A. An anti-IL-17a fynomer binds to an anti-IL-6R antibody, as described in WO2015141862A1.
In certain embodiments, the multispecific antibody is formed by coupling the first antibody or antigen-binding fragment thereof with an additional antibody or antigen-binding fragment thereof or an antibody mimetic, wherein each antibody or antigen-binding fragment thereof or antibody analog retains its original binding specificity, and wherein the first antibody or antigen-binding fragment thereof is the antibody or antigen-binding fragment thereof of the invention. In certain embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody or a tetraspecific antibody.
The antibody of the invention can be prepared by various methods known in the art, e.g., genetic engineering recombinant technology. For example, obtaining a DNA molecule encoding the heavy and light chain genes of the antibody of the invention by chemical synthesis or PCR amplification, inserting the obtained DNA molecule into an expression vector and then transfecting a host cell; then, culturing the transfected host cell under particular conditions to express the antibody of the invention.
The antigen-binding fragments of the invention can be obtained by hydrolyzing an intact antibody molecule (See Morimoto et al., J. Biochem. Biophys. Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). In addition, these antigen-binding fragments can also be produced directly by recombinant host cells (reviewed in Hudson, Curr. Opin. Immunol. 11: 548-557 (1999); Little et al., Immunol. Today, 21: 364-370 (2000)). For example, Fab′ fragments can be obtained directly from host cells; Fab′ fragments can be chemically coupled to form F (ab′)2 fragments (Carter et al., Bio/Technology, 10: 163-167 (1992)). Furthermore, Fv, Fab or F (ab′)2 fragments can also be isolated directly from the recombinant host cell culture medium. Other technologies for preparing these antigen-binding fragments are well known to those of ordinary skill in the art.
Thus, in another aspect, the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of the invention, or a heavy chain variable region and/or a light chain variable region thereof, or one or more CDRs thereof. In some embodiments, the nucleotide sequence may be substituted in accordance with the codon degeneracy which is known in the art. In certain embodiments, the nucleotide sequence is codon optimized.
In certain embodiments, the isolated nucleic acid molecule of the invention comprises: (i) a first nucleic acid and a second nucleic acid encoding a heavy chain variable region and a light chain variable region of the antibody or antigen-binding fragment thereof of the invention, respectively, or (ii) a first nucleic acid encoding a heavy chain variable region and a heavy chain constant region, and a second nucleic acid encoding a light chain variable region and a light chain constant region, respectively, of the antibody or antigen-binding fragment thereof of the invention, or (iii) a first nucleic acid and a second nucleic acid encoding a heavy chain and a light chain of the antibody or antigen-binding fragment thereof of the invention, respectively. In certain embodiments, the first and second nucleic acids comprise a nucleic acid of a degenerate sequence of, or a substantially identical sequence to, any one of the first and second nucleic acids in the above (i)-(iii). The degenerate sequence or substantially identical sequence refers to a sequence having at least about 85%, 90%, 954, 99% or greater sequence identity or having one or more nucleotide substitutions or differing by not more than 3, 6, 15, 30 or 45 nucleotides, as compared to the nucleic acid molecules in (i)-(iii). For example, the isolated nucleic acid molecule may comprise a nucleotide sequence set forth in SEQ ID NO: 85, SEQ ID NO: 87, SEQ ID NO: 89, SEQ ID NO: 46 or a degenerate sequence thereof or a sequence substantially identical thereto.
In certain embodiments, the invention provides an isolated nucleic acid molecule, comprising a nucleic acid molecule encoding a heavy chain of antibody, and/or a nucleic acid molecule encoding a light chain of antibody, wherein the nucleic acid molecule encoding the heavy chain of antibody has a sequence selected from the group consisting of: (a) a nucleotide sequence set forth in SEQ ID NO: 85 or a degenerate sequence thereof, or (b) a sequence substantially identical to the nucleotide sequence of (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity or having one or more nucleotide substitutions compared to the nucleotide sequence of (a)), or (c) a sequence which differs by not more than 3, 6, 15, 30 or 45 nucleotides from the nucleotide sequence of (a); and/or, the nucleic acid molecule encoding the light chain of antibody has a sequence selected from the group consisting of: (d) a nucleotide sequence set forth in SEQ ID NO: 87 or a degenerate sequence thereof, or (e) a sequence substantially identical to the nucleotide sequence of (d) (e.g. a sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity or having one or more nucleotide substitutions compared to the nucleotide sequence of (d)), or (f) a sequence which differs by not more than 3, 6, 15, 30 or 45 nucleotides from the nucleotide sequence of (d).
In certain embodiments, the isolated nucleic acid molecule of the invention comprises a nucleic acid molecule encoding a heavy chain of antibody set forth in SEQ ID NO: 85, and/or a nucleic acid molecule encoding a light chain of antibody set forth in SEQ ID NO: 87.
In certain embodiments, the invention provides an isolated nucleic acid molecule, comprising a nucleic acid molecule encoding a heavy chain of antibody, and/or a nucleic acid molecule encoding a light chain of antibody, wherein the nucleic acid molecule encoding a heavy chain of antibody has a sequence selected from the group consisting of: (a) a nucleotide sequence set forth in SEQ ID NO: 89 or a degenerate sequence thereof, or (b) a sequence substantially identical to the nucleotide sequence of (a) (e.g., a sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity or having one or more nucleotide substitutions compared to the nucleotide sequence of (a)), or (c) a sequence which differs by not more than 3, 6, 15, 30 or 45 nucleotides from the nucleotide sequence of (a); and/or, the nucleic acid molecule encoding a light chain of antibody has a sequence selected from the group consisting of: (d) a nucleotide sequence set forth in SEQ ID NO: 46 or a degenerate sequence thereof, or (e) a sequence substantially identical to the nucleotide sequence of (d) (e.g. a sequence having at least about 85%, 90%, 95%, 99% or greater sequence identity or having one or more nucleotide substitutions compared to the nucleotide sequence of (d)), or (f) a sequence which differs by not more than 3, 6, 15, 30 or 45 nucleotides from the nucleotide sequence of (d).
In certain embodiments, the isolated nucleic acid molecule of the invention comprises a nucleic acid molecule encoding a heavy chain of antibody set forth in SEQ ID NO: 89, and/or a nucleic acid molecule encoding a light chain of antibody set forth in SEQ ID NO: 46.
In another aspect, a vector (e.g., a cloning vector or an expression vector) comprising the isolated nucleic acid molecule of the invention is provided. In certain embodiments, the vector of the invention is, for example, a plasmid, a cosmid, a phage, a lentivirus, or the like. In certain embodiments, the vector is capable of expressing the antibody or antigen-binding fragment thereof of the invention in vivo in a subject (e.g., a mammal, e.g., a human).
In another aspect, a host cell comprising the isolated nucleic acid molecule of the invention or a vector of the invention is provided. The host cell may be a eukaryotic cell (e.g., a mammalian cell, an insect cell, a yeast cell) or a prokaryotic cell (e.g., Escherichia coli). Suitable eukaryotic cells include, but not limited to, NS0 cells, Vero cells, Hela cells, COS cells, CHO cells, HEK293 cells, BHK cells, and MDCKII cells. Suitable insect cells include, but not limited to, Sf9 cells. In certain embodiments, the host cell of the invention is a mammalian cell, such as CHO (e.g., CHO-K1, CHO-S, CHO DXB11, CHO DG44).
In another aspect, a method for preparing the antibody or antigen-binding fragment thereof of the invention is provided, comprising culturing the host cell of the invention under conditions allowing for expression of the antibody or antigen-binding fragment thereof, and recovering the antibody or antigen-binding fragment thereof from the cultured host cell culture.
In another aspect, one or more chimeric antigen receptors (CAR) and cells carrying or expressing the CAR are provided, the cells carrying or expressing the CAR comprising or expressing any antibody or antigen-binding fragment (e.g., scFv), nucleic acid or vector of the invention.
The cell receiving CAR is called as host cell. A host cell carrying or expressing CAR is called as CAR cell. In certain embodiments, the host cell is selected from an immune cell, preferably, the immune cell is selected from the group consisting of T lymphocytes, NK cells, monocytes, macrophages or dendritic cells, and any combination thereof.
Preparation of CAR cells can be referred to the technologies or conditions described in the literatures in the art, for example, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition (translated by Huang Peitang et al.), or can be carried out according to the product instructions.
In another aspect, an oncolytic virus comprising the isolated nucleic acid molecule of the invention or a vector of the invention is provided.
It is characterized in that a nucleic acid molecule encoding one or several antibodies or antigen-binding fragments thereof of the invention is inserted or carried into the oncolytic virus. The oncolytic virus carrying the nucleic acid molecule of the invention expresses one or more anti-PD-1 antibodies or antigen-binding fragments thereof.
Preparation of a modified oncolytic virus can be referred to the technologies or conditions described in the literatures in the art, for example, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition (translated by Huang Peitang et al.), or can be carried out according to the product instructions.
The oncolytic virus carrying the nucleic acid molecule of the invention can be effective in delivering an anti-PD-1 antibody or antigen-binding fragment thereof, realizing the function of expressing the anti-PD-1 antibody or antigen-binding fragment thereof in vivo while realizing the virus function of lysing tumor cells, thus playing dual roles in both lysing tumor cells and enhancing anti-tumor immunity.
Therefore, the oncolytic virus modified by the invention can be used to prepare a medicament for preventing and/or treating tumors; the oncolytic virus modified by the invention can be used to prevent and/or treat tumors.
In another aspect, a fusion protein comprising the monoclonal antibody or antigen-binding fragment thereof of the invention and a non-immunoglobulin portion which is a polypeptide such as a cytokine (e.g., IL-2, IL-15, IL-7, IL-12, IL-18, IL-21, TNF-α, or IFNγ) is provided.
In another aspect, the invention provides a pharmaceutical composition comprising the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the CAR cell, the multispecific antibody, the conjugate, the oncolytic virus or the fusion protein of the invention, and a pharmaceutically acceptable carrier and/or excipient.
In certain embodiments, the pharmaceutical composition of the invention comprises the antibody or antigen-binding fragment thereof of the invention, and a pharmaceutically acceptable carrier and/or excipient.
In certain embodiments, the pharmaceutical composition of the invention comprises the host cell of the invention, which comprises an isolated nucleic acid molecule or vector described above, and a pharmaceutically acceptable carrier and/or excipient.
In certain embodiments, the pharmaceutical composition of the invention comprises the multispecific antibody of the invention, and a pharmaceutically acceptable carrier and/or excipient.
In certain embodiments, the pharmaceutical composition of the invention comprises the conjugate of the invention, and a pharmaceutically acceptable carrier and/or excipient.
In certain embodiments, the pharmaceutical composition of the invention comprises the CAR cell of the invention, and a pharmaceutically acceptable carrier and/or excipient.
In certain embodiments, the pharmaceutical composition of the invention comprises the oncolytic virus of the invention, and a pharmaceutically acceptable carrier and/or excipient.
In certain embodiments, the pharmaceutical composition of the invention comprises the fusion protein of the invention, and a pharmaceutically acceptable carrier and/or excipient.
In certain embodiments, the pharmaceutical composition may also comprise an additional pharmaceutical active agent. In certain embodiments, the additional pharmaceutically active agent is a chemotherapeutic, an anti-tumor agent, an anti-infection agent, or an anti-autoimmune disease agent, an anti-angiogenesis agent, or a tyrosine kinase inhibitor.
In certain embodiments, the antibody or antigen-binding fragment thereof of the invention and the additional pharmaceutically active agent are provided as separate components or as components in the same composition. Thus, the antibody or antigen-binding fragment thereof of the invention can be administered in combination or separately, simultaneously, or sequentially with the additional pharmaceutically active agent.
In certain embodiments, the pharmaceutical composition may also comprise an additional pharmaceutical active agent. The additional pharmaceutically active agent is selected from the group consisting of an anti-tumor agent, a radiopharmaceutical, an agent associated with an immune-regulatory effect, or an agent associated with an autoimmune disease. The anti-tumor agent comprises a chemotherapeutic agent, an antibody associated with an immune-regulatory effect, or an anti-tumor biomacromolecule agent. The chemotherapeutic agent comprises an anti-angiogenesis agent or a tyrosine kinase inhibitor. In certain embodiments, the chemotherapeutic agent includes, but not limited to: for example, 5-fluorouracil, actinomycin-D, daunorubicin, mitomycin, vincristine and so on, or any combination thereof.
In certain embodiments, the radiopharmaceutical includes, but not limited to, carbon-11, carbon-14, chromium-51, cobalt-57, cobalt-58, erbium-169, fluorine-18, gallium-67, gold-198, indium-ill, indium-113m, iodine-123, iodine-125, iodine-131, and the like.
In certain embodiments, the antibody associated with an immune-regulatory effect refers to an antibody useful in preventing, inhibiting, and/or treating a tumor, such as an anti-CTLA-4 antibody, an anti-HER2 antibody, an anti-EGFR antibody, an anti-LAG-3 antibody, or a TIM-3 antibody, and the like; preferably, the anti-LAG-3 antibody is AB12T (Sichuan Kelun Biotech Biopharmaceutical Co., Ltd., WO2019141092); and the TIM-3 antibody is AB12S3 (Sichuan Kelun Biotech Biopharmaceutical Co., Ltd., WO2019206095).
In certain embodiments, the agent associated with an immune-regulatory effect refers to an agent useful in preventing, inhibiting and/or treating inflammation or infection, or having an immune-regulatory effect, such as an anti-IL-1 antibody, an anti-IL-6 antibody, an anti-TNF-α antibody, and the like.
In certain embodiments, the agent associated with an autoimmune disease refers to an agent useful in preventing, inhibiting and/or treating an autoimmune disease, such as an anti-IL-1 antibody, an anti-IL-6 antibody, an anti-TNF-α antibody, an anti-CD20 antibody, an anti-B-lymphocyte stimulator (BLyS) antibody, and the like.
In certain embodiments, the tyrosine kinase inhibitor includes, but not limited to, imatinib, sorafenib, sunitinib, lapatinib, dasatinib.
In certain embodiments, the pharmaceutical composition is administered concurrently, separately, or sequentially with other treatments or therapeutic agents, such as before, simultaneously with, or after tumor treatment.
In another aspect, the antibody or antigen-binding fragment thereof, nucleic acid, vector, host cell, CAR cell, multispecific antibody, oncolytic virus, fusion protein or conjugate in the pharmaceutical composition of the invention is sufficient to produce at least one of the following biological activities in a subject:
In another aspect, the invention provides the use of the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the CAR cell, the multispecific antibody, the conjugate, the oncolytic vims, or the fusion protein of the invention in the preparation of a medicament for:
In certain embodiments, when used for preparing a medicament, the host cell of the invention comprises the isolated nucleic acid molecule or the vector described above.
In certain embodiments, when the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the CAR cell, the multispecific antibody, the oncolytic virus, the fusion protein, the conjugate, the pharmaceutical composition, or the pharmaceutical composition of the invention in combination with an additional pharmaceutically active agent (e.g., an anti-tumor agent, a radiopharmaceutical, an agent associated with an immune-regulatory effect, or an agent associated with an autoimmune disease) is used for preparing a medicament, the medicament is used for preventing and/or treating a tumor, an infection, or an autoimmune disease in a subject (e.g., a human).
In certain embodiments, the subject is a mammal. In certain embodiments, the subject is a human.
In certain embodiments, the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the cell, the multispecific antibody, the oncolytic virus, the conjugate, the fusion protein, the agent, the pharmaceutical composition, or the pharmaceutical composition of the invention in combination with an additional pharmaceutically active agent (e.g., an anti-tumor agent, a radiopharmaceutical, an agent associated with an immune-regulatory effect, or an agent associated with an autoimmune disease) is used for preventing and/or treating a tumor. In certain embodiments, the tumor is selected from one or more in the tissues from the group consisting of: skin, bone marrow, blood, lymph, head and neck, brain, lung, breast, stomach, liver, pancreas, gallbladder, intestine, ovary, prostate, adrenal gland, kidney, bladder, uterus, cervix, testis, penis, and soft tissue.
In certain embodiments, the tumor is selected from one or more in the group consisting of lung cancer, liver cancer, ovarian cancer, cervical cancer, skin cancer, colon cancer, rectal cancer, glioma, bladder cancer, breast cancer, kidney cancer, esophageal cancer, gastric cancer, oral squamous cell cancer, urethral epithelial cell cancer and pancreatic cancer, and head and neck tumors.
In certain embodiments, the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the CAR cell, the multispecific antibody, the oncolytic virus, the fusion protein, the conjugate, the pharmaceutical composition, or the pharmaceutical composition of the invention together with an additional pharmaceutically active agent (e.g., an anti-tumor agent, a radiopharmaceutical, an agent associated with an immune-regulatory effect, or an agent associated with an autoimmune disease) is used for preventing and/or treating a disease or condition associated with an infection. In certain embodiments the infection includes, but not limited to, a bacterial infection, a pathogen infection, a fungal infection, and a viral infection. In certain embodiments, the antibody or antigen-binding fragment thereof, the vector, the host cell, the multispecific antibody, the conjugate is used for preventing and/or treating a viral infection, such as a chronic viral infection, an infection of hepatitis B virus, hepatitis C virus, or HIV, etc., or preventing and/or treating other chronic infections, such as tuberculosis.
In certain embodiments, the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the CAR cell, the multispecific antibody, the oncolytic virus, the fusion protein, the conjugate, the pharmaceutical composition, or the pharmaceutical composition of the invention together with an additional pharmaceutically active agent (e.g., an anti-tumor agent, a radiopharmaceutical, an agent associated with an immune-regulatory effect, or an agent associated with an autoimmune disease) is used for preventing and/or treating a disease associated with autoimmunity. In certain embodiments, the disease includes, but not limited to, hyperthyroidism, diabetes, myasthenia gravis, ulcerative colitis, gastritis, pemphigus, primary biliary cirrhosis, multiple sclerosis, lupus erythematosus, rheumatoid arthritis, and the like.
In another aspect, the invention provides a method for at least one of in vivo/in vitro (1) inhibiting or blocking the binding of PD-1 to PD-L1 or PD-L2, (2) down-regulating or eliminating the activity of PD-1, (3) reducing or relieving the immunosuppression of PD-1 or PD-L1 or PD-L2, (4) increasing the production of IFNγ or IL-2 in T lymphocytes and (5) enhancing the ability of T lymphocytes to kill tumor cells, comprising: administrating to a cell or a subject any one of the described antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the multispecific antibody, the conjugate, the pharmaceutical composition, or the pharmaceutical composition together with an additional pharmaceutically active agent (e.g., an anti-tumor agent, a radiopharmaceutical, an agent associated with an immune-regulatory effect, or an agent associated with an autoimmune disease).
Optionally, a chemotherapeutic, an anti-tumor agent, an anti-angiogenesis agent, a tyrosine kinase inhibitor, or a PD-1 pathway inhibitor is administered concurrently with, before or after administration of the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the multispecific antibody, the conjugate, or the pharmaceutical composition.
In certain embodiments, the subject is a mammal; preferably, the subject is a human.
Thus, in another aspect, the invention provides a method for preventing and/or treating a tumor, an infection or an autoimmune disease in a subject, comprising administering to the subject in need thereof an effective amount of the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the CAR cell, the multispecific antibody, the oncolytic virus, the conjugate, the fusion protein, the pharmaceutical composition, or the pharmaceutical composition of the invention together with an additional pharmaceutically active agent (e.g., an anti-tumor agent, a radiopharmaceutical, an agent associated with an immune-regulatory effect, or an agent associated with an autoimmune disease).
The antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the CAR cell, the multispecific antibody, the oncolytic virus, the conjugate, the fusion protein or the pharmaceutical composition of the invention can be formulated as any dosage form known in the medicines, such as tablet, pill, suspension, emulsion, solution, gel, capsule, powder, granule, elixir, lozenge, suppository, injection (including injectable solution, sterile injectable powder and concentrated injectable solution), inhalant, spray, etc. The preferred dosage form depends on the intended administration route and therapeutic use. The pharmaceutical composition of the invention should be sterile and stable under production and storage conditions, and can be prepared into an injection.
In addition, the antibody or antigen-binding fragment thereof of the invention may be present in a unit dose form in the pharmaceutical composition for convenient administration.
The pharmaceutical composition of the invention may include a “therapeutically effective amount” or a “prophylactically effective amount” of the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the CAR cell, the multispecific antibody, the oncolytic virus, the fusion protein, or the conjugate of the invention. A “prophylactically effective amount” means an amount sufficient to prevent, stop, or delay the occurrence of a disease. A “therapeutically effective amount” means an amount sufficient to cure or at least partially prevent a disease and its complications in a patient who has suffered from the disease, for example 0.1 mg/mL to 5000 mg/mL.
In the invention, the subject may be a mammal, such as a human.
The antibody or antigen-binding fragment thereof of the invention is capable of specifically binding PD-1, and thus can be used to detect the presence or level of PD-1 in a sample.
Thus, in another aspect, the invention provides a kit comprising an antibody or antigen-binding fragment thereof of the invention. In certain embodiments, the antibody or antigen-binding fragment thereof of the invention carries a detectable marker. In a preferred embodiment, the kit further comprises a second antibody that specifically recognizes the antibody or antigen-binding fragment thereof of the invention. Preferably, the second antibody further comprises a detectable marker.
In the invention, the detectable marker may be any substance detected by fluorescence, spectroscopy, photochemistry, biochemistry, immunology, electricity, optics, or chemistry. Especially preferred is that such markers am suitable for immunoassays (e.g., enzyme-linked immunoassay, radioimmunoassay, fluorescence immunoassay, chemiluminescence immunoassay, etc.). Such markers are well known in the art and include, but not limited to, enzymes (e.g. horseradish peroxidase, alkaline phosphatase, β-galactosidase, urease, glucose oxidase, etc.), radionuclides (e.g., 3H, 125I, 35S, 14C or 32P), fluorescent dyes (e.g., fluorescein isothiocyanate (FITC), fluorescein, tetramethyl rhodamine isothiocyanate (TRITC), phycoerythrin (PE), Texas Red, rhodamine, quantum dots or cyanine dye derivatives (e.g., Cy7, Alexa 750)), acridine ester compounds, magnetic beads (e.g., Dynabeads®), calorimetric markers such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads, and biotin used to bind avidin (e.g. streptavidin) modified by the above markers. The use of such markers was taught in the patents including, but not limited to, U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241, all of which are incorporated herein by reference. Markers encompassed by the invention can be detected by a method known in the art. For example, a radioactive marker may be detected using a photographic film or a scintillation calculator, and a fluorescent marker may be detected using a photodetector to detect the emitted light. An enzyme marker may be generally detected by providing a substrate for the enzyme and detecting the reaction product produced by the effect of the enzyme on the substrate, and a calorimetric marker may be detected by a simple visual coloring marker. In some embodiments, the detectable markers described above may be linked to the recombinant protein of the invention via linkers of different lengths to reduce the potential steric hindrance.
In another aspect, the invention provides a method for detecting the presence or level of PD-1 in a sample, comprising the step of utilizing the antibody or antigen-binding fragment thereof of the invention. In a preferred embodiment, the antibody or antigen-binding fragment thereof of the invention still carries a detectable marker. In another preferred embodiment, the method further comprises detecting an antibody or antigen-binding fragment thereof of the invention using an agent with a detectable marker. The method may be used for a diagnostic purpose or a non-diagnostic purpose (e.g., PD-1/PD-L1 pathway studies, drug screening, histochemical analysis, etc.). In certain embodiments, the sample for a non-diagnostic purpose is a cell sample, such as a cell line or an ex vivo cell culture.
In another aspect, the invention provides a method for detecting the presence or level of PD-1 in a sample, comprising contacting the sample with the antibody or antigen-binding fragment thereof of the invention under conditions allowing for the formation of a complex between the antibody or antigen-binding fragment thereof and PD-1, and detecting the formation of the complex.
In another aspect, use of the antibody or antigen-binding fragment thereof of the invention for preparing a kit for detecting the presence or level of PD-1 in a sample is provided. In another aspect, the invention provides a diagnostic or therapeutic kit comprising one or more of the antibody or antigen-binding fragment thereof, the nucleic acid, the vector, the host cell, the multispecific antibody, the conjugate, the fusion protein or the pharmaceutical composition of the invention. Optionally, the diagnostic or therapeutic kit further includes instructions for using the same.
The antibody or antigen-binding fragment of the invention has a high binding affinity to PD-1 and has a strong specificity. Therefore, the antibody or antigen-binding fragment of the invention is suitable for preventing and/or treating a tumor, an infection, or an autoimmune disease. The humanized and IgG1-1D mutant anti-PD-1 humanized antibody of the invention not only retains the functions and properties of the parent murine antibody, but also has a higher humanization degree, so it can be administered safely to a human subject without triggering an immunogenic reaction. Therefore, the antibody or antigen-binding fragment of the invention is of great clinical value.
In the invention, unless defined otherwise, all scientific and technical terms used herein have the same meaning as those commonly understood by one of ordinary skill in the art to which this invention belongs. In addition, the cell culture, biochemistry, nucleic acid chemistry, immunology laboratory and other operation steps used herein are conventional steps widely used in corresponding fields. Meanwhile, in order to better understand the invention, definitions and explanations of related terms am provided below.
There is no rule of singular and plural in Chinese grammar, the nouns used herein refer to both singular and plural, which can be particularly determined according to the context, therefore, when the nouns used herein are translated into English, “one or more” can be preceded.
As used herein, the term “antibody” refers to an immunoglobulin molecule that typically consists of two pairs of polypeptide chains, each having a light chain (LC) and a heavy chain (HC). Light chains am classified as either kappa (κ) or lambda (λ) light chains. Heavy chains can be classified as μ, δ, γ, α, or ε, and the isotypes of antibody can be defined as IgM, IgD, IgG, IgA and IgE, respectively. Within light and heavy chains, variable and constant regions are joined by a “J” segment of about 12 or more amino acids, the heavy chain also comprising a “D” segment of about 3 or mom amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2 and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain CL. The constant domain is not directly involved in the binding of an antibody to an antigen, but exhibits various effector functions, such as mediating the binding of an immunoglobulin to a host tissue or factor, including various cells of the immune system (e.g., effector cells) and a first component (C1q) of the classical complement system. The VH and VL regions can also be subdivided into hypervariable regions (called complementary determining region (CDR)) interspersed with relatively conservative regions called framework regions (FR). Each VH and VL consists of three CDRs and four FRs arranged from amino terminal to carboxyl terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain/light chain pair form antigen-binding sites, respectively. The distribution of amino acids in various regions or domains may follow the definitions described in Kabat, Sequences of Proteins of Immunological Interest (National Institutions of Health, Bethesda, Md. (1987 and 1991)), or Chothia & Lesk (1987) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883.
As used herein, the term “complementary determining region” or “CDR” refers to amino acid residues in the variable region of an antibody which are responsible for antigen binding. The precise boundaries of these amino acid residues may be defined according to various numbering systems known in the art, such as the definitions in the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutions of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987)) J. Mol. Biol. 196: 901-917; Chothia et al. (1989) Nature 342: 878-883) or the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003). For a given antibody, a person skilled in the art will readily identify CDRs defined by each numbering system. Also, the correspondence between different numbering systems is well known to those skilled in the art (see, for example, Lefranc et al., Dev. Comparat. Immunol. 27: 55-77, 2003).
In the invention, the CDRs contained in the antibody or antigen-binding fragment thereof of the invention may be determined according to various numbering systems known in the art. In certain embodiments, the CDRs contained in the antibody or antigen-binding fragment thereof of the invention am preferably determined by a Kabat or IMGT numbering system.
As used herein, the term “framework region” or “FR” residues refer to those amino acid residues in the variable region of an antibody other than the CDR residues as defined above.
As used herein, the term “germline antibody gene” is an immunoglobulin sequence encoded by a non-lymphocyte that does not undergo maturation processes of genetic rearrangement and maturation leading to the expression of a specific immunoglobulin. An advantage provided by various embodiments of the invention is derived from a consensus that germline antibody genes retain more important amino acid sequence structures showing characteristics of individual animal species than mature antibody genes. Therefore, when applied therapeutically to this species, it is less recognized as an exogenous substance by this species.
The term “antibody” is not limited by any particular method for producing the same. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody may be an antibody of different isotypes, such as an IgG (e.g., an IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM antibody.
As used herein, the term “antigen-binding fragment” of an antibody refers to a polypeptide fragment of an antibody, such as a polypeptide fragment of a full-length antibody, which retains the ability to specifically bind to the same antigen as bound by the full-length antibody and/or competes with the full-length antibody for specific binding to the antigen, which is also known as an “antigen-binding part”. In general, see Fundamental Immunology, Ch. 7 (Paul, W., ed., 2nd Edition, Raven Press, N.Y. (1989), which is incorporated herein by reference in its entirety for all purposes. Antigen-binding fragments of an antibody can be produced by recombinant DNA technologies or by enzymatic or chemical cleavage of an intact antibody. Non-limiting examples of antigen-binding fragments include Fab, Fab′, F (ab′)2, Fd, Fv, dAb and complementary determining region (CDR) fragments, single chain antibody (e.g. scFv), chimeric antibody, diabody, linear antibody, nanobody (e.g. by technology from Ablynx), domain antibody (e.g. by technology from Domantis), and a polypeptide comprising at least a portion of an antibody sufficient to confer the specific antigen-binding ability to the polypeptide. Engineering modified antibody variants are reviewed in Holliger et al., 2005; Nat Biotechnol, 23: 1126-1136.
As used herein, the term “full-length antibody” means an antibody consisting of two “full-length heavy chains” and two “full-length light chains”. Wherein, “full-length heavy chain” refers to a polypeptide chain which, from N-terminal to C-terminal, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain and a heavy chain constant region CH3 domain; and, when the full-length antibody is an IgE isotype, it optionally further includes a heavy chain constant region CH4 domain. Preferably, a “full-length heavy chain” is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 from N-terminal to C-terminal. A “full-length light chain” is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) from N-terminal to C-terminal. Two pairs of full-length antibody chains am linked by a disulfide bridge between CL and CH1 and a disulfide bridge between HRs of two full-length heavy chains. The full-length antibody of the invention may be derived from a single species, such as a human; and can also be a chimeric antibody or a humanized antibody. The full-length antibody of the invention comprises two antigen-binding sites formed by VH and VL pairs, respectively, which sites specifically recognize/bind the same antigen.
As used herein, the term “Fd fragment” means an antibody fragment consisting of VH and CH1 domains; the term “dAb fragment” means an antibody fragment consisting of a VH domain (Ward et al., Nature 341: 544 546 (1989)); the term “Fab fragment” means an antibody fragment consisting of VL, VH, CL and CH1 domains; the term “F(ab′)2 fragment” means an antibody fragment comprising two Fab fragments linked by a disulfide bridge on the hinge region; the term “Fab′ fragment” means a fragment obtained by reducing the disulfide bridge linking two heavy chain fragments in the F(ab′)2 fragment, which consists of a complete light chain and a Fd fragment (composed of VH and CH1 domains) of heavy chain.
As used herein, the term “Fv fragment” means an antibody fragment consisting of VL and VH domains of a single-arm of antibody. The Fv fragment is generally considered to be the smallest antibody fragment that can form a complete antigen-binding site. It is generally thought that six CDRs confer antigen-binding specificity to an antibody. However, even one variable region (such as a Fd fragment, which contains only three antigen-specific CDRs) can recognize and bind antigen, although possibly having a lower affinity than a complete binding site.
As used herein, the term “Fc fragment” means an antibody fragment formed by linking the second and third constant regions of the first heavy chain of an antibody to the second and third constant regions of the second heavy chain of the antibody via a disulfide bridge. Fc fragment of an antibody has many different functions, but does not involve in antigen binding.
As used herein, the term “scFv” refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are linked by a linker (see, for example, Bird et al., Science 242: 423-426 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988); and Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Ed. Roseburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994)). Such scFv molecules may have a general structure: NH2-VL-Linker-VH-COOH or NH2-VH-Linker-VL-COOH. A suitable linker in the art consists of repeated GGGGS amino acid sequences or variants thereof. For example, a linker of amino acid sequence (GGGGS)4 or a variant thereof can be used (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444-6448). Other linkers that may be used in the invention am described in Alfthan et al. (1995), Protein Eng. 8: 725-731; Choi et al. (2001), Eur. J. Immunol. 31: 94-106; Hu et al. (1996), Cancer Res. 56: 3055-3061; Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol. In some cases, a disulfide bridge may also be present between VH and VL of scFv. As used herein, the term “di-scFv” refers to an antibody fragment formed by linking two scFvs.
As used herein, the term “diabody” means that the VH and VL domains am expressed on a single polypeptide chain but the used linker is too short to allow for pairing between two domains of the same chain, thus forcing the domain to pair with a complementary domain of the other chain and producing two antigen-binding sites (see, for example, Holliger P. et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993), and Poljak R. J. et al., Structure 2:1121-1123 (1994)).
As used herein, “antibody mimetic” refers to specifically binds to an antigen as an antibody, but without antibody structure. They are usually artificial peptides or proteins having a molar mass of about 3 to 20 kDa. Examples are designed ankyrin repeat protein (DARPin) and fynomer. The designed ankyrin repeat protein (DARPin) can be linked to an IgG antibody, a scFv-Fc antibody fragment or combination thereof, as described in CN104341529A. An anti-IL-17a fynomer binds to an anti-IL-6R antibody, as described in WO2015141862A1.
Each of the above antibody fragments retains the ability to specifically bind to the same antigen as bound by the full-length antibody, and/or competes with the full-length antibody for specific binding to an antigen.
An antigen-binding fragment (e.g., the antibody fragment described above) may be obtained from a given antibody (e.g., the antibody provided by the invention) using conventional technologies known to those skilled in the art (e.g., recombinant DNA technologies or enzymatic or chemical cleavage methods), and the antigen-binding fragment of the antibody is specifically screened in the same manner as for an intact antibody.
Herein, unless clearly indicated otherwise in the context, reference to the term “antibody” includes not only an intact antibody but also an antigen-binding fragment thereof.
As used herein, the terms “monoclonal antibody”, “McAb” and “mAb” have the same meaning and are used interchangeably, which refers to an antibody or a fragment thereof derived from a population of highly homologous antibody molecules, that is, a population of identical antibody molecules except for natural mutations that may occur spontaneously. A monoclonal antibody has a high specificity for a single epitope on an antigen. Compared to a monoclonal antibody, polyclonal antibodies usually contain at least two or more different antibodies, which usually recognize different epitopes on an antigen. Furthermore, the modifier “monoclonal” simply indicates that the features of an antibody are obtained from a population of highly homologous antibodies and cannot be understood as requiring any particular method to prepare the antibody.
A monoclonal antibody of the invention can be prepared by various technologies, such as hybridoma technology (see, for example, Kohler et al. Nature, 256: 495, 1975), recombinant DNA technology (see, for example, U.S. Pat. No. 4,816,567), or phage antibody library technology (see, for example, Clackson et al. Nature 352: 624-628, 1991, or Marks et al. J. Mol. Biol. 222: 581-597, 1991).
For example, a monoclonal antibody can be prepared as follows. Firstly, a mouse or other suitable host animal is immunized with an immunogen (added with an adjuvant if necessary). After immunized, the animal will produce lymphocytes that secrete antibodies that specifically bind to the immunogen in vivo. In addition, lymphocytes can also be obtained by immunization in vitro. The lymphocytes of interest are collected and fused with myeloma cells using a suitable fusion reagent, such as PEG, to obtain hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996). The hybridoma cells prepared as above can be inoculated into a suitable culture medium for growth, which then can be used for detecting the production of monoclonal antibodies against a specific antigen. Methods for determining the binding specificity of monoclonal antibodies produced by hybridoma cells include, for example, immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA). For example, the affinity of monoclonal antibodies can be determined by Scatchard analysis described in Munson et al., Anal. Biochem. 107: 220 (1980). After the specificity, affinity and reactivity of the antibodies produced by hybridoma are determined, the cell lines of interest can be subcloned by the standard limited dilution method described in Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103, Academic Press, 1996. The suitable culture medium can be DMEM or RPMI-1640. In addition, hybridoma cells can also be grown in vivo in animals in the form of ascites tumors. Monoclonal antibodies secreted by subcloned cells can be isolated from the cell culture medium, ascites, or serum by using traditional immunoglobulin purification methods, such as protein A agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography, or the like.
Monoclonal antibodies can also be obtained by genetic engineering recombination technologies. DNA molecules encoding heavy chain and light chain genes of a monoclonal antibody can be isolated from hybridoma cells by PCR amplification using nucleic acid primers specifically binding to the heavy chain and light chain genes of the monoclonal antibody. The obtained DNA molecules are inserted into an expression vector with which host cells (such as E. coli cells, COS cells, CHO cells, or other myeloma cells that do not produce immunoglobulins) are then transfected, and are cultured under suitable conditions to obtain the recombinantly expressed antibody of interest.
Antibodies can be purified by well-known technologies such as affinity chromatography using protein A or protein G. Subsequently or alternatively, a specific antigen (a target molecule recognized by an antibody) or its antigenic epitope can be immobilized on a column, and an immunologically specific antibody can be purified by immune-affinity chromatography. For purification of immunoglobulins, reference may be made to, for example, D. Wilkinson (The Scientist, published by The Scientist, Inc., Philadelphia Pa., Vol. 14, No. 8 (Apr. 17, 2000), pp. 25-28).
As used herein, “% identity” between two sequences is a function of the number of identical positions shared by the two sequences (i.e., % homology=the number of identical positions/the total number of positions×100), while considering the number of gaps and the length of each gap which needs to be introduced when the two sequences are optimally aligned. Sequence alignment and determination of % identity between two sequences can be accomplished by a mathematical algorithm. For example, the % identity between two amino acid sequences can be determined by the algorithm of E. Meyers and W. Miller (Computer. App. Biosci., 4:11-17 (1988)).
As used herein, a sequence having “% identity” retains important biological activities, such as antibody-binding specificity, of the sequence which it is aligned with or it is derived from. A sequence having a substitution, deletion or addition of one or more amino acids or any combination thereof retains important biological activities, such as antibody-binding specificity, of the sequence which it is aligned with or it is derived from. A nucleotide sequence having “% identity” or a nucleotide sequence differing by not more than 3, 6, 15, 30 or 45 nucleotides can achieve similar functions as the sequence which it is aligned with or it is derived from, e.g. all the expressed proteins can specifically bind to the same antigen or molecule.
As used herein, the term “chimeric antibody” means such an antibody where one part of its light chain or/and heavy chain is derived from an antibody (which can be derived from a specific species or belong to a specific antibody class or subclass), and the other part of the light chain or/and heavy chain is derived from another antibody (which may be derived from the same or different species or belong to the same or different antibody class or subclass), which in any case retains binding activity to a target antigen (U.S. Pat. No. 4,816,567 to Cabilly et al.; Morrison et al., Proc. Natl. Acad. Sci. USA, 81: 6851 6855 (1984)).
As used herein, the term “humanized antibody” refers to a genetically engineered non-human antibody whose amino acid sequence is modified to increase the homology with the sequence of a human antibody. Generally, all or part of the CDR regions of a humanized antibody are derived from a non-human antibody (donor antibody), and all or part of the non-CDR regions (e.g., FR in the variable region and/or the constant region) are derived from a human immunoglobulin (acceptor antibody). Humanized antibody usually retain the expected properties of the donor antibody, including but not limited to antigen specificity, affinity, reactivity, ability to improve immune cell activity, ability to enhance immune response, etc. Donor antibody may be mouse, rat, rabbit, or non-human primate (e.g., cynomolgus monkey) antibody having desired properties (e.g., antigen specificity, affinity, reactivity, ability to enhance immune cell activity and/or ability to enhance immune response).
Humanized antibodies are particularly advantageous, because they not only retain the expected properties of non-human donor antibodies (e.g., murine antibodies), but also effectively reduce the immunogenicity of non-human donor antibodies (e.g., murine antibodies) in a human subject. However, due to the matching problem between the CDRs of the donor antibody and the FRs of the acceptor antibody, the expected properties of humanized antibodies (e.g., antigen specificity, affinity, reactivity, ability to improve immune cell activity and/or ability to enhance immune response) are generally lower than those of non-human donor antibodies (e.g. mouse antibodies).
Thus, although researchers in the art have developed a deep research and made some progress on the humanization of antibodies (see, for example, Jones et al., Nature, 321: 522 525 (1986); Reichmann et al., Nature, 332: 323 329 (1988); Presta, Curr. Op. Struct. Biol., 2: 59 3 596 (1992); And Clark, Immunol. Today 21: 397 402 (2000)), nevertheless, the detailed guidance is not provided in the prior art on how to humanize a given donor antibody sufficiently so that the produced humanized antibody not only has a humanization degree as high as possible, but also retain the expected properties of the donor antibody as much as possible. To obtain a humanized antibody not only having a high humanization degree (for example, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% humanization), but also retaining the expected properties of a particular donor antibody, those skilled in the art have to exploit, investigate and modify the particular donor antibody by expensing a lot of creative works.
In the invention, in order to make a humanized antibody retain the properties of a donor antibody as much as possible (including, for example, antigen specificity, affinity, reactivity, ability to improve immune cell activity and/or ability to enhance immune response), the framework region (FR) of the humanized antibody of the invention comprises both amino acid residues of a human acceptor antibody and corresponding amino acid residues of a non-human donor antibody.
In the present application, the expected properties of the antibody of the invention include: (1) specific recognizing/binding PD-1 (in particular human PD-1); (2) inhibiting and/or blocking the binding of PD-1 to PDL-1; (3) inhibiting and/or blocking the intracellular signaling mediated by the binding of PD-1 to PDL-1; (4) improving the activity of immune cells; (5) enhancing immune response; (6) reducing or eliminating antibody-dependent cell-mediated phagocytosis (ADCP) and/or antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC). The humanized antibody of the invention retains one or more of the above-mentioned expected properties of the parent antibody (a murine antibody or mouse-human chimeric antibody).
The chimeric antibody or humanized antibody of the invention can be prepared according to the sequence of the murine monoclonal antibody prepared as above. The DNA encoding heavy and light chains can be obtained from a target mouse hybridoma and engineered using standard molecular biology technologies to include a non-mouse (e.g., human) immunoglobulin sequence.
For preparing a chimeric antibody, a mouse immunoglobulin variable region can be linked to a human immunoglobulin constant region using methods known in the art (see, for example, U.S. Pat. No. 4,816,567 to Cabilly et al.). For example, the DNA encoding a VH is operably linked to another DNA molecule encoding a heavy chain constant region to obtain a full-length heavy chain gene, or the DNA encoding a VL is operably linked to another DNA molecule encoding a light chain constant region CL to obtain a full-length light chain gene (as well as a Fab light chain gene). Sequences of human heavy chain constant region genes and light chain constant region genes are known in the art (see, for example, Kabat, E. A. et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242), and DNA fragments containing these regions can be obtained by standard PCR amplification. The heavy chain constant region may be an IgG1 (e.g., uniprot ID P01857), IgG2 (e.g., uniprot ID P01859), IgG3 (e.g., uniprot ID P01860), IgG4 (e.g., uniprot ID P01861), IgA, IgE, IgM, or IgD constant region, generally preferably an IgG1 or IgG4 constant region. Light chain constant regions can be κ or λ constant regions, generally preferably a K constant region.
For preparing a humanized antibody, a mouse CDR region can be inserted into a human framework sequence using methods known in the art (see U.S. Pat. No. 5,225,539 to Winter, U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and 6,180,370 to Queen et al.; and Lo, Benny, K. C., editor, in Antibody Engineering: Methods and Protocols, volume 248, Humana Press, New Jersey, 2004). Alternatively, transgenic animals can also be utilized, which am incapable of producing endogenous immunoglobulins after immunization but capable of producing an intact human antibody library (see, for example, Jakobovits et al., 1993, Proc. Natl. Acad. Sci. USA 90: 2551; Jakobovits et al., 1993, Nature 362: 255-258; Bruggermann et al., 1993, Year in Immunology 7:33; and Duchosal et al., 1992, Nature 355: 258; Lonberg et al. (1994) Nature 368 (6474): 856-859; WO02/43478). Other methods of antibody humanization include phage display technology (Hoogenboom et al., 1991, J. Mol. Biol. 227: 381; Marks et al., J. Mol. Biol. 1991, 222: 581-597; Vaughan et al., 1996, Nature Biotech 14: 309).
As used herein, the term “humanization degree” is an indicator for evaluating the number of non-human amino acid residues in a humanized antibody. The humanization degree of humanized antibodies can be predicted, for example, by DomainGapAlign from the IMGT website for the homology of variable region sequences with human V domains.
As used herein, the term “specifically binding” refers to non-random binding reaction between two molecules, such as reaction between an antibody and an antigen against which it is. The strength or affinity of the specific binding interaction can be indicated by the equilibrium dissociation constant (KD) of the interaction. In the invention, the term “KD” refers to the dissociation equilibrium constant of specific antibody-antigen interaction, which is used to describe the binding affinity between an antibody and an antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding, and the higher the affinity between the antibody and the antigen. In certain embodiments, an antibody specifically binding to an antigen (or an antibody specific for an antigen) means that the antibody binds to the antigen with an affinity (KD) of less than about 10−9 M, for example less than about 10−9 M, 10−10, 10−11 M or 10−12 M or less. In some embodiments, when KD≤10×10−8 M (preferably KD≤5×10−9 M), the antibody or antigen-binding fragment thereof of the invention is considered as specifically binding to PD-1.
The specific binding properties between two molecules can be measured using methods well known in the art. One of the methods involves measuring the rate of formation and dissociation of antigen-binding site/antigen complexes. Both “binding rate constant” (ka or kon) and “dissociation rate constant” (kdis or koff) can be calculated from the concentration and the actual rates of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis/kon is equal to the dissociation constant KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). KD, kon and kdis values can be measured by any effective method. In certain embodiments, the dissociation constant can be measured by bioluminescence interferometry (e.g., ForteBio Octet assay). In addition, the dissociation constant can also be measured by surface plasmon resonance technology (such as Biacore) or Kinexa.
As used herein, the term “vector” refers to a nucleic acid vehicle into which a polynucleotide can be inserted. When enabling the expression of a protein encoded by the inserted polynucleotide, the vector is called as expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, so that the carried genetic material elements can be expressed in host cells. Vectors am well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); phages, such as λ phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but not limited to, retroviruses (including lentivirus), adenoviruses, adeno-associated viruses, herpesviruses (e.g., herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papovaviruses (e.g., SV40). A vector may contain elements controlling expression, including but not limited to a promoter sequence, a transcription initiation sequence, an enhancer sequence, a selection element, and a reporter gene. In addition, the vector may also contain an origin of replication.
As used herein, the term “host cell” refers to a cell into which a vector can be introduced, including, but not limited to, prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells or human cells.
As used herein, the term “identity” is used to refer to the sequence matching degree between two polypeptides or two nucleic acids. When a position in two sequences for comparison is occupied by the same base or amino acid monomer subunit (for example, a position in each of two DNA molecules is occupied by adenine, or a position in each of two polypeptides is occupied by lysine), then the molecules are identical at that position. “Percentage identity” between two sequences is a function of the number of matching positions shared by the two sequences divided by the number of positions to be compared and ×100. For example, if six positions of the ten positions in two sequences match, then the two sequences are 60% identical. For example, the DNA sequences CTGACT and CAGGTT have 50% identity (3 positions of the total 6 positions match). Typically, the two sequences am compared and aligned for maximum identity. Such an alignment can be realized by using, for example, the method of Needleman et al. (1970) J. Mol. Biol. 48: 443-453, which can be conveniently performed by a computer program such as Align program (DNAstar, Inc). Additionally, the percentage identity between two amino acid sequences can be determined by the algorithm of Needleman and Wunsch (J Mol Biol. 48: 444-453 (1970)) in the GAP program incorporated in the GCG software package (available in www.gcg.com), using either a Blossum Matrix 62 or PAM250, and a gap weight of 16, 14, 12, 10, 8, 6 or 4 and a length weight of 1, 2, 3, 4, 5 or 6.
As used herein, the term “conservative substitution” means an amino acid substitution that does not adversely affect or alter the expected properties of a protein/polypeptide comprising an amino acid sequence. For example, a conservative substitution may be introduced by standard technologies known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution includes a substitution of an amino acid residue with an amino acid residue having a similar side chain, e.g., with a residue that is physically or functionally similar to the corresponding amino acid residue (e.g., having similar size, shape, charge, chemical property, including the ability to form covalent or hydrogen bonds, etc.). Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids having basic side chains (e.g., lysine, arginine and histidine), acidic side chains (e.g. aspartic acid, glutamic acid), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g. alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g. tyrosine, phenylalanine, tryptophan, histidine). Therefore, it is preferred to replace a corresponding amino acid residue with another amino acid residue from the same side chain family. Methods for identifying conservative substitution of amino acids am well known in the art (see, for example, Brummell et al., Biochem. 32: 1180-1187 (1993); Kobayashi et al., Protein Eng. 12 (10): 879-884 (1999); and Burks et al., Proc. Natl Acad. Set USA 94: 412-417 (1997), which are incorporated herein by reference).
The compilation of twenty conventional amino acids involved herein follows the conventional usage. See, for example, Immunology-A Synthesis (2nd Edition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In the invention, the terms “polypeptide” and “protein” have the same meaning and are used interchangeably. Also, in the invention, amino acids are generally expressed by single-letter and three-letter abbreviations well known in the art. For example, alanine can be expressed as A or Ala.
As used herein, the term “pharmaceutically acceptable carrier and/or excipient” means a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and active ingredient, which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995) and include, but not limited to, pH modulators, surfactants, adjuvants, ionic strength enhancers, diluents, osmotic pressure maintaining reagents, delayed absorption reagents, preservatives. For example, the pH modulators include, but not limited to, phosphate buffers. The surfactants include but not limited to cationic, anionic, or nonionic surfactants, such as Tween-80. The ionic strength enhancers include, but not limited to, sodium chloride. The preservatives include, but not limited to, various antibacterial and antifungal agents, such as p-hydroxybenzoate, trichloro-tert-butyl alcohol, phenol, sorbic acid, etc. The osmotic pressure maintaining reagents include, but not limited to, sugars, NaCl and the like. The delayed absorption reagents include, but not limited to, monostearates and gelatin. Diluents include, but not limited to, water, aqueous buffers (e.g., buffer saline), alcohols and polyols (e.g., glycerol), etc. The preservatives include, but not limited to, various antibacterial and antifungal agents, such as thiomersalate, 2-phenoxyethanol, p-hydroxybenzoate, trichloro-tert-butyl alcohol, phenol, sorbic acid, etc. The stabilizers have a meaning commonly understood by those skilled in the art, which can stabilize the desired activity of the active ingredient in drug, including but limited to, sodium glutamate, gelatin, SPGA, sugars (such as sorbitol, mannitol, starch, sucrose, lactose, dextran, or glucose), amino acids (such as glutamic acid, glycine), proteins (such as dried whey, albumin, or casein) or degradation products thereof (such as lactalbumin hydrolysate).
As used herein, the term “prevention” refers to a process for preventing or delaying the onset of a disease or condition or symptom (e.g., a tumor, an infection, or an autoimmune disease) in vivo in a subject. As used herein, the term “treatment” refers to a process for achieving a beneficial or desired clinical result. For purposes of the invention, beneficial or desired clinical result includes, but not limited to, alleviation of a symptom, diminishment of extent of disease, stabilizing (i.e., not worsening) state of a disease, delay or slowing of disease progression, amelioration or palliation of disease state, and remission (whether partial or all) of symptoms, no matter detectable or undetectable. In addition, “treatment” can also mean prolonging survival as compared to the expected survival (without treatment).
As used herein, the term “subject” refers to a mammal, such as a primate mammal, such as a human. In some embodiments, the subject (e.g., human) has a tumor, an infection, or an autoimmune disease, or is at a risk of suffering from such disease.
As used herein, the term “effective amount” refers to an amount sufficient to achieve or at least partially achieve a desired effect. For example, an effective amount for preventing a disease (e.g., a tumor, an infection, or an autoimmune disease) is an amount sufficient to prevent, stop, or delay the onset of the disease (e.g., a tumor, an infection, or an autoimmune disease); a therapeutic effective amount refers to an amount sufficient to cure or at least partially prevent a disease and its complications of a patient who have already suffered from the disease. To determine such effective amount is within the scope of capability of those skilled in the art. For example, the amount effective for a therapeutic use will depend on the severity of the disease to be treated, the general state of the patient's own immune system, the general condition of the patient such as age, weight and gender, route of administration, and other treatments administered concurrently, etc.
As used herein, the term “immune cell” includes cells that have hematopoietic origins and play a role in an immune response, for example, lymphocytes, such as B cells and T cells; natural killer cells; myeloid cells, such as monocytes, macrophages, eosinophils, mast cells, basophils, and granulocytes.
As used herein, the term “immune response” means the action of immune cells (such as lymphocytes, antigen presenting cells, phagocytes or granulocytes) and soluble macromolecules (including antibodies, cytokines and complements) produced by immune cells or liver, which leads to selective damage, destruction or removal from human body of invasive pathogens, pathogen-infected cells or tissues, cancer cells, or normal human cells or tissues in the case of an autoimmune or pathological inflammation. In the invention, the term “antigen-specific T cell response” refers to an immune response produced by T cells when the T cells are stimulated by an antigen specific to the T cells. Non-limiting examples of responses produced by T cells in response to antigen-specific stimulation include proliferation of T cells and production of cytokines (e.g., IL-2).
As used herein, the term “effector function” refers to those biological activities which contribute to the Fc region of an antibody (Fc region of a natural sequence or Fc region of an amino acid sequence variant) and vary with antibody isotypes. Examples of effector functions of an antibody include, but not limited to: Fc receptor binding affinity, antibody-dependent cell-mediated cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cell phagocytosis (ADCP), down-regulation of cell surface receptors (such as B cell receptors), B cell activation, cytokine secretion, half-lives/clearance rates of antibodies and antigen-antibody complexes, etc. Methods for altering effector functions of an antibody are known in the art, for example by introducing mutations in the Fc region.
As used herein, the term “antibody-dependent cell-mediated cytotoxicity (ADCC)” means a type of cytotoxicity, wherein Ig binds to the Fc receptor (FcR) present on cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, or macrophages), enabling these cytotoxic effector cells to specifically bind target cells to which antigens are attached, and then killing the target cells by secreting cytotoxins. Methods for detecting the ADCC activity of an antibody am known in the art, for example, evaluated by determining the binding activity between the antibody to be tested and the Fc receptor (e.g., CD16a).
As used herein, the term “complement-dependent cytotoxicity (CDC)” refers to a type of cytotoxicity that activates a complement cascade by binding the complement component C1q to the antibody Fc. Methods for detecting the CDC activity of an antibody are known in the art, for example, evaluated by determining the binding activity between the antibody to be tested and the Fc receptor (e.g., C1q).
The terms “cancer” and “tumor” am used interchangeably and refer to a large class of diseases characterized by uncontrolled growth of abnormal cells in vivo. Unregulated cell division may lead to the formation of malignant tumors or cells that invade neighboring tissues, which may metastasize to distant sites of the body through the lymphatic system or bloodstream. Cancer includes benign and malignant cancers and dormant tumors or micrometastases. Cancer also includes hematological malignancies.
Exemplary tumors include, but not limited to, solid tumors, hematological tumors (e.g., leukemia, lymphoma, myeloma, such as multiple myeloma), and metastatic, refractory, or recurrent lesions of cancer, for example, including but not limited to, esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (e.g., non-small cell lung cancer), liver cancer, stomach cancer, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thoracic adenocarcinoma, cholangiocarcinoma, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (e.g., multiple myeloma), sarcoma, glioblastoma, neuroglioblastoma, and leukemia.
The term “hematological malignancy” includes lymphoma, leukemia, myeloma or lymphoid malignant tumor, and splenic cancer and lymph node tumor. Exemplary lymphoma includes B-cell lymphoma and T-cell lymphoma. B-cell lymphoma includes, for example, Hodgkin's lymphoma. T-cell lymphoma includes, for example, cutaneous T-cell lymphoma. Hematological malignancy also includes leukemia, such as secondary leukemia or acute lymphoblastic leukemia. Hematological malignancy also includes myeloma (e.g., multiple myeloma) and other hematological and/or B-cell or T-cell related cancers.
The term “pharmaceutically acceptable” means, when a molecule entity, a fragment of a molecule or a composition is properly administered to an animal or human, they will not produce unfavorable, allergic, or other adverse reactions. Specific examples of materials that may be used as pharmaceutically acceptable carrier or component thereof include sugars (e.g., lactose), starch, cellulose and derivatives thereof, vegetable oils, gelatin, polyols (e.g., propylene glycol), alginic acid, and the like.
Compared with the prior art, the technical solutions of the invention have the following beneficial effects.
(1) The antibody of the invention not only specifically recognizes/binds PD-1 and blocks the binding of PD-1 to PDL-1 or PD-L2, but also enhances the activity of immune cells and stimulates immune response in vitro/in vivo. Therefore, the antibody of the invention has a potential to prevent and/or treat a tumor, an infection, or an autoimmune disease.
(2) The antibody (especially humanized antibody) of the invention not only retains the functions and properties of the parental murine antibody, thereby having a potential to prevent and treat a tumor, an infection or an autoimmune disease; but also has an extremely high humanization degree, so it can be safely administered to a human subject without triggering an immunogenic reaction. Therefore, the antibody (especially humanized antibody) of the invention is of great clinical value.
The sequence information related to the invention is described in the following table, and the specific sequences are shown in the sequence listing.
Embodiments of the invention will be described in detail below in combination with the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples am only intended to illustrate the invention and are not intended to limit the scope of the invention. The various objects and advantageous aspects of the invention will become implementable to those skilled in the art from the following detailed description of the drawings and preferred embodiments. The examples without specific technologies or conditions illustrated therein can be referred to the technologies or conditions described in the literatures in the art, for example, see Sambrook et al., Molecular Cloning: A Laboratory Manual, Third Edition (translated by Huang Peitang et al.), or can be carried out according to the product instructions. The reagents or instruments employed without denoting manufacturers am conventional products that can be purchased from the market.
The following three materials were used as antigens for immunization: (i) a plasmid DNA containing a nucleic acid sequence coding for an intact human PD-1. The nucleotide sequence encoding the intact human PD-1 of the amino acid sequence of NP_005009.2 was obtained by codon optimization and synthesis (Shanghai Sangon Biotech), which was then cloned into a pcDNA3.1 vector, the plasmid was extracted by an endotoxin-free plasmid extraction kit (purchased from OMEGA) and dissolved in a normal saline injection. (ii) A recombinant fusion protein of the extracellular domain of human PD-1. The human PD-1-ECD-mFc protein were obtained by fusing the extracellular domain (ECD) of PD-1 with a mouse Fc (mFc) amino acids (AB097847.1), codon optimizing and synthesizing the nucleotide sequence by Nanjing Genscript, molecular cloning and lentivirus infecting the HEK293F cells, the supernatant was then purified by a ProA column, the purity of the recombinant protein was verified by SDS-PAGE and SEC, and the activity of the recombinant protein human PD-1-mFc was verified by ELISA. (iii) CHO-S cells expressing the full-length human PD-1 on membrane. The CHO-hPD-1 cell line was obtained by molecular cloning, infecting CHO-S cells with lentivirus and screening under pressure followed by FACS assay.
Preparation of the control antibody for the experiments: the amino acid sequences of pembrolizumab (IMGT ID: 472), sintilimab (IMGT ID: 859) and tirelizumab (IMGT ID: 757) were from the IMGT website, each of which was codon optimized and synthesized into an expression vector pcCHO1.0 by Nanjing Genscrip, and transiently transfected into CHO-S cells by PEI, and the obtained cells expressed the antibodies, the supernatant were then purified, and denominated as the control antibody 1, the control antibody 2 and the control antibody 3, respectively.
The plasmid DNA, the recombinant fusion protein of human PD-1 extracellular domain and the CHO-hPD-1 cells were used as antigens to immunize various strains of mice such as B6C57 mice, Balb/c mice, and the like (all purchased from Beijing Vital River) aged 7-8 weeks. During immunization, serum titer of the anti-PD-1 antibody was detected by ELISA every two weeks. After the single-cell suspension of mice spleen and lymph node was mixed, SP2/0 myeloma cells were then added in a quantity ratio of 1:1 and mixed evenly. The cell mixed solution was washed and resuspended with electrofusion buffer (Zhuhai Qiwen Biotechnology) wherein the cell density in the suspension is 1×106 cells/mL. After electrofusion by a BTX 2000 electrofusion device, the cell suspension was immediately transferred from the fusion chamber to a sterile centrifuge tube containing complete culture medium and incubated in an incubator at 37° C. for at least 1 hour. The cell suspension was then mixed and plated into 96-well plates at a density of 2×104 cells per well, for a total of about 300 plates. The fused cells were cultured at 37° C. and 5% CO2. After hybridoma clones were cultured for 5 days, the hybridoma clones in all the 96-well plates were thoroughly exchanged for complete culture medium, and when growing for 7 days, the hybridoma clones were screened.
Hybridoma screening was performed by ELISA and competitive FACS. The specific experimental steps of ELISA were as follows: the extracellular domain of human PD-1 and that of monkey PD-1 (NP_001107830) were fused to corresponding tags, respectively, antigens such as unman PD-1-His, human PD-1-hFc and monkey PD-1-hFc were constructed by molecular cloning and homologous recombination, expressed and purified, which were then diluted to 1 μg/mL with CBS solution, and coated on a 96-well ELISA plate (purchased from Guangzhou Jiete Bio) at 4° C. overnight. After washing, 100 μL of phosphate buffer (PBS) containing 2% BSA was used for blocking at 37° C. for 1 hour, followed by adding 20 μL of hybridoma clone supernatant and incubating at 37° C. for 2 hours. After washing, a goat anti-mouse secondary antibody (FC) labeled with horseradish peroxidase diluted in phosphate buffer (PBS) containing 2% BSA in a ratio of 1:15000 was added, followed by incubating at 37° C. for 1 hour. After washing, the TMB chromogen solution was used for developing, and the absorbance value was read using a microplate reader at 450 nm.
The specific experimental steps of competitive FACS were as follows: competitive FACS screening were performed on human and monkey binding positive hybridoma clones after ELISA screening. CHO-hPD-1 cells expressing the full-length human PD-1 were centrifugated and washed with phosphate buffer (PBS). The cell density was adjusted to 1×107 cells/mL with phosphate buffer (PBS), the resultant solution was then added into a V-bottomed 96-well plate at 50 μL/well, and the supernatant was removed by centrifugation. 50 μL of the supernatant of positive hybridoma clones was taken to be added to a V-bottomed 96-well plate together with 50 μL of FITC-labeled human PD-L1-mFc recombinant fusion protein which was diluted to 300 nM/mL with phosphate buffer (PBS) containing 0.5% BSA, followed by thoroughly mixing, and incubating at 4° C. in dark for 40 min. After centrifugation and washing with phosphate buffer (PBS) twice, the results were analyzed by flow cytometry (Beckman) and FlowJo software.
Through the screening described in 1.3, the hybridoma clones 42G1, 29C6, 91D5, 14A4 and 35E6 were selected for subcloning, the anti-PD-1 antibodies produced by which were denominated as 42G1, 29C6, 91D5, 14A4 and 35E6, respectively. Specifically, after serial dilution, hybridoma cells in wells were subjected to cell counting, individual wells having 60-70 hybridoma cells were selected for resuspending and plating in a 96-well plate, and the plated cells were cultured at 37° C., 5% CO2. After culturing for 7 days, ELISA and FACS assays were performed using the same method as in Example 1.3, 1-2 positive single clones were selected from each plate for expansion culture in serum-free medium, then murine antibodies were collected for the next functional verification.
In order to verify the binding affinity of murine anti-PD-1 antibody to human PD-1-hFc and monkey PD-1-hFc, ELISA was used. The specific experimental steps are as follows: human PD-1-hFc and monkey PD-1-hFc were diluted to 1 μg/mL with CBS solution, and then coated on a 96-well ELISA plate (purchased from Guangzhou Jiete Bio) at 4° C. overnight. After washing, 100 μL of phosphate buffer (PBS) containing 2% BSA was used for blocking at 37° C. for 1 hour, and then 100 μL of murine anti-PD-1 antibodies 42G1, 29C6 and 91D5 were added (starting at 10 μg/mL, 4-fold serial dilution, 2 replicates), then allowed for incubating at 37° C. for 2 hours. After washing, a goat anti-mouse secondary antibody (FC) labeled with horseradish peroxidase diluted in phosphate buffer (PBS) containing 2% BSA in a ratio of 1:15000 was added, followed by incubating at 37° C. for 1 hour. After washing, the TMB chromogen solution was used for developing, and the absorbance value was read using a microplate reader at 450 nm. The data were imported into Graphprism to calculate the EC50 value.
The results were shown in Table 1, EC50 of each of murine antibodies 29C6, 42G1 and 91D5 for human PD-1-hFc and monkey PD-1-hFc were 30-70 pM, showing a good binding affinity of murine antibodies to human or monkey PD-1.
Competitive ELISA was used to verify blocking of PD-1/PD-L1 by murine anti-PD-1 antibody. The specific experimental steps were as follows: human PD-1-hFc antigens were diluted to 1 μg/mL with CBS solution, then coated on a 96-well ELISA plate (purchased from Guangzhou Jiete Bio) at 4° C. overnight. After washing, 100 μL of phosphate buffer (PBS) containing 2% BSA was used for blocking at 37° C. for 1 hour, and then 100 μL of murine anti-PD-1 antibodies 42G1, 29C6 and 91D5 containing 0.8 μg/mL biotinylated PD-L1-mFc were added (starting at 10 μg/mL, 2-fold serial dilution, 2 replicates), and allowed for incubating at room temperature for 2 hours. After washing, streptavidin labeled with horseradish peroxidase diluted in phosphate buffer (PBS) containing 2% BSA in a ratio of 1:15000 was added, followed by incubating at room temperature for 1 hour. After washing, the TMB chromogen solution was used for developing, and the absorbance value was read using a microplate reader at 450 nm, and the data were imported into Graphprism to calculate the IC50 value.
The results were shown in Table 1, IC50 of each of murine antibodies 29C6, 42G1 and 91D5 for blocking the binding of PD-1-hFc to PD-L1-mFc were at the level of nM, showing a good activity of blocking PD-1/PD-L1 binding.
In order to verify the binding of anti-human PD-1 murine antibody to PD-1 on the cell membrane surface, FACS technology was used to detect the affinity between murine antibodies and CHO-hPD-1 cells.
The specific experimental steps were as follows: CHO-hPD-1 cells were centrifuged at 500 g for 3 minutes, and then resuspended with PBS (containing 1% BSA) to 6×106 cells/mL, and 50 μL was taken to be added to corresponding 96-well plates; murine antibodies 42G1, 29C6 and the control antibody 1 were diluted with PBS (containing 1% BSA) (starting at 1 μM, 3-fold serial dilution, 11 concentration points), and then 50 μL was added to corresponding 96-well plates, and there was no antibody in the blank control well; the cells and antibodies were gently mixed, and left at 4° C. for 1 hour, washed with PBS twice, and resuspended with 50 μL of PBS (containing 1% BSA); wherein, 2.5 μL of fluorescent secondary antibody Alexa Fluor 488 anti-human IgG Fc was added to the sample of the control antibody 1, 2.5 μL of FITC anti-mouse IgG was added to the samples of the 42G1 and 29C6 murine antibodies, respectively, and left at 4° C. for 1 hour, washed with PBS for 3 times, and then detected by flow cytometry (manufactured by Beckman, Model: CytExpert), and the data were imported into Graphprism to calculate EC50 values.
The experimental results were shown in
2.4 Determination of In Vivo Drug Efficacy of Anti-PD-1 Murine Antibody in hPD-1 Transgenic Mice
In order to verify the anti-tumor effect of anti-human PD-1 murine antibody in vivo, a subcutaneous tumorigenesis model of hPD-1 (Beijing Vitalstar) transgenic mice was used. The assay was as follows: MC38 tumor cells (purchased from ATCC) were cultured using RPMI1640 culture medium containing 10% fetal bovine serum at 37° C., 5% CO2. Mouse PD-L1 can bind to human PD-1 normally, and anti-human PD-1 antibody can inhibit the growth of tumor cells after blocking the binding of mouse PD-L1 to human PD-1. MC38 cells in exponential growth phase were collected, and resuspended to a suitable concentration with PBS and inoculated subcutaneously to female hPD-1 mice to establish a colon cancer model. When reaching an average volume of about 100 mm3, the mice were randomly divided into the following groups according to tumor size: the group of Human IgG (negative control), the group of murine antibody 42G1, the group of murine antibody 29C6 and the group of the control antibody 2, respectively, wherein administered dosage is 1 mg/kg and injected intraperitoneally twice a week for 3 weeks. After administration, the tumor volume and body weight of mice were observed and measured regularly. The experimental index was to investigate the effect of drugs on tumor growth, and the specific index was T/C % or tumor growth inhibition % (TGI %). The tumor diameter was measured by a vernier caliper twice a week, and the formula of calculating the tumor volume (V) was: V=½×a×b2, wherein, a and b denote the length and the width, respectively. T/C %=T/C×100, wherein C and T represent the tumor volume or the tumor weight of the solvent group and the treatment group, respectively. Tumor growth inhibition (TGI %)=(C−T)/C×100, wherein C and T represent the tumor volume or the tumor weight of the solvent group and the treatment group, respectively.
The results were shown in
The murine antibody sequence was acquired according to the method of Gary C. Howard et al. (Basic Methods in Antibody Production and Character, CRC press, 2000), the candidate hybridoma RNAs were extracted and amplified by RT-PCR to obtain the light and heavy variable regions of the murine antibodies, which were then constructed to T vectors for sequencing. Hybridoma RNAs were extracted according to the instructions of the TRIzol RNA Isolation Reagents kit, which was then dissolved in 5 μL of DEPC treated water, and the RNA concentration was detected. According to the instructions of the RevertAid First Strand cDNA Synthesis Kit, the total cDNA was obtained by reverse transcription using an oligo(dT)18 primer. Referring to the method of Gary C. Howard et al., as well as the sequence analysis of all murine antibodies with IMGT, the upstream primers of multiple pairs for variable regions were designed by selecting the regions of high homology, and the downstream primers were designed by CH1 homologous sequences, the light and heavy chain variable regions of the antibodies were obtained by PCR amplification by using primer pool, and the PCR product was constructed to pUCm-T vector, after identification and sequencing analysis, the variable regions and CDR sequences of the antibodies were shown in Table 2.
The chimeric antibodies were constructed by fusion the amino acid sequences of the light chain variable regions of 14A4, 29C6, 35E6 and 91D5 to the amino acid sequence of the light chain kappa constant region (SEQ ID NO: 84), respectively, and fusion the amino acid sequences of the heavy chain variable regions of 14A4, 29C6, 35E6 and 91D5 to the amino acid sequence of the heavy chain constant region of IgG4 (SEQ ID NO: 82), respectively. The cDNA was synthesized after codon optimization and ligated to the plasmid PPT5 (Shanghai sangon Co., Ltd.). PPT5 vectors carrying the heavy and light chains of each chimeric antibody were simultaneously transfected into CHO-S-E cells (prepared by Sichuan Kelun Biotech Biopharmaceutical Co., Ltd.), and the culture supernatant was purified by a protein A column (MabSelect SuRe, GE) to obtain chimeric antibodies, which were denominated as 14A4-CH, 29C6-CH, 35E6-CH, and 91D5-CH, respectively.
The specific experimental steps were as follows: human PD-1-hFc and monkey PD-1-hFc antigens were diluted to 1 μg/mL with CBS solution, then coated on a 96-well ELISA plate (purchased from Guangzhou Jiete Bio) at 4° C. overnight. After washing, 100 μL of phosphate buffer (PBS) containing 2% BSA was used for blocking at 37° C. for 1 hour, followed by adding 100 μL of chimeric antibodies 14A4-CH, 91D5-CH, 35E6-CH, 29C6-CH and the control antibody 1 (starting at 10 μg/mL, 4-fold serial dilution), respectively, and incubating at 37° C. for 2 hours. After washing, a goat anti-human secondary antibody (FC) labeled with horseradish peroxidase diluted in phosphate buffer (PBS) containing 2% BSA in a ratio of 1:15000 was added, followed by incubating at 37° C. for 1 hour. After washing, the TMB chromogen solution was used for developing, and the absorbance value was read using a microplate reader at 450 nm, and the data were imported into Graphprism to calculate EC50 values.
The results were shown in Table 3 below, the binding affinity EC50 of each of chimeric antibodies 14A4-CH, 91D5-CH, 35E6-CH, 29C6-CH, and the control antibody 1 to human PD-1-hFc and monkey PD-1-hFc were in a range from 0.05 to 0.3 nM.
The specific experimental steps were as follows: human PD-1-hFc was diluted to 1 μg/mL with CBS solution, coated on a 96-well ELISA plate (purchased from Guangzhou Jiete Bio) at 4° C. overnight. After washing, 100 μL of phosphate buffer (PBS) containing 2% BSA was used for blocking at 37° C. for 1 hour. 50 μL of chimeric antibodies 14A4-CH, 91D5-CH, 35E6-CH, 29C6-CH and the positive control antibody 1 was added (starting at 20 μg/mL, 2-fold dilution), respectively, simultaneously 50 μL (1.6 μg/mL) of hPD-L1-mFc (hPD-L1 amino acid sequence NP_054862.1) or hPD-L2-mFc (hPD-L2 amino acid sequence AAI13679) was added per well, followed by incubating at room temperature for 2 hours. After washing, a goat anti-mouse secondary antibody labeled with HRP diluted in phosphate buffer (PBS) containing 2% BSA in a ratio of 1:15000 was added, followed by incubating at room temperature for 1 hour. After washing, the TMB chromogen solution was used for developing, and the absorbance value was read using a microplate reader at 450 nm, and the data were imported into Graphprism to calculate ICs values.
The results were shown in Table 3 below. It can be seen from IC50 that, the activity of each of the chimeric antibodies 14A4-CH, 91D5-CH, 35E6-CH, 29C6-CH for blocking PD-1/PD-L1 binding was comparable to that of the control antibody 1, and the activity of each of the chimeric antibodies for blocking PD-1/PD-L2 binding was comparable to that of the control antibody 1.
A competitive FACS technology was used to detect the activity of anti-PD-1 chimeric antibody for blocking PD-L1/CHO-hPD-1 binding. The specific experimental steps were as follows: CHO-hPD-1 cells were centrifuged at 500 g for 3 minutes, and resuspended with PBS (containing 1% BSA) to 6×106 cells/mL, 50 μL of which was added to corresponding 96-well plates; chimeric antibodies 14A4-CH, 91D5-CH, 35E6-CH, 29C6-CH and the control antibody 1 were diluted with PBS (containing 1% BSA), and diluted to 1 μM with PBS containing 0.4 μg/mL PD-L1-mFc-Bio, then 3-fold diluted to obtain 9 concentration points, and then 50 μL was added to corresponding 96-well plates, and there was no antibody in the blank control well; the cells and the antibodies were gently mixed, left at 4° C. for 1 hour, washed with PBS twice, resuspended with 50 μL of PBS (containing 1% BSA); 2.5 μL of APC streptavidin fluorescent secondary antibody was added to each of the samples, left at 4° C. for 1 hour, followed by washing with PBS for 3 times, and each sample was detected by flow cytometry (manufactured by Beckman, Model: CytExpert). The data were imported into Graphprism to calculate IC50 values.
The experimental results were shown in Table 3 below and
The dynamic affinity of candidate chimeric antibodies 4A4-CH, 91D5-CH, 35E6-CH, 29C6-CH and the control antibody 1 to human PD-1-His were measured by Octet ForteBio® for detecting the dynamic affinity of an antibody to an antigen. The specific experimental steps were as follows: a ProA biosensor was firstly allowed for binding to the antibody to be tested until the response signal value reached 0.5 nm, then binding to human PD-1-His protein (500, 250, 125, 62, 31, 16 and 0 nM) for 80 seconds, and then dissociating for 120 seconds, the measured results were analyzed by 1:1 model and global fitting. As shown in Table 4, the association rates of each of the chimeric antibodies 14A4-CH, 91D5-CH, 35E6-CH and 29C6-CH were at the same level compared to that of the control antibody 1 (as shown by kon values), while the dissociation rates of each of the chimeric antibodies were slower than that of the control antibody 1 by one order of magnitude (as shown by kdis values), the affinity of each of the chimeric antibodies were stronger than that of the control antibody 1 by 4.3, 11.7, 15.4 and 7.3 times, respectively.
The cell bioactivity of blocking PD-1/PD-L1 binding was detected by the reporter gene assay. The CHO-PD-L1-CD3 cell line stably expresses PD-L1 and anti-CD3-scFv, used as target cell; the Jurkat-PD-1-NFAT cell line stably expresses PD-1 and luciferase, used as effector cell, wherein the luciferase gene is regulated by a NFAT element (transcription factor) (IL-2 promoter). The binding of PD-1 to PD-L1 can block the transduction of CD3 downstream signaling, thus inhibiting the expression of luciferase, when PD-1 antibody or PDL-1 antibody is added, this blocking effect is reversed, and luciferase can be expressed, so fluorescence signal can be detected (see Lan Wang, Chuanfei Yu et. al., Development of a robust reporter gene assay to measure the bioactivity of anti-PD-1/anti-PD-L1 therapeutic antibodies. J Pharm Biomed Anal 2017 Oct. 25; 145: 447-453).
Specific experimental steps were as follows: Jurkat-PD-1-NFAT cells (purchased from Promega) and CHO-S-OKT3-PD-L1 cells (purchased from Promega) were centrifuged at 200 g for 5 min, and resuspended with the detection buffer RPMI1640+1% FBS. CHO-S-OKT3-PD-L1 cells were plated at 5×105 cells/40 μL/well. Jurkat-PD-1-NFAT cells were added at 1.25×106/40 μL/well to the wells previously plated with CHO-S-OKT3-PDL1 cells, followed by adding 20 μL of chimeric antibodies 14A4-CH, 91D5-CH, 35E6-CH, 29C6-CH and the control antibody 1 (starting at 100 μM, 2-fold serial dilution), respectively. After incubation for 5 hours, they were taken out and placed at room temperature for 20 min, added with 20 μL of detection reagent Bright-Glo Luciferase (purchased from Promega Company), oscillated at room temperature for 5 min, and measured using a microplate reader (BMG Company, PHEARstar FS). The experimental data were curve-fitted by Graphprism to calculate EC50. The results were shown in Table 5. The cell activities of each of chimeric antibodies 14A4-CH, 91D5-CH, 35E6-CH, 29C6-CH and the control antibody 1 for blocking PD-1/PD-L1 binding were at the level of nM.
The Tm value of anti-PD-1 chimeric antibody was determined by DSF (Differential Scanning Fluorimetry). The specific experimental steps were as follows: each of anti-PD-1 chimeric antibodies 14A4-CH, 91D5-CH, 35E6-CH and 29C6-CH were diluted to 1 mg/mL with PBS, and 12.5 μL of each was taken and added with 2.5 μL of 40×SYPRO Orange dye (Life Technologies Co., Ltd., Cat. No. 4306737) and 5 μL of ddH2O. Then each sample was added to a Q-PCR system (AB Applied Biosystems ABI, 7500) for reaction, the Q-PCR parameter setting: Target (ROX), the program (25° C., 2 min; 1% rate, 95%° C.; 95° C., 2 min).
The results were shown in
4.8 Determination of In Vivo Drug Efficacy of Anti-Human PD-1 Chimeric Antibody in hPD-1 Transgenic Mice
The model was established using the same method as in Example 2.4. When reaching the average volume of about 100 mm3, the mice were randomly divided into the following groups according to tumor size: the group of Human IgG4 (negative control), the group of chimeric antibody 91D5-CH, the group of 35E6-CH antibody, the group of the positive control antibody 1 and the group of the control antibody 3, respectively, each group was injected intraperitoneally with a dosage of 0.5 mg/kg, twice a week for 3 weeks. After administration, the tumor volume and body weight of mice were observed and measured regularly, the specific results were shown in
The murine antibodies 29C6, 35E6 and 91D5 were humanized using antibody CDR-grafted methods. In brief, the humanization modification involves the following steps: aligning the amino acid sequence of a murine monoclonal antibody with that of a human germline antibody to find out the sequence with high homology and better physical and chemical properties for serving as the human germline framework sequence; analyzing and investigating the affinity of HLA-DR to select the human germline framework sequence with low affinity; and grafting six CDRs of the murine antibody to the selected heavy chain and light chain framework sequences, respectively.
Specifically, the heavy chain and light chain CDR regions of murine antibodies 29C6, 35E6 and 91D5 were transplanted into the FR frameworks of corresponding humanization templates. The heavy chain humanization template of 29C6 was the human germline gene sequence IGHV1-18*01 (see IMGT accession number M99641), and the light chain humanization template of 29C6 was the human germline gene sequence IGKV3-11*01 (see IMGT accession number X01668); the heavy chain humanization template of 35E6 was the human germline gene sequence IGHV4-59*01 (see IMGT accession number AB019438), and the light chain humanization template of 35E6 was the human germline gene sequence IGKV3-11*01 (see IMGT accession number X01668); the heavy chain humanization template of 91D5 was the human germline gene sequence IGHV1-18*01 (see IMGT accession number M99641), and the light chain humanization template of 91D5 was the human germline gene sequence IGKV1-39*01 (see IMGT accession number X59315).
Furthermore, the framework amino acid sequences of variable regions and neighboring regions thereof were analyzed by molecular docking using computer simulation techniques, to investigate the stereoscopic combination mode. By calculating electrostatic forces, van der Waals forces, hydrophilicity and hydrophobicity as well as entropy values, the key amino acids in the amino acid sequence of the murine antibody that can interact with PD-1 and maintain the spatial conformation were analyzed, and retained in the transplanted antibody. Namely, a series of back mutations were carried out on the amino acid residues in the FR regions of the humanization template, so that the humanized antibody could retain the antigen-binding ability of the murine antibody as much as possible.
According to the above methods, based on the CDRs of murine antibody 29C6, two humanized antibodies were constructed, denominated as 29C6-HZ1 and 29C6-HZ2, respectively; based on the CDRs of murine antibody 35E6, one humanized antibody was constructed, denominated as 35E6-HZ1; based on the CDRs of murine antibody 91D5, two humanized antibodies were constructed, denominated as 91D5-HZ1 and 91D5-HZ2, respectively. The heavy chain constant region of each humanized antibody was human IgG4 heavy chain constant region (SEQ ID NO: 82), and the light chain constant region thereof was human light chain kappa constant region (SEQ ID NO: 84).
The amino acid sequences of the variable regions and constant regions of the humanized antibodies above were shown in Table 6.
To verify the affinity of the humanized PD-1 antibody to PD-1-His antigen, ELISA was employed. The specific experimental steps were as follows: human PD-1-his was diluted to 1 μg/mL with CBS solution, then coated on a 96-well ELISA plate (purchased from Guangzhou Jiete Bio) at 4° C. overnight. After washing, 100 μL of phosphate buffer (PBS) containing 2% BSA was used for blocking at 37° C. for 1 hour, followed by adding 100 μL of chimeric antibodies 29C6-CH, 35E6-CH, 91D5-CH and humanized antibodies 29C6-HZ1, 29C6-HZ2, 35E6-HZ1, 91D5-HZ1, 91D5-HZ2 (starting at 10 μg/mL, 3-fold gradient dilution, 2 replicates), and incubating at 37° C. for 2 hours. After washing, a goat anti-mouse secondary antibody (FC) labeled with horseradish peroxidase diluted in phosphate buffer (PBS) containing 2% BSA in a ratio of 1:15000 was added, followed by incubating at 37° C. for 1 hour. After washing, the TMB chromogen solution was used for developing, and the absorbance value was read using a microplate reader at 450 nm. The data were imported into Graphprism to calculate EC50 values.
The results were shown in Table 7, in whole, the humanization had little effect on affinity, and retained a good binding affinity to human PD-1-His.
The dynamic affinity of anti-human PD-1 humanized antibodies to PD-1-His was determined using Octet ForteBio®, including chimeric antibodies 29C6-CH and 91D5-CH, humanized antibodies 29C6-HZ2, 35E6-HZ1, 91D5-HZ1, 91D5-HZ2 and the control antibody 1, and specific experimental steps refer to Example 4.5. The experimental results were shown in Table 8, and the KD values of each of chimeric antibodies and humanized antibodies were at the level of nM, and stronger than that of the control antibody 1.
The activity of anti-human PD-1 humanized antibodies for blocking human PD-1/human PD-L1 binding and human PD-1/human PD-L2 binding was detected by competitive ELISA, including chimeric antibodies 29C6-CH, 91D5-CH and 35E6-CH, as well as humanized antibodies 29C6-HZ1, 29C6-HZ2, 35E6-HZ1, 91D5-HZ1 and 91D5-HZ2, and the control antibody 1, and see Example 4.3 for specific experimental steps and the result analysis. The results were shown in Table 9. All the antibodies had a good activity of blocking the binding of human PD-1/human PD-L1 or human PD-1/human PD-L2.
Cytological functions of anti-human PD-1 humanized antibodies were detected using a luciferase reporter system, including humanized antibodies 29C6-HZ2, 35E6-HZ1, 91D5-HZ2 and the control antibody 1, and see Example 4.6 for specific experimental steps. The results were shown in
In this experiment, the level of IL-2 cytokines secreted by SEB-stimulated PBMC cells under the effect of anti-PD-1 humanized antibodies 91D5-HZ2 and 29C6-HZ2, the control antibody 1, the control antibody 3 and the IgG control antibody, respectively, was measured by HTRF assay to evaluate the biological activity of these antibodies.
PBMC cells were prepared from human fresh blood from two donors A and B, respectively, by using a density gradient centrifugation. Fresh PBMC cells were resuspended in a medium containing 1 ng/mL SEB and adjusted to a cell density of 5×105/mL, then evenly distributed into a 96-well plate at 100 μL per well (5×104 cells/well). The antibodies were diluted with 3-fold gradient, and added at 100 μL per well. The antibody concentration gradient started at 1 μM, 3-fold dilution, 2 replicates for each concentration of each antibody. After 72 hours, the cell supernatant was collected by centrifugation, and the secretion of IL-2 in the supernatant was detected by the IL-2 HTRF kit (CisBio).
The results were shown in
Since PD-1 is mainly expressed in activated T cells, at present, IgG4 subtype is used for all the PD-1 antibodies on market, in order to prevent PD-1 positive T cells from being killed. Many studies have shown that human IgG4 antibody subtypes have moderate ADCC, almost without CDC effector function. However, natural IgG4 has been found to be less stable under pressure conditions (such as in acidic buffers, or the like) (Boning Liu, Huaizhu Guo, Jin Xu et. al., Acid-induced aggregation property of nivolumab is dependent on the Fc. Mabs, 2016, 1942-0862).
The IgG1 subtype having better stability, and the hinge region mutation can weak ADCC and CDC functions, meanwhile eliminate the affinity of FcγRI, and avoid the clearance of T cells by monocytic myeloid-derived suppressor cells (MDSC); on this basis, mutation of Fc constant region can enhance the affinity of FcRn and prolong the half-life in vivo, to obtain a better drug efficacy. The inventors used the constant region of mutant IgG1 subtype Fc, including L234A, L235A, D265A, N434A point mutations (EU numbering, SEQ ID NO: 83) to remove FcR binding and prolong the affinity of FcRn. As shown in Table 10, the nucleic acid coding sequences of heavy chain sequence (amino acid SEQ ID NO: 86) and light chain sequence (amino acid SEQ ID NO: 88) of 91D5-HZ2-IgG1-1D, and of heavy chain sequence (amino acid SEQ ID NO: 90) and light chain sequence (amino acid SEQ ID NO: 11) of 35E6-HZ1-IgG1-1D were constructed into pTT5 expression vectors, respectively, and transiently transfected into CHO-S cells by PEI for expression, after purification by ProA, 91D5-HZ2-IgG1-1D and 35E6-HZ1-IgG1-1D antibodies were obtained.
The affinities of 91D5-HZ2-IgG1-1D, 35E6-HZ1-IgG1-1D, the control antibody 1 and the control antibody 3 for unman PD-1-mFc and monkey PD-1-hFc were determined by ELISA. See Example 4.2 and Example 6.1 for specific experimental steps, and the results were shown in Table 11, wherein, the ELISA binding affinity of each of the four PD-1 antibodies to human PD-1-mFc and monkey PD-1-hFc was at the level of nM. For the binding activity to human PD-1-mFc, 93D5-HZ2-IgG1-2D was slightly stronger than the control antibody 1 and the control antibody 3, and 35E6-HZ-IgG1-1D was comparable to the control antibody 1 and the control antibody 3.
The dynamic affinities of IgG1-1D mutant anti-PD-1 humanized antibodies for PD-1-His were detected by Octet ForteBio®, including 91D5-HZ2-IgG1-1D, 35E6-HZ1-IgG1-1D, the control antibody 1 and the control antibody 3, and see Example 4.5 for specific experimental steps. The experimental results were shown in Table 12, wherein, the dynamic affinities of the four antibodies were all at the level of nM, and the affinities of 91D5-HZ2-IgG1-1D and 35E6-HZ1-IgG1-1D were about 4-5 times stronger than those of the control antibody 1 and the control antibody 3, indicating they had a stronger binding activity.
To verify the binding of IgG1-1D mutant anti-PD-1 humanized antibody to PD-1 on the cell membrane surface, FACS technology was used to detect the affinity of murine antibodies to CHO-hPD-1 cells.
The specific experimental steps were as follows: CHO-hPD-1 cells were centrifuged at 500 g for 3 minutes, and resuspended with PBS (containing 1% BSA) to 6×106 cells/mL, and then 50 μL was taken to corresponding 96-well plates; FITC labeled 91D5-HZ2-IgG1-1D, 35E6-HZ1-IgG1-1D, the control antibody 1 and the control antibody 3 were diluted with PBS (containing 1% BSA) (starting at 1 μM, 4-fold gradient dilution, 11 concentration points), and then 50 μL was added to corresponding 96-well plates, 1 μM FITC-labeled IgG negative control antibody was used as negative control, followed by placing at 4° C. for 1 h; washing with PBS for 3 times, and detected by flow cytometry (manufacturer: Beckman, Model: CytExpert), and the data were imported into Graphprism to calculate the EC50 values.
The experimental results were shown in
To verify that IgG1-1D mutant anti-PD-1 humanized antibody blocks the binding of CHO-hPD-1 to PD-L1, FACS technology was used for detection, and see Example 4.4 for the specific experimental steps.
The experimental results were shown in
The nonspecific binding of IgG1-1D mutant anti-PD-1 humanized antibody to HUVEC and HEK293T epithelial cells was detected by FACS. In brief, the density of HUVEC or HEK293T cells was set to be 2×105 cells per well. Test antibodies 91D5-HZ2-IgG1-1D, 35E6-HZ1-IgG1-1D, the control antibody 1 and the control antibody 3 were labeled by FITC and diluted to 500 nM, then incubated with HUVEC or HEK293T cells at 4° C. for 1 hour, respectively. After washing, the cells were detected and analyzed by flow cytometry (manufacturer Beckman, model: CytExpert). The results showed that no nonspecific binding was observed.
The nonspecific binding of IgG1-1D mutant anti-PD-1 humanized antibody to BTLA-his, PD-L1-his and ICOS was detected by ELISA. The specific experimental steps were as follows: human PD-1-his, human BTLA-his, human ICOS and human PD-L1-his (purchased from Novoprotein) were diluted with CBS solution to 1 μg/mL, then coated on a 96-well ELISA plate (purchased from Guangzhou Jiete Bio) at 4° C. overnight. After washing, 100 μL of phosphate buffer (PBS) containing 2% BSA was used for blocking at 37° C. for 1 hour, followed by adding 100 μL of 91D5-HZ2-IgG1-1D, 35E6-HZ1-IgG1-1D and the control antibody 1 (10 μg/mL and 1 μg/mL), setting 2 replicates, incubated at 37° C. for 2 hours. After washing, a goat anti-human IgG secondary antibody labeled with horseradish peroxidase diluted in phosphate buffer (PBS) containing 2% BSA in a ratio of 1:15000 was added, followed by incubating at 37° C. for 1 hour. After washing, the TMB chromogen solution was used for developing, and the absorbance value was read using a microplate reader at 450 nm. The data were imported into Graphprism to calculate EC50 values. The experimental results were shown in
The Tm values of 91D5-HZ2-IgG1-1D, 35E6-HZ1-IgG1-1D, the control antibody 1 and the control antibody 3 were detected by DSF (Differential Scanning Fluorimetry), and see Example 4.7 for specific experimental steps. The results were shown in
Cytological functions of IgG1-1D mutant anti-PD-1 humanized antibodies were determined by a luciferase reporter system, and see Example 4.6 for specific experimental steps. The experimental results were shown in
The binding of antibody Fc to CD64 (i.e., FcγRI) is one important aspect in exerting ADCP function, and the dynamic affinity of IgG1-1D mutant anti-PD-1 humanized antibodies to CD64 (purchased from ACRO) was detected by Octet ForteBio®, including 91D5-HZ2-IgG1-1D, 35E6-HZ1-IgG1-1D, the control antibody 1 and the control antibody 3. The specific experimental steps were as follows: by using SA biosensor (purchased from Pall), firstly immobilizing CD64 protein diluted to 4 μg/mL with PBST, the antibody was diluted to 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 1.5625 nM, 0 nM with PBST (pH7.0); allowing for binding for 90 seconds, then dissociating for 120 seconds, and the data were analyzed by 1:1 model and global fitting.
The experimental results were shown in
The antibody Fc can bind to FcRn so that it cannot be easily cleared in vivo, thus prolonging the half-life in vivo. The dynamic affinities of IgG1-1D mutant anti-PD-1 humanized antibodies to FcRn (purchased from ACRO) were measured by Octet ForteBio®, including 91D5-HZ2-IgG1-1D, 35E6-HZ1-IgG1-1D, the control antibody 1 and the control antibody 3. The specific experimental steps were as follows: by using a SA biosensor (purchased from Pall), firstly immobilizing FcRn protein diluted to 3 μg/mL with PBST; diluting the antibody to 200 nM, 100 nM, 50 nM, 25 nM, 12.5 nM, 6.25 nM, 3.125 nM, 0 nM with PBST (pH6.0); allowing for binding for 90 seconds, then dissociating for 120 seconds, and the data were analyzed by 1:1 model and global fitting.
The experimental results were shown in Table 13, wherein, the affinities of 91D5-HZ2-IgG1-1D and 35E6-HZ1-IgG1-1D for FcRn were at the level of E-09 (M), and the affinities of the control antibody 1 and the control antibody 2 for FcRn were at the level of E-08 (M). The affinity of IgG1-1D mutant anti-PD-1 humanized antibody was 2-3 times stronger than that of the control antibody, and its half-life in vivo may be prolonged.
The binding of the antibody Fc to CD32a is one important aspect in exerting ADCP function, and the ADCP function of IgG1-1D mutant anti-PD-1 humanized antibody was detected by a luciferase reporter system. Specific experimental steps were as follows: Jurkat-NFAT/CD32a and CHO-S-PD-1 cells (Sichuan Kelun Biotech Biopharmaceutical Co., Ltd.) were taken and centrifuged, resuspended with RPMI1640+1% FBS medium, then counted and diluted to 2×106 cells/mL and 1×106 cells/mL, respectively, and 50 μL of cell suspension was added per well; 91D5-HZ2-IgG1-1D, 35E6-HZ1-IgG1-1D, the control antibody 1, the control antibody 3 and the murine PD-1 antibody 29C6 were diluted (starting at 1.2 pM, 2-fold dilution, 2 replicates), and added to corresponding wells at 50 μL/well; after incubation at 37° C. for 5 hours, 20 μL of one-glo detection reagent (purchased from Promega) was added per well for detection by a microplate reader.
The results were as shown in
The binding of the antibody Fc to CD16a is one important aspect in exerting ADCC function, and the ADCC function of IgG1-1D mutant anti-PD-1 humanized antibody was detected by a luciferase reporter system. Specific experimental steps were as follows: Jurkat-NFAT/CD16a and CHO-S-PD-1 cells (Sichuan Kelun Biotech Biopharmaceutical Co., Ltd.) were taken and centrifuged, resuspended with RPMI1640+1% FBS medium, then counted and diluted to 2×106 cells/mL and 1×106 cells/mL, respectively, 50 μL of cell suspension was added per well; 91D5-HZ2-IgG1-1D, 35E6-HZ1-IgG1-1D, the control antibody 1, the control antibody 3 were diluted (starting at 450 μg/mL, 10-fold dilution, 2 replicates), and the murine PD-1 antibody 42G1 (450 μg/mL), which were added to corresponding wells at 50 μL/well; after incubation at 37° C. for 5 hours, 20 μL of one-glo detection reagent (purchased from Promega) was added per well for detection by a microplate reader.
The results were as shown in
The inhibitory effects of 35E6-HZ1-IgG1-1D and 91D5-HZ2-IgG1-1D antibodies on the proliferation of subcutaneous transplantation tumor in human immune system reconstructed mice or transgenic mice were evaluated in Examples 8.1 to 8.3. Specifically, in the invention, the antibodies 35E6-HZ1-IgG1-1D and 91D5-HZ2-IgG1-1D were administered to a Hu-PBMC-B-NDG mouse model transplanted subcutaneously with human non-small cell lung cancer HCC827 and a hPD-1/hTIM3 transgenic mouse model transplanted subcutaneously with mouse colon cancer cell line MC38 by tail intravenous injection or intraperitoneal injection, twice a week for 3 weeks. The changes in tumor volume and body weight of animals were measured twice a week, and the efficacy (tumor growth inhibition) of the antibody of the invention on tumor-bearing mice was calculated.
Test drug: taking an appropriate amount of 35E6-HZ1-IgG1-1D or 91D5-HZ2-IgG1-1D antibody, calculating the dosage volume according to 10 μL/g, and diluting the mother solution into corresponding volume of dosing solution with normal saline according to the dosage to be administered. The same dose of human anti-chicken lysozyme immunoglobulin Human IgG1 (Sichuan Kelun Biotech Biopharmaceutical Co., Ltd.) and human immunoglobulin Human IgG (Chengdu Rongsheng Pharmaceuticals Co., Ltd.) were used as negative control, and Keytruda (purchased from Merck, Cat. No. S001195) was used as positive control, and AB12T8 (prepared with reference to WO2019141092) was for combined administration.
Experimental animals and cell lines: B-NDG mice (Biocytogen Jiangsu Co., Ltd), hPD-1/hTIM-3 transgenic mice (Jiangsu GemPharmatech Co., Ltd.), mouse colon cancer cell line MC38 (Nanjing Cobioer Gene Technology Co., Ltd), human non-small cell lung cancer cell line HCC827 (ATCC), and human peripheral blood mononuclear lymphocyte PBMC (AllCELLS).
The tumor-bearing mice with an average tumor volume of 70-150 mm3 were randomly divided into groups (the number of groups was determined according to the number of samples). Human anti-chicken lysozyme immunoglobulin Human IgG1, intravenous human immunoglobulin Human IgG or the antibody of the invention was administered according to the groups, respectively, twice a week for 3 weeks, wherein the route of administration was intraperitoneal injection or tail vein injection, and the dosing volume was 10 mL/kg. The tumor diameter was measured by a vernier caliper twice a week after administration, and the tumor volume was calculated according to the following formula: V=0.5 a×b2, wherein a and b denote the long and short diameter of the tumor, respectively. Animal mortality was observed and recorded daily.
The tumor growth inhibition TGI (%) was calculated by the following formula to evaluate the tumor inhibition effect of the antibody of formula (I): TGI (%)=[1−(VTend−VT0)/(VCend−VC0)]*100%, wherein, VTend: the mean value of tumor volume at the end of the experiment in the treated group, VT0: the mean value of tumor volume at the beginning of dosing in the treated group, VCend: the mean value of tumor volume at the end of the experiment in the negative control group, VC0: the mean value of tumor volume at the beginning of dosing in the negative control group, and the relative tumor proliferation rate T/C (%) was calculated by the following formula to evaluate the anti-tumor efficacy of the antibody of formula (I): T/C=(VTend/VT0)/(VCend/VC0).
8.1 Determination of In Vivo Drug Efficacy of 35E6-HZ1-IgG1-1D in Human Immune System Reconstructed Mice hPBMC-B-NDG-HCC827 Model 1
HCC827 (non-small cell lung cancer) cells were cultured with RPMI1640 culture medium containing 10% fetal bovine serum at 37° C., 5% CO2. HCC827 cells in exponential growth phase were collected, then resuspended to a suitable concentration with PBS and inoculated subcutaneously into female B-NDG mice to establish a non-small cell lung cancer model. PBMC was thawed in the next day, resuspended to a suitable concentration with PBS, and then inoculated intraperitoneally into tumor-bearing mice to rebuild their immune systems. When tumors reaching the average volume of about 97 mm3, the mice were randomly divided into the following groups according to tumor size: human anti-chicken lysozyme immunoglobulin Human IgG1 (negative control) group, 35E6-HZ1-IgG1-1D antibody group and Keytnda group, injected via tail vein, twice a week for three weeks. After dosing, the tumor volume and body weight of mice were observed and measured regularly. The specific results were shown in Table 14, as shown in
HCC827 cells were cultured with RPMI1640 culture medium containing 10% fetal bovine serum at 37° C., 5% CO2. HCC827 cells in exponential growth phase were collected, then resuspended to a suitable concentration with PBS and inoculated subcutaneously into female B-NDG mice to establish a non-small cell lung cancer model. Five days after tumor cells were inoculated, PBMC was thawed, then resuspended to a suitable concentration with PBS, and then inoculated intraperitoneally into the tumor-bearing mice to rebuild their immune systems. When tumors reaching an average volume of about 91 mm3, the mice were randomly divided into the following groups according to tumor size: the group of human anti-chicken lysozyme immunoglobulin Human IgG1 (negative control), the group of single 91D5-HZ2-IgG1-1D antibody, and the group of 91D5-HZ2-IgG1-1D antibody in combination with LAG3 monoclonal antibody (AB12T, Sichuan Kelun Biotech Biopharmaceutical Co., Ltd.), respectively, which were injected by tail vein, twice a week for 2 weeks. After administration, the tumor volume and body weight of mice were observed and measured regularly, and the specific results were shown in Table 15, as shown in FIG. 18-19. Compared to Human IgG1 negative control group, the group of single 91D5-HZ2-IgG1-1D antibody had a good tumor inhibition trend, with TGI of 69.13%, and the group of 91D5-HZ2-IgG1-1D antibody in combination with LAG3 monoclonal antibody had further enhanced tumor inhibition effect, with TGI of 83.40%. Neither animal death nor significant animal weight loss nor obvious dug toxicity was observed in all treatment groups during the observation period, showing a good tolerance in the mice during the treatment.
MC38 (mouse colon cancer cell line) cells were cultured with RPMI1640 culture medium containing 10% fetal bovine serum at 37° C., 5% CO2. MC38 cells in exponential growth phase were collected, resuspended to a suitable concentration with PBS, and inoculated subcutaneously into hPD-1/hTIM-3 transgenic mice to establish a colon cancer model. When tumors reaching an average volume of about 83 mm3, the mice were randomly divided into the following groups according to tumor size: the group of intravenous human immunoglobulin Human IgG (negative control), the group of 91D5-HZ2-IgG1-1D antibody, the group of 35E6-HZ1-IgG1-1D antibody and the group of Keytruda, which were intraperitoneally injected, twice a week for 3 weeks. After administration, the tumor volume and body weight of mice were observed and measured regularly, and the specific results were shown in Table 16, as shown in
Although specific embodiments of the invention have been described in detail, those skilled in the art will understand that various modifications and changes may be made to the details in accordance with all the published teachings, which are also included in the scope of the invention. All the invention is given by the appended claims and any equivalents thereof.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201910939944.8 | Sep 2019 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2020/116417 | 9/21/2020 | WO |
