CN116964093A - Binding agents and methods of use thereof - Google Patents

Binding agents and methods of use thereof Download PDF

Info

Publication number
CN116964093A
CN116964093A CN202280013079.4A CN202280013079A CN116964093A CN 116964093 A CN116964093 A CN 116964093A CN 202280013079 A CN202280013079 A CN 202280013079A CN 116964093 A CN116964093 A CN 116964093A
Authority
CN
China
Prior art keywords
binding agent
kir
seq
tregs
binding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202280013079.4A
Other languages
Chinese (zh)
Inventor
C·克雷恩
K·斯维德里克
S·朱利安
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Mozart Treatment Co
Original Assignee
Mozart Treatment Co
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Mozart Treatment Co filed Critical Mozart Treatment Co
Priority claimed from PCT/US2022/014881 external-priority patent/WO2022169825A1/en
Publication of CN116964093A publication Critical patent/CN116964093A/en
Pending legal-status Critical Current

Links

Abstract

The present application provides binding agents that specifically bind to cd8+kir+t regulatory cells and their use in the treatment of a disease or disorder (e.g., an inflammatory disease, an autoimmune disease, cancer, or an infectious disease).

Description

Binding agents and methods of use thereof
Statement regarding sequence listing
The sequence listing relevant to the present application is provided in text format in place of paper copies and is incorporated by reference into this specification. The text file containing the sequence listing is named 670151_403wo_sequence_listing. The text file 85KB, created at 2022, 1 month and 20 days, is being submitted electronically via the EFS Web.
Background
The immune system includes the innate and adaptive immune systems. The adaptive immune system has many cell subtypes, including T cell subsets and B cell subsets. T cell subsets include T cells of various types, including naive T lymphocytes and effector T lymphocytes, such as cytotoxic T cells and helper T cells, and regulatory T cells. The activity of these T cell types is achieved by a balance between the activity of effector T cells and the regulation of regulatory T cells. While effector T cells promote inflammation, regulatory T cells are generally thought to regulate inflammation. Tregs therefore play an important role in autoimmune pathogenesis by maintaining self tolerance, limiting autoimmunity, and controlling expansion and activation of autoreactive cd4+ T effector cells. Disruption of the balance between effector T cells and regulatory T cells may lead to inappropriate activation or suppression of immune responses, loss of self tolerance, autoimmune disorders, and cancer. The mechanism and regulation by regulatory T cells to maintain immune system balance has only just begun to be understood.
Disclosure of Invention
Provided herein are binding agents that modulate the activity of cd8+kir+t regulatory cells (tregs) and methods of use thereof. The binding agent is bispecific or multispecific and specifically binds to an antigen expressed on the surface of a cd8+kir+ Treg. In some embodiments, the cd8+kir+treg is MHC class I-restricted. In some embodiments, the cd8+kir+treg is not MHC Qa-1 restricted. Methods of treating autoimmune diseases, infectious diseases, and cancers using the binding agents are also provided.
In some embodiments, a binding agent is provided comprising a first binding domain that specifically binds to a first antigen selected from antigens expressed on cd8+kir+t regulatory cells (tregs) other than KIR proteins; and a second binding domain that specifically binds to an inhibitory KIR protein expressed on the surface of the cd8+kir+ Treg, wherein the binding agent binds to the cd8+kir+ Treg.
In some embodiments, the first antigen is selected from the group consisting of: CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4 and 41BB. In some embodiments, the first antigen is selected from the group consisting of: CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4 and 41BB. In some embodiments, the first antigen is selected from the group consisting of CD3, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB, and HLA-DR. In some embodiments, the first antigen is selected from the group consisting of CD3, CD5, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB, and HLA-DR. In some embodiments, the first antigen is selected from the group consisting of LAG-3/CD223, TIM-3, PD-1, S1000A8/9, and TLT2. In some embodiments, the first antigen is selected from the group consisting of CD103 (ITGAE), CD166, CD177, CXCR3, and S1000A8/9. In some embodiments, the first antigen is selected from CCR7, CXCR3, and CXCR5. In some embodiments, the first antigen is selected from PD-1, CXCR3, and ICOS. In some embodiments, the first antigen is selected from the group consisting of CD3, CD5, and CD8. In some embodiments, the first antigen is selected from CD3 and CD8.
In some embodiments, the binding agent is a bispecific antibody, diabody antibody, antibody Fc fusion protein, scFv1-scFv2, scFv12-Fc-scFv22, igG-scFv, DVD-Ig, trifunctional antibody/tetravalent body tumor, diad IgG, scFv2-Fc, tandAb, scFv-HSA-scFv, scFv-VHH, fab-scFv-Fc, fab-VHH-Fc, dAb-IgG, igG-VHH, tandem scFv-Fc, (scFv 1) 2 -Fc-(VHH) 2 BiTe, DART, cross mab mab, anticalin, affibody, avimer, DARPin, adnectin, scFv-Fc, single arm tandem scFv-Fc or DART-Fc. In some embodiments, either the first binding domain or the second binding domain is selected from an antibody or antigen binding portion thereof, and the other binding domain is an antibody fragment. In some embodiments, the antigen binding portion is Fab, fab ', F (ab') 2 Fv, scFv or single domain antibodies (also known as VHH, VNAR, sdAb or nanobodies). In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region. In some embodiments, the second binding domain comprises a heavy chain variable region and a light chain variable region.
In some embodiments, the first binding domain specifically binds CD3 or a subunit of CD3, optionally CD3 epsilon. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL amino acid sequences being selected from the group consisting of amino acid sequence pairs shown in seq id nos:
a. SEQ ID NO. 1 and SEQ ID NO. 2, respectively;
b. SEQ ID NO 9 and SEQ ID NO 10, respectively;
c. SEQ ID NO. 17 and SEQ ID NO. 18, respectively;
d. SEQ ID NO. 25 and SEQ ID NO. 26, respectively;
e. SEQ ID NO 33 and SEQ ID NO 34, respectively;
f. SEQ ID NO 41 and SEQ ID NO 34, respectively;
g. SEQ ID NO 45 and SEQ ID NO 34, respectively;
h. SEQ ID NO. 49 and SEQ ID NO. 50, respectively;
i. SEQ ID NO 57 and SEQ ID NO 58, respectively;
j. SEQ ID NO. 65 and SEQ ID NO. 66, respectively; and
k. SEQ ID NO. 65 and SEQ ID NO. 166, respectively.
In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable region comprising hCDR1, and hCDR3, respectively, and CDR1, CDR2, and CDR3, the CDRs having amino acid sequences selected from the group of amino acid sequences shown in seq id nos:
a. SEQ ID NO 3 to SEQ ID NO 8, respectively;
b. SEQ ID NO. 11 to SEQ ID NO. 16, respectively;
c. SEQ ID NO 19 through SEQ ID NO 24, respectively;
d. SEQ ID NO 27 through SEQ ID NO 32, respectively;
e. SEQ ID NO 35 to SEQ ID NO 40, respectively;
f. SEQ ID NO. 42 to SEQ ID NO. 44 and SEQ ID NO. 38 to SEQ ID NO. 40, respectively;
g. 46 to 48 and 38 to 40, respectively;
h. SEQ ID NO. 51 to SEQ ID NO. 56, respectively;
i. 59 to 64, respectively;
j. SEQ ID NO 67 to SEQ ID NO 72, respectively; and
k. SEQ ID NOS 67-69 and 167-169, respectively.
In some embodiments, the first binding domain specifically binds CD8 or a subunit of CD8, optionally CD8 a. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL amino acid sequences being selected from the group consisting of amino acid sequence pairs shown in seq id nos:
a. SEQ ID NO 73 and SEQ ID NO 74, respectively; and
b. SEQ ID NO. 81 and SEQ ID NO. 82, respectively;
or the first binding domain comprises a VHH chain having an amino acid sequence selected from the group of amino acid sequences shown in seq id no:
c.SEQ ID NO:89；
d, SEQ ID NO. 93; and
e.SEQ ID NO:97。
in some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable region comprising hCDR1, and hCDR3, respectively, and CDR1, CDR2, and CDR3, the CDRs having amino acid sequences selected from the group of amino acid sequences shown in seq id nos:
a. 75 to 80 respectively; or (b)
b. 83 to 88 respectively;
or the first binding domain comprises a VHH chain having hCDR1, hCDR2 and hCDR3, the VHH CDR having an amino acid sequence selected from the group of amino acid sequences shown in the group consisting of seq id nos:
c. SEQ ID NO 90 to SEQ ID NO 92, respectively;
d. 94 to 96 respectively; and
e. 98 to 100, respectively.
In some embodiments, the first binding domain specifically binds ICOS or a subunit of ICOS. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), VH and VL having the amino acid sequences of SEQ ID No. 170 and SEQ ID No. 171, respectively.
In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region comprising hCDR1, and hCDR3 having the amino acid sequences of SEQ ID NOS 172-174, respectively, and lCDR1, lCDR2, and lCDR3 having the amino acid sequences of SEQ ID NOS 175-177, respectively.
In some embodiments, the first binding domain specifically binds to PD-1 or a subunit of PD-1. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL having the amino acid sequences of SEQ ID NO:178 and SEQ ID NO:179, respectively.
In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region comprising hCDR1, hCDR1 and hCDR3 having the amino acid sequences of SEQ ID NOs 180-182, respectively, and ilcdr 1, lCDR2 and ilcdr 3 having the amino acid sequences of SEQ ID NOs 183-185, respectively.
In some embodiments, the first binding domain specifically binds CXCR3 or a subunit of CXCR 3. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), VH and VL having the amino acid sequences of SEQ ID No. 186 and SEQ ID No. 187, respectively.
In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region comprising hCDR1, and hCDR3 having the amino acid sequences of SEQ ID NOS: 188-190, respectively, and lCDR1, lCDR2, and lCDR3 having the amino acid sequences of SEQ ID NOS: 191-193, respectively.
In some embodiments, the first binding domain specifically binds CD5 or a subunit of CD 5. In some embodiments, the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), VH and VL having the amino acid sequences of SEQ ID No. 194 and SEQ ID No. 195, respectively.
In some embodiments, the first binding domain comprises a heavy chain variable region and a light chain variable region comprising hCDR1, and hCDR3 having the amino acid sequences of SEQ ID NOS 196-198, respectively, and lCDR1, lCDR2, and lCDR3 having the amino acid sequences of SEQ ID NOS 199-201, respectively.
In some embodiments, the second binding agent specifically binds an inhibitory KIR protein selected from KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2, and KIR2DL3, or a combination thereof. In some embodiments, the second binding agent specifically binds KIR2DL1/2/3 or KIR2DL1/2. In some embodiments, the second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL having amino acid sequences selected from the group consisting of amino acid sequence pairs shown below:
a. SEQ ID NO. 101 and SEQ ID NO. 102, respectively;
b. SEQ ID NO 109 and SEQ ID NO 110, respectively;
c. SEQ ID NO 117 and SEQ ID NO 118, respectively;
d. SEQ ID NO. 125 and SEQ ID NO. 126, respectively;
e. SEQ ID NO 133 and SEQ ID NO 134, respectively;
f. 141 and 142, respectively;
g. 149 and 150, respectively; and
h. SEQ ID NO. 157 and SEQ ID NO. 158, respectively.
In some embodiments, the second binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable region comprising hCDR1, and hCDR3, respectively, and CDR1, CDR2, and CDR3, the CDRs having amino acid sequences selected from the group of amino acid sequences shown in seq id nos:
a. 103 to 108 respectively;
b. SEQ ID NO 111 through SEQ ID NO 116, respectively;
c. 119 to 124 respectively;
d. 127 to 132 respectively;
e. SEQ ID NO. 135 to SEQ ID NO. 140, respectively;
f. SEQ ID NO 143 to SEQ ID NO 148, respectively;
g. 151 to 156 respectively; and
h. 159 to 164, respectively.
In some embodiments, the binding agent does not comprise an Fc domain. In some embodiments, the binding agent comprises an Fc domain. In some embodiments, the Fc domain is selected from the group consisting of IgG1 and IgG4 Fc domains. In some embodiments, the binding agent has substantially no effector function activity. In some embodiments, the Fc domain is an IgG1 Fc domain. In some embodiments, the Fc domain is an IgG1 Fc null.
In some embodiments, the binding agent is divalent or tetravalent. In some embodiments, the binding agent is bispecific.
Also provided is a pharmaceutical composition comprising a binding agent of any of the embodiments described herein and a pharmaceutically acceptable carrier.
Nucleic acids encoding the binding agents of any of the embodiments described herein are also provided. Further provided is a vector comprising any of the embodiments of the nucleic acids described herein. Further provided are cell lines comprising any of the embodiments of the nucleic acids or vectors described herein.
In some embodiments, a method of treating an autoimmune disease is provided comprising administering to a subject in need thereof any embodiment of a binding agent or pharmaceutical composition described herein in an amount effective to reduce the number or activity of pathogenic immune cells and thereby improve the symptoms of an autoimmune disease in the subject.
In some embodiments, a method of inhibiting an immune response mediated by pathogenic immune cells is provided, comprising contacting cd8+ kir+ T regulatory cells (tregs) with an amount effective to activate or stimulate cd8+ kir+ tregs (activated tregs) according to any embodiment of the binding agents or pharmaceutical compositions described herein, thereby reducing the number or activity of pathogenic immune cells.
In some embodiments, a method of inhibiting an immune response to an autoantigen is provided, comprising administering any embodiment of a binding agent or pharmaceutical composition described herein to a subject in need thereof in an amount effective to activate or stimulate cd8+kir+ tregs, thereby reducing the number or activity of pathogenic immune cells that respond to the autoantigen.
In some embodiments, a method of inhibiting an immune response to an antigen is provided, comprising administering any embodiment of a binding agent or pharmaceutical composition described herein to a subject in need thereof in an amount effective to activate or stimulate cd8+kir+ tregs, thereby reducing the number or activity of pathogenic immune cells that are responsive to the antigen.
In some embodiments of these methods of treating an autoimmune disease or suppressing an immune response, cd8+ kir+ tregs are contacted with a binding agent in vivo. In some embodiments, the cd8+kir+ tregs are contacted ex vivo with a binding agent. In some embodiments, the activated cd8+kir+ tregs are administered to a subject in need thereof in an effective amount. In some embodiments, the pathogenic immune cells are autoreactive cd4+ T cells, autoantibody-producing B cells, or autoantigen presenting dendritic cells. In some embodiments, the pathogenic immune cells are cells presenting autoantigens. In some embodiments, the titer of the autoantigen is reduced in the subject.
In some embodiments, the subject has an autoimmune disease. In some embodiments, the autoimmune disease is selected from the group consisting of: celiac disease, crohn's disease, juvenile idiopathic arthritis, inflammatory Bowel Disease (IBD), insulin dependent diabetes mellitus (IDDM or type 1 diabetes), lupus nephritis, myasthenia gravis, myocarditis, multiple Sclerosis (MS), pemphigus/pemphigoid, rheumatoid Arthritis (RA), scleroderma/systemic sclerosis, sjogren's syndrome (SjS), systemic Lupus Erythematosus (SLE), and ulcerative colitis.
In some embodiments of the method of treating an autoimmune disease or suppressing an immune response, the binding agent specifically binds to an inhibitory KIR protein on CD8 and cd8+kir+ tregs. In some embodiments, the binding agent specifically binds to an inhibitory KIR protein on CD3 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to an inhibitory KIR protein on CD5 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to an inhibitory KIR protein on PD-1 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to inhibitory KIR proteins on ICOS and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to inhibitory KIR proteins on CXCR3 and cd8+ kir+ tregs. In some embodiments, the cd8+kir+treg is MHC class I-restricted. In some embodiments, the CD8+KIR+Treg is not MHC HLA E (Qa-1 b) restricted.
In some embodiments of the method of treating an autoimmune disease or suppressing an immune response, the method further comprises administering an immunosuppressant to the subject. In some embodiments, wherein administration of the binding agent to the subject results in improved therapeutic outcome in the subject. In some embodiments, the improved therapeutic result is a reduced frequency and severity of disease onset, a reduced systemic inflammatory cytokine, or a reduced self-reporting of symptoms associated with the autoimmune disease.
In some embodiments of the method of treating an autoimmune disease or suppressing an immune response, the binding agent is administered intravenously. In some embodiments, the binding agent is administered subcutaneously. In some embodiments, the binding agent is administered at a dose of about 0.01mg/kg to about 20 mg/kg. In some embodiments, the binding agent has substantially no effector function activity.
In some embodiments, there is provided a use of a binding agent or pharmaceutical composition described in any of the embodiments herein for treating an autoimmune disease in a subject by activating or stimulating cd8+kir+ tregs. In some embodiments, there is provided the use of a binding agent or pharmaceutical composition described in any of the embodiments herein for reducing the immune response of pathogenic immune cells by activating or stimulating cd8+kir+ tregs. In some embodiments, there is provided the use of a binding agent or pharmaceutical composition described in any of the embodiments herein for reducing autoantibody titers by activating or stimulating cd8+kir+ tregs.
In some embodiments, a method of treating cancer is provided, comprising administering to a subject in need thereof any of the embodiments of the binding agents or pharmaceutical compositions described herein, wherein the binding agent has substantially no effector function activity, in an amount effective to activate or stimulate cd8+kir+t regulatory cells (tregs) and thereby ameliorate symptoms of cancer.
In some embodiments, a method of stimulating an immune response against an antigen associated with cancer (cancer antigen) is provided, comprising contacting a cd8+ kir+ T regulatory cell (Treg) with a binding agent or pharmaceutical composition according to any embodiment herein in an amount effective to activate or stimulate the cd8+ kir+ Treg (activated Treg), thereby increasing the immune response against the cancer antigen, wherein the binding agent has substantially no effector function activity.
In some embodiments, a method of treating cancer is provided, comprising administering to a subject in need thereof any of the embodiments of the binding agents or pharmaceutical compositions described herein in an amount effective to deplete cd8+kir+ tregs and thereby ameliorate symptoms of the cancer, wherein the binding agent has effector function activity including at least ADCC.
In some embodiments, a method of stimulating an immune response against an antigen associated with cancer (cancer antigen) is provided, comprising contacting cd8+kir+t regulatory cells (tregs) with a binding agent or pharmaceutical composition according to any embodiment herein in an amount effective to deplete cd8+kir+tregs, thereby increasing the immune response against the cancer antigen, wherein the binding agent has effector function activity including at least ADCC.
In some embodiments of the method of treating cancer or stimulating an immune response against an antigen associated with cancer, cd8+kir+ tregs are contacted with the binding agent in vivo. In some embodiments, the cd8+kir+ tregs are contacted ex vivo with a binding agent. In some embodiments, the activated cd8+kir+ tregs are administered to a subject in need thereof in an effective amount. In some embodiments, the increased immune response includes cancer cytopenia or immunosuppressive immune cell depletion. In some embodiments, the number of cancer cells in the subject is reduced.
In some embodiments, the cancer is selected from the group consisting of: carcinoma, lymphoma, blastoma, sarcoma, myeloma, and leukemia. In some embodiments, the cancer is selected from the group consisting of: solid tumors such as breast cancer, cervical cancer, ovarian cancer, lung cancer, CRC (and other bowel cancers), skin cancer, esophageal cancer, adenocarcinoma, bladder cancer, and prostate cancer; lymphoma.
In some embodiments, the binding agent specifically binds to an inhibitory KIR protein on CD8 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to an inhibitory KIR protein on CD3 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to an inhibitory KIR protein on CD5 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to an inhibitory KIR protein on PD-1 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to inhibitory KIR proteins on CXCR3 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to inhibitory KIR proteins on ICOS and cd8+ kir+ tregs. In some embodiments, the cd8+kir+treg is MHC class I-restricted. In some embodiments, the CD8+KIR+Treg is not MHC HLA E (Qa-1 b) restricted.
In some embodiments, the method further comprises administering a chemotherapeutic agent to the subject. In some embodiments, the method further comprises administering an immunotherapy to the subject. In some embodiments, the method further comprises administering an immunotherapy, such as an immune checkpoint inhibitor, to the subject. In some embodiments, wherein administration of the binding agent to the subject results in improved therapeutic outcome in the subject. In some embodiments, the improved therapeutic result is a partial response or a complete response. In some embodiments, the improved therapeutic result is remission
In some embodiments, the binding agent is administered intravenously. In some embodiments, the binding agent is administered subcutaneously. In some embodiments, the binding agent is administered at a dose of about 0.01mg/kg to about 20 mg/kg.
In some embodiments, there is provided a use of a binding agent or pharmaceutical composition as described in any of the embodiments herein for treating cancer in a subject by activating or stimulating cd8+kir+ tregs, wherein the binding agent has substantially no effector function activity.
In some embodiments, there is provided the use of a binding agent or pharmaceutical composition described in any of the embodiments herein for reducing immunosuppression of immunosuppressive immune cells by activating or stimulating cd8+kir+ tregs, wherein the binding agent has substantially no effector function activity.
In some embodiments, there is provided a use of a binding agent or pharmaceutical composition as described in any of the embodiments herein for reducing tumor burden in a subject by activating or stimulating cd8+kir+ tregs, wherein the binding agent has substantially no effector function activity.
In some embodiments, there is provided the use of any of the embodiments of the binding agents or pharmaceutical compositions described herein for treating cancer in a subject by depleting cd8+kir+ tregs, wherein the binding agent has effector function activity including at least ADCC.
In some embodiments, there is provided the use of any of the embodiments of the binding agents or pharmaceutical compositions described herein for depleting cd8+kir+ tregs, wherein the binding agent has effector function activity including at least ADCC.
In some embodiments, there is provided the use of any of the embodiments of a binding agent or pharmaceutical composition described herein for reducing tumor burden in a subject, wherein the binding agent has effector function activity comprising at least ADCC.
In some embodiments, a method of treating an infection is provided, comprising administering any embodiment of a binding agent or pharmaceutical composition described herein to a subject in need thereof in an amount effective to activate or stimulate cd8+kir+ tregs and thereby ameliorate symptoms of the infection.
In some embodiments, a method of stimulating an immune response against an infected cell resulting from an infection is provided, comprising contacting a cd8+ kir+ T-regulatory cell (Treg) with a binding agent or pharmaceutical composition according to any embodiment herein in an amount effective to activate or stimulate the cd8+ kir+ Treg (activated Treg), thereby generating the immune response against the infected cell.
In some embodiments, the cd8+kir+ tregs are contacted with the binding agent in vivo. In some embodiments, the cd8+kir+ tregs are contacted ex vivo with a binding agent. In some embodiments, the activated cd8+kir+ tregs are administered to a subject in need thereof in an effective amount.
In some embodiments of treating an infection or stimulating an immune response to an infected cell, the immune response comprises an infectious cytopenia or an immunosuppressive immunocytopenia selected from the group consisting of: CD4 regulatory T cells and tolerogenic DCs. In some embodiments, the number of infected cells in the subject is reduced. In some embodiments, the infection is selected from the group consisting of bacterial disease, systemic fungal disease, rickettsiosis, parasitic disease, and viral disease. In some embodiments, the infection is selected from the group consisting of: HIV infection, hepatitis C Virus (HCV) infection, human Papilloma Virus (HPV) infection, epstein Barr Virus (EBV) infection, coronavirus infection such as SARS-COV2 infection (Covid-19), cytomegalovirus (CMV) infection, and influenza virus infection.
In some embodiments, the binding agent specifically binds to an inhibitory KIR protein on CD8 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to an inhibitory KIR protein on CD3 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to an inhibitory KIR protein on CD5 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to an inhibitory KIR protein on PD-1 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to inhibitory KIR proteins on CXCR3 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to inhibitory KIR proteins on ICOS and cd8+ kir+ tregs. In some embodiments, the cd8+kir+treg is MHC class I-restricted. In some embodiments, the CD8+KIR+Treg is not MHC HLA E (Qa-1 b) restricted.
In some embodiments, the method further comprises administering an antimicrobial or antiviral agent to the subject. In some embodiments, wherein administration of the binding agent to the subject results in improved therapeutic outcome in the subject. In some embodiments, the improved therapeutic result is a reduction in infection. In some embodiments, the improved therapeutic result is a reduction in infected cells. In some embodiments, the binding agent is administered intravenously. In some embodiments, the binding agent is administered subcutaneously. In some embodiments, the binding agent is administered at a dose of about 0.01mg/kg to about 20 mg/kg. In some embodiments, the binding agent has substantially no effector function activity.
In some embodiments, there is provided a use of a binding agent or pharmaceutical composition described in any of the embodiments herein for treating an infection in a subject by activating or stimulating cd8+kir+ tregs.
In some embodiments, there is provided the use of any of the embodiments of the binding agents or pharmaceutical compositions described herein for stimulating an immune response by activating or stimulating cd8+kir+ tregs and thereby inhibiting immunosuppressive immune cells.
In some embodiments, there is provided a use of a binding agent or pharmaceutical composition described in any of the embodiments herein for reducing infection in a subject by activating or stimulating cd8+kir+ tregs.
In some embodiments, a method of reducing or preventing the onset of Graft Versus Host Disease (GVHD) after transplantation is provided, comprising administering any embodiment of a binding agent or pharmaceutical composition described herein to a subject in need thereof in an amount effective to activate or stimulate cd8+kir+ tregs and thereby reduce or ameliorate at least one symptom of GVHD. In some embodiments, the binding agent has substantially no effector function activity.
In some embodiments, a method of treating a subject receiving a graft is provided, comprising contacting cd8+kir+t regulatory cells (tregs) with a binding agent or pharmaceutical composition according to any embodiment herein in an amount effective to activate or stimulate cd8+kir+tregs (activated tregs), thereby reducing or inhibiting GVHD. In some embodiments, the binding agent has substantially no effector function activity.
In some embodiments, a method of treating a subject receiving a transplant is provided comprising administering any embodiment of the binding agents or pharmaceutical compositions described herein to a subject in need thereof in an amount effective to deplete cd8+kir+ tregs and thereby ameliorate symptoms of GVHD. In some embodiments, the binding agent has effector function activity including at least ADCC.
In some embodiments, a method of inhibiting GVHD against a transplant is provided comprising contacting cd8+kir+t regulatory cells (tregs) with an amount of a binding agent or pharmaceutical composition according to any embodiment herein effective to deplete cd8+kir+tregs, thereby reducing GVHD or symptoms thereof. In some embodiments, the binding agent has effector function activity including at least ADCC.
In some embodiments, the cd8+kir+ tregs are contacted with the binding agent in vivo. In some embodiments, the cd8+kir+ tregs are contacted ex vivo with a binding agent. In some embodiments, the activated cd8+kir+ tregs are administered to a subject in need thereof in an effective amount.
In some embodiments, reducing GVHD comprises reducing cd4+ T cell activity in GVHD. In some embodiments, the implant is selected from the following: organ grafts, hematopoietic stem cell grafts, cord blood stem cell grafts, induced pluripotent stem cell-derived progenitor or differentiated cell grafts, and bone marrow grafts. In some embodiments, the graft is a hematopoietic stem cell graft, umbilical cord blood stem cell graft, induced pluripotent stem cell-derived progenitor cell, or differentiated cell graft, or bone marrow graft. In some embodiments, the implant is allogeneic.
In some embodiments, the binding agent specifically binds to an inhibitory KIR protein on CD8 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to an inhibitory KIR protein on CD3 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to an inhibitory KIR protein on CD5 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to an inhibitory KIR protein on PD-1 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to inhibitory KIR proteins on CXCR3 and cd8+ kir+ tregs. In some embodiments, the binding agent specifically binds to inhibitory KIR proteins on ICOS and cd8+ kir+ tregs. In some embodiments, the cd8+kir+treg is MHC class I-restricted. In some embodiments, the CD8+KIR+Treg is not MHC HLA E (Qa-1 b) restricted.
In some embodiments, an immunosuppressant is also administered to the subject.
In some embodiments, wherein administration of the binding agent to the subject results in improved therapeutic outcome in the subject. In some embodiments, the improved therapeutic result is a decrease in symptoms associated with GVHD, a decrease in systemic inflammatory cytokines, a decrease in pathology in tissues affected by GVHD, a decrease in symptom self-reports associated with an immune response associated with an adverse effect on host tissues, an improvement or extension of graft implantation, a decrease in one or more symptoms, and/or prevention, extension or slowing of the occurrence or progression of graft rejection, or extension of graft implantation with the use of a broad spectrum immunosuppressant (e.g., corticosteroid).
In some embodiments, the binding agent is administered intravenously. In some embodiments, the binding agent is administered subcutaneously.
In some embodiments, there is provided the use of any of the binding agents or pharmaceutical compositions described herein for treating graft-related GVHD in a subject. In some embodiments, the binding agent has substantially no effector function activity.
In some embodiments, there is provided the use of any of the binding agents or pharmaceutical compositions described herein for treating graft-related GVHD in a subject by activating or stimulating cd8+kir+ tregs. In some embodiments, the binding agent has substantially no effector function activity.
In some embodiments, there is provided the use of any of the binding agents or pharmaceutical compositions described herein for reducing GVHD associated with a graft by activating or stimulating cd8+kir+ tregs. In some embodiments, the binding agent has substantially no effector function activity.
In some embodiments, there is provided the use of any of the binding agents or pharmaceutical compositions described herein for reducing GVHD of a graft. In some embodiments, the binding agent has substantially no effector function activity.
In some embodiments, there is provided the use of any of the binding agents or pharmaceutical compositions described herein for treating graft-related GVHD in a subject by depleting cd8+kir+ tregs. In some embodiments, the binding agent has effector function activity including at least ADCC.
In some embodiments, there is provided the use of any of the binding agents or pharmaceutical compositions described herein for depleting cd8+kir+ tregs. In some embodiments, the binding agent has effector function activity including at least ADCC.
In some embodiments, there is provided the use of any of the binding agents or pharmaceutical compositions described herein for depleting cd8+kir+ tregs in a subject who has received an implant to reduce GVHD. In some embodiments, the binding agent has effector function activity including at least ADCC.
These and other aspects of the invention will be more fully understood by reference to the following detailed description, non-limiting examples of specific embodiments and the accompanying drawings.
Drawings
FIG. 1 shows various forms of IgG-scFv bispecific antibodies.
Figure 2 shows various forms of certain bispecific antibodies.
Figure 3 shows various forms of other bispecific antibodies.
Figures 4A-4D show the effect of Ly49 blocking on cd8+kir+t regulatory cells.
FIG. 5 shows the experimental design for assessing Ly49 blocking in a mouse Experimental Autoimmune Encephalomyelitis (EAE) model.
Figure 6 shows disease severity (as measured by clinical scores) 7 to 27 days after blocking immunization with MOG, mog+sp, mog+ly49 alone.
Figures 7A to 7C show the characteristics of T cells that are prevalent in celiac disease patients. Increased prevalence of cd8+kir+ T cells in celiac patients (fig. 7A); cd8+ T cell percentage increase with intracellular ifnγ and perforin (fig. 7B); and the percentage of cd8+ T cells with intracellular granzyme B increased (fig. 7C).
Figures 8A-8B show that celiac patients have more cd8+kir+ T cells (figure 8A) and cd8+kir+icos+ T cells (figure 8B) than healthy controls.
Fig. 9A-9B show that the re-stimulation of gluten peptides from cd8+kir+t cells of celiac patients increased degranulation (fig. 9A, left panel) and granzyme B levels (fig. 9B, right) compared to unstimulated cells or cells stimulated with control influenza peptide. Gluten peptide restimulation also resulted in a decrease in reactive cd4+ T cells compared to those of unstimulated cells or restimulated with control influenza peptide (fig. 9B).
Fig. 10A-10B show that KIR blockade of cd8+ tregs ("KIR blockade") results in increased intracellular granzyme B levels (fig. 10A) and increased degranulation (CD 107) (fig. 10B).
FIG. 11 shows an increase in cytolytic activity of CD8+ T cells, a decrease in CD4+ T cell activation and an increase in CD4+ T cell death in PBMC of celiac disease patients receiving KIR blocking and gluten re-stimulation treatment.
Figure 12 shows that KIR blocking of cd8+cd16+ T cells reduced activation and proliferation of CD 4T cells (CD 69) in three celiac patient samples.
Fig. 13 shows the percentage of cd8+kir+ Treg cells (upper panel) and cd8+cd39+ Treg cells (lower panel) in samples from celiac disease, crohn's disease, multiple Sclerosis (MS), systemic Lupus Erythematosus (SLE), ulcerative Colitis (UC), or type 1 diabetes (T1D) patients or healthy subjects.
Figure 14 shows the expression of cd8+kir+ Treg cell upper surface markers CD39, KLRG1, NKG2D, NKG2C, KLRB, CXCR3 and CD122 isolated from PBMC samples of celiac patients.
Figure 15 shows granzyme B, perforin and IL-10 produced by cd8+ Treg cells isolated from PBMC samples of celiac patients.
Figure 16 shows RANTES and tnfβ produced by cd8+kir+ Treg cells isolated from PBMC samples of celiac patients after stimulation with anti-CD 3 antibodies.
FIG. 17 shows that co-culture of CD4+ T cells with CD8+ KIR+ Treg cells after prolamin stimulation reduced IL-17A and IL-23 production.
Figure 18 shows co-culture with cd8+kir+ Treg cells and altered cd4+ T cell activation and ifnγ by gluten stimulation.
Figure 19 shows co-culture with cd8+kir+ Treg cells and stimulation with prolamin increases anti-inflammatory cytokines in cd4+ T cells. IL-10 production by CD4+ T cells is indicated by staining with intracellular cytokines.
Figure 20 shows an increase in cd8+ Treg cell response following repeated antigen exposure.
FIG. 21 shows the selective expression of the inhibitory KIR proteins KIR2DL1/2/3 and KIR3DL1 from CD8+ Treg cells of PBMC of celiac patients.
Figure 22 shows cd8+kir+t cell increase in celiac patients.
Figure 23 shows the presence of cd8+kir+t cells in intestinal tissue of celiac patients.
Figure 24 shows the interaction of granzyme positive cd8+ T cells with cd4+ T cells in intestinal tissue of celiac disease patients. Granzyme B appears white, cd8+ T cells appear green, cd4+ T cells appear ocher, and interactions between cd8+ T cells and cd4+ T cells appear yellow.
Figure 25 shows cd8+ Treg cell increase in peripheral blood 6 days after challenge with gluten (6D).
Figure 26 shows cd8+ Treg cell increase in tissue biopsies 14 days after challenge with gluten (14D).
Figure 27 shows cd4+ T cell numbers, costimulatory molecule expression, proliferation and KIR2DL expressing T cells in celiac patient tissues 14 days after gluten challenge (relative to gluten challenge prior to matched patient tissues).
FIG. 28 shows an example of a bispecific KIR binding agent and its expected effect on activation signal intensity.
FIG. 29A shows bispecific antibodies with a CD8 binding domain and a binding domain targeting KIR2DL 1/2/3. After administration of anti-CD 8 scFv/KIR FAB-Fc (at doses of 10. Mu.g/mL, 1. Mu.g/mL or 0.1. Mu.g/mL) or monoclonal KIR blocking (20. Mu.g), FIG. 29B shows degranulation and FIG. 29C shows granzyme B levels.
FIG. 30 shows a dose-dependent decrease in pro-inflammatory cytokines after administration of anti-CD 8 scFv/KIR FAB-Fc (at doses of 10. Mu.g/mL or 1. Mu.g/mL).
FIG. 31 shows CD4+ T cell death following administration of anti-CD 8 scFv/KIR FAB-Fc (at doses of 10. Mu.g/mL, 1. Mu.g/mL or 0.1. Mu.g/mL) or monoclonal KIR blocking (20. Mu.g).
FIG. 32 shows CD4+ T cell death following administration of anti-CD 8 scFv/KIR FAB-Fc (at doses of 10. Mu.g/mL, 1. Mu.g/mL, or 0.1. Mu.g/mL).
FIG. 33 shows preferential binding of bispecific blocking agents (anti-KIR 2DL1/2/3 and anti-CD 8) to CD8+ T cells relative to NK cells.
FIG. 34 shows preferential binding of bispecific blocking agents (anti-KIR 2DL1/2/3 and anti-CD 8) to CD8+ T cells relative to NK cells and CD4+ T cells.
Detailed Description
Definition of the definition
For convenience, certain terms in the description, examples and claims are defined herein. Unless otherwise indicated or implied by the context, the following terms and phrases have the following meanings. These definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed invention since the scope of the invention is limited only by the claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
As used herein, the terms "a" and "an" are to be understood as "one", "at least one" or "one or more", unless otherwise indicated. As used herein, the singular terms shall include the plural and the plural terms shall include the singular unless the context requires otherwise.
Throughout the specification and claims, the word "comprise" and the like should be interpreted in an inclusive sense, rather than an exclusive or exhaustive sense, unless the context clearly requires otherwise; that is, in the sense of "including but not limited to".
The terms "reduce", "reduced" and "inhibit" refer generally herein to a statistically significant amount of reduction relative to a reference.
The terms "increased" or "enhanced" or "activated" refer herein to a statistically significant increase relative to a reference.
As used herein, the term "isolated" or "partially purified" in the context of a nucleic acid, polypeptide, or protein refers to a nucleic acid, polypeptide, or protein that is separated from at least one other component present in the nucleic acid (e.g., the nucleic acid or polypeptide or protein) that is present with the nucleic acid, polypeptide, or protein found in its natural source and/or that will be present with the nucleic acid, polypeptide, or protein when expressed by a cell, or that is secreted in the context of a secreted polypeptide and protein. Chemically synthesized nucleic acids, polypeptides, or proteins, or nucleic acids synthesized using in vitro transcription/translation, are considered "isolated". The term "purified" or "substantially purified" refers to an isolated nucleic acid, polypeptide, or protein that is at least 95% by weight of a subject nucleic acid, polypeptide, or protein, including, for example, at least 96%, at least 97%, at least 98%, at least 99% or more.
As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to refer to a series of amino acid residues, each of which is interconnected by a peptide bond between the α -amino and carboxyl groups of adjacent residues. The terms "protein" and "polypeptide" also refer to polymers of protein amino acids, including modified amino acids (e.g., phosphorylated, glycosylated, etc.) and amino acid analogs, regardless of their size or function. "proteins" and "polypeptides" are generally used to refer to relatively large polypeptides, while the term "peptide" is generally used to refer to small polypeptides, but these terms overlap in their use in the art. The terms "protein" and "polypeptide" are used interchangeably herein when referring to the encoded gene product or fragment thereof. Thus, exemplary polypeptides or proteins include the above-described gene products, naturally-occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs.
CD3 epsilon is a protein expressed on T cells, including regulatory T cells. CD3 epsilon polypeptides include, but are not limited to, those having the amino acid sequence shown in NP 000724.1; the sequence is incorporated herein by reference.
CD5 is a protein expressed on T cells and B cells. CD5 polypeptides include, but are not limited to, those having the amino acid sequences shown in np_055022.2 and np_ 001333385.1; these sequences are incorporated herein by reference.
CD8 a is a protein expressed on T cells, including regulatory T cells. CD8 a polypeptides include, but are not limited to, those having the amino acid sequences set forth in np_001759.3, NP001139345.1, np_741969.1, np_001369627.1, np_757362.1, np_001171571.1, np_742100.1, np_742099.1, and np_ 004922; these sequences are incorporated herein by reference.
KIR3DL1 is a protein expressed on NK cells and on some T cells. It is also known as CD158E1, KIR2DL5B, KIR DL1/S1, NKAT-3, NKAT3, NKB1 and NKB1B. KIR3DL1 polypeptides include, but are not limited to, those having the amino acid sequences shown in np_037421.2 and np_ 001309097; these sequences are incorporated herein by reference.
KIR3DL2 is a protein expressed on NK cells and on some T cells. It is also known as 3DL2, CD158K, KIR-3DL2, NKAT-4, NKAT4B and p140.KIR3DL2 polypeptides include, but are not limited to, those having the amino acid sequences shown in np_006728.2 and np_ 001229796.1; these sequences are incorporated herein by reference.
KIR2DL1 is a protein expressed on NK cells and on some T cells. It is also known as CD158A, KIR-K64, KIR221, KIR2DL3, NKAT-1, NKAT1 and p58.1.KIR2DL1 polypeptides include, but are not limited to, those having the amino acid sequence shown in np_ 055033.2; the sequence is incorporated herein by reference.
KIR2DL2 is a protein expressed on NK cells and on some T cells. It is also known as CD158B1, CD158B, NKAT-6, NKAT6 and p58.2.KIR2DL2 polypeptides include, but are not limited to, those having the amino acid sequence shown in np_ 055034.2; the sequence is incorporated herein by reference.
KIR2DL3 is a protein expressed on NK cells and on some T cells. It is also known as CD158B2, CD158B, GL183, KIR-023GB, KIR-K7B, KIR-K7c, KIR2DL, KIR2DS5, KIRCL23, NKAT2A, NKAT B and p58.KIR2DL3 polypeptides include, but are not limited to, those having the amino acid sequence shown in np_ 056952.2; the sequence is incorporated herein by reference.
CD27 is also known as TNF receptor superfamily member 7, S152, LPFS2, T14, TNFRSF7 and Tp55.CD27 polypeptides include, but are not limited to, those having the amino acid sequence shown in np_ 001233.2; the sequence is incorporated herein by reference.
CD38 is also known as ADP ribose cyclase/cyclic ADP ribose hydrolase 1, ADPRC1 and ADPRC 1.CD38 polypeptides include, but are not limited to, those having the amino acid sequence shown in np_ 001766.2; the amino acid sequence is incorporated herein by reference.
CD39 is also known as exonucleoside triphosphate bisphosphate hydrolase 1, SPG64 ATPDase and NTPDase-1. Which encodes plasma membrane proteins that hydrolyze extracellular ATP and ADP to AMP. CD39 polypeptides include, but are not limited to, those having the amino acid sequences disclosed in np_001307845.1, np_001157651.1, np_001157650.1, np_001091645.1, np_001767.3, np_001299583.1, np_001157655.1, np_001157654.1, and np_ 001157653.1; these amino acid sequences are incorporated herein by reference.
CD40L or CD40 ligand is also known as CD154, HIGM1, IGM, IMD3, T-BAM, TNFSF5, TRAP, gp39, and hCD40L. It is expressed on the surface of T cells. CD40L polypeptides include, but are not limited to, those 1 having the amino acid sequence shown in np_ 000065.1; the sequence is incorporated herein by reference.
CD45 is known as the C protein tyrosine phosphatase receptor, B220, CD45R, GP180, L-CA, LCA, LY and T200. It has many subtypes including CD45RA, CD45Rb and CD45RO. CD45RA and CD45Rb are expressed on naive T cells. CD45RO is expressed on memory T cells. CD45R0 polypeptides include, but are not limited to, those having the amino acid sequences disclosed in P08575-4. CD45RA polypeptides include, but are not limited to, those having the amino acid sequences disclosed in P08575-8. CD45RB polypeptides include, but are not limited to, those having the amino acid sequences disclosed in P08575-9. See UniProtKB database. These sequences are incorporated herein by reference.
CD73 is also known as 5' extracellular nucleotidase, CALJA, CD73, E5NT, NT5, NTE, eN and eNT. CD73 polypeptides include, but are not limited to, those disclosed in np_001191742.1 and np_ 002517.1; these amino acid sequences are incorporated herein by reference.
CD103 or integrin subunit αe (ITGAE), also known as sumenae. CD103 polypeptides include, but are not limited to, those having the amino acid sequence disclosed in np_ 002199.3; the amino acid sequence is incorporated herein by reference.
CD122 or interleukin 2 receptor subunit beta, also known as IL15RB, IMD63, and P70-75.CD122 polypeptides include, but are not limited to, those having the amino acid sequences disclosed in np_001333152.1, np_001333151.1 and np_ 000869.1; these amino acid sequences are incorporated herein by reference.
CD166 or activated leukocyte adhesion molecule (ALCAM), also known as MEMD. CD166 polypeptides include, but are not limited to, those having the amino acid sequences set forth in np_001618.2, np_001230209.1, np_001230210.1, and np_ 001230212.1; these amino acid sequences are incorporated herein by reference.
CD177 is also known as HNA-2a, HNA2A, NB1, NB1 GP, PRV-1 and PRV1.CD177 polypeptides include, but are not limited to, those having the amino acid sequence shown in np_ 065139.2; the amino acid sequence is incorporated herein by reference.
CCR7 or C-C motif chemokine receptor 7, also known as BLR2, CC-CKR-7, CCR-7, CD197, CDw197, CMKBR7 and EBI1.CCR7 polypeptides include, but are not limited to, those having the amino acid sequences set forth in np_001829.1, np_001288643.1, np_001288645.1, np_001288646.1 and np_ 001288647.1; these amino acid sequences are incorporated herein by reference.
CXCR3 or C-X-C motif chemokine receptor 3, also known as GPR9, migR, CD182, CD183, mig-R, CKR-L2, CMKAR3 and IP10-R. CXCR3 polypeptides include, but are not limited to, those having the amino acid sequences set forth in np_001495.1 and np_ 001136269.1; these amino acid sequences are incorporated herein by reference.
CXCR5 or C-X-C motif chemokine 5, also known as BLR1, CD185 and MDR15.CXCR5 polypeptides include, but are not limited to, those having the amino acid sequences set forth in np_001707.1 and np_ 116743.1; these amino acid sequences are incorporated herein by reference.
HLA-DR is a class II histocompatibility antigen consisting of two chains. HLA-DR alpha chain polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP-061984.2. HLA-DR β chain polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP-002116.2, NP-072049.2, NP-001346123.1, and NP-001346122.1. These amino acid sequences are incorporated herein by reference.
ICOS or inducible T cell costimulators, also known as AILIM, CD278 and CVID1.ICOS polypeptides include, but are not limited to, those having the amino acid sequence shown in NP 036224.1; the amino acid sequence is incorporated herein by reference.
LAG-3 or CD223, also known as lymphocyte activation 3.LAG-3 polypeptides include, but are not limited to, those having the amino acid sequence shown in np_ 002277.4; the amino acid sequence is incorporated herein by reference.
OX-40 is also known as TNF receptor superfamily member 4 or TNFRSF4, ACT35, CD134, IMD16, and TXGP1L. OX-40 polypeptides include, but are not limited to, those having the amino acid sequence shown in np_ 003318.1; the amino acid sequence is incorporated herein by reference.
PD-1 is also known as programmed cell death protein 1.PD-1 polypeptides include, but are not limited to, those having the amino acid sequence set forth in NP-005009.2; the amino acid sequence is incorporated herein by reference.
S1000A8/9 or S100A8 and S100A9 are Ca belonging to the S100 family, respectively 2+ Binding proteins. S100A8 or S100-A8 is also known as 60B8AG, CAGA, CFAG, CGLA, CP-10, L1Ag, MA387, MIF, MRP8, NIF and P8. S100A8 polypeptides include, but are not limited to, those having the amino acid sequences set forth in np_001306125.1, np_001306126.1, np_001306127.1, np_001306130.1, and np_ 002955.2. S100A9 or S100-A9, also known as 60B8AG, CAGB, CFAG, CGLB, L1AG, LIAG, MAC387, MIF, MRP14, NIF and P14. S100A9 polypeptides include, but are not limited to, those having the amino acid sequence set forth in np_ 002956.1. These amino acid sequences are incorporated herein by reference.
TIM-3, also known as hepatitis A Virus cell receptor 2 (HAVCR 2), also known as CD366, HAVcr-2, KIM-3, SPTCL, TIM3, TIMD-3 and TIMD3.TIM-3 polypeptides include, but are not limited to, those having the amino acid sequence shown in np_ 116171.3; the amino acid sequence is incorporated herein by reference.
TLT-2 or myeloid cell triggering receptor-like protein 2 (TREML 2), also known as C6orf76 or dJ238O23.1.TLT-2 polypeptides include, but are not limited to, those having the amino acid sequence shown in NP-079083.2; the amino acid sequence is incorporated herein by reference.
2B4 or CD244, also known as NAIL, NKR2B4, nmrk and SLAMF4.2B4 polypeptides include, but are not limited to, those having the amino acid sequences set forth in NP-057466.1, NP-001160135.1, or NP-001160136.1; these amino acid sequences are incorporated herein by reference.
41BB or TNF receptor superfamily member 9 (TNFSF 9), also known as ILA, 4-1BB, CD137 and CDw 137. 41BB polypeptides include, but are not limited to, those having the amino acid sequence shown in np_ 001552.2; the amino acid sequence is incorporated herein by reference.
As used herein, "epitope" refers to amino acids conventionally bound by an immunoglobulin VH/VL pair, such as antibodies and other binding agents described herein. Epitopes can be formed on polypeptides by contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes formed by consecutive amino acids are typically retained upon exposure to denaturing solvents, whereas epitopes formed by three folds are typically lost upon treatment with denaturing solvents. Epitopes typically comprise at least 3, more typically at least 5, about 9, or about 8-10 amino acids with unique spatial conformations. Epitopes define the minimum binding site for an antibody or other binding agent and thus represent the specific target for an antibody, antigen-binding portion thereof, or other immunoglobulin-based binding agent. In the case of single domain antibodies, an epitope represents a structural unit bound by a variable domain in isolation.
As used herein, "specifically binds" refers to a binding agent (e.g., an antibody or antigen-binding portion thereof) as described herein with KD 10 -5 M (10000 nM) or less, e.g. 10 -6 M、10 -7 M、10 -8 M、10 -9 M、10 -10 M、10 -11 M、10 -12 M or less binding to the target. Specific binding may be affected by, for example, the affinity and avidity of the antibody or other binding agent, and the concentration of the target polypeptide. One of ordinary skill in the art can determine the binding agents described herein using any suitable method, such as titration of the binding agents in a suitable cell binding assaySuitable conditions for the antibodies and other binding agents to selectively bind the target antigen. Binding agents that specifically bind to the target are not replaced by a non-similar competitor. In certain embodiments, a binding agent (e.g., an antibody or antigen binding portion thereof) is said to specifically bind its target when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.
In some embodiments, a binding agent (e.g., an antibody or antigen binding portion thereof) or other binding agent as described herein is at 10 -5 M (10000 nM) or less, e.g. 10 -6 M、10 -7 M、10 -8 M、10 -9 M、10 -10 M、10 -11 M、10 -12 M or lower dissociation constant (KD) specifically binds to the target polypeptide. In some embodiments, an antibody or antigen binding portion thereof or other binding agent as described herein is used to bind to a polypeptide from about 10 -5 M to 10 -6 The dissociation constant (KD) of M specifically binds to a target polypeptide. In some embodiments, an antibody or antigen binding portion thereof or other binding agent as described herein is used to bind to a polypeptide from about 10 -6 M to 10 -7 The dissociation constant (KD) of M specifically binds to a target polypeptide. In some embodiments, an antibody or antigen binding portion thereof or other binding agent as described herein is used to bind to a polypeptide from about 10 -7 M to 10 -8 The dissociation constant (KD) of M specifically binds to a target polypeptide. In some embodiments, an antibody or antigen binding portion thereof or other binding agent as described herein is used to bind to a polypeptide from about 10 -8 M to 10 -9 The dissociation constant (KD) of M specifically binds to a target polypeptide. In some embodiments, an antibody or antigen binding portion thereof or other binding agent as described herein is used to bind to a polypeptide from about 10 -9 M to 10 -10 The dissociation constant (KD) of M specifically binds to a target polypeptide. In some embodiments, an antibody or antigen binding portion thereof or other binding agent as described herein is used to bind to a polypeptide from about 10 -10 M to 10 -11 The dissociation constant (KD) of M specifically binds to a target polypeptide. In some embodiments, an antibody or antigen binding portion thereof or other binding agent as described herein is used to bind to a polypeptide from about 10 -11 M to 10 -12 The dissociation constant (KD) of M specifically binds to a target polypeptide. In some embodiments, an antibody or anti-antibody thereof as described herein Primary binding moieties or other binding agents to less than 10 -12 The dissociation constant (KD) of M specifically binds to a target polypeptide.
As used herein, the term "consisting essentially of … …" refers to those elements required for a given embodiment. The term allows for the presence of elements that do not materially affect the basic and novel or functional characteristics of this embodiment.
The term "consisting of … …" refers to compositions, methods and their respective components as described herein, which do not include any elements not listed in the detailed description.
Except in the examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as being modified in all instances by the term "about". The term "about" when used in connection with a percentage may represent +/-1%.
The term "statistical significance" or "significance" refers to statistical significance, generally referring to the difference of two standard deviations (2 SD) above or below a reference value.
Other terms are defined in the description of various aspects of the invention.
Modulation of CD8+KIR+T regulatory cells
Provided herein are binding agents comprising a binding domain that specifically binds to an antigen expressed on cd8+kir+t regulatory cells (tregs). In some embodiments, the cd8+kir+treg is MHC class I-restricted. In some embodiments, the CD8+KIR+Treg is not MHC Qa-1 (HLA-E) restricted. Methods of using the binding agents for the treatment of autoimmune diseases, infectious diseases, and cancers are also provided.
The binding agent comprises a first binding domain that specifically binds to a T cell antigen expressed on cd8+ kir+ tregs but not KIR proteins, and a second binding domain that specifically binds to an inhibitory KIR protein expressed on cd8+ kir+ tregs. In some embodiments, the first binding domain specifically binds an antigen selected from the group consisting of: CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4 and 41BB. In some embodiments, the first binding domain specifically binds an antigen selected from the group consisting of: CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4 and 41BB. In some embodiments, the first binding domain specifically binds an antigen subunit selected from the group consisting of: CD3, CD8, CD40L, CD122, HLA-DR, OX-40, S1000A8/9 and 41BB/CD137.
In some embodiments, the first antigen is selected from a functional agonist capable of activating cd8kir+ tregs. In some embodiments, for example, such antigens are CD3, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB, and HLA-DR. In some embodiments, for example, such antigens are CD3, CD5, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB, and HLA-DR. In some embodiments, the first binding domain has agonist activity when bound to such antigen.
In some embodiments, the first antigen is selected from a functional antagonist that reduces the functional inhibition of cd8kir+ tregs. In some embodiments, for example, such antigens are LAG-3/CD223, TIM-3, PD-1, S1000A8/9 and TLT2. In some embodiments, the first binding domain has antagonist activity (e.g., blocking activity) when bound to such antigen.
In some embodiments, the first antigen is a tethered moiety to enhance the specificity of the binding agent for cd8kir+ tregs. In some embodiments, for example, such antigens are CD3, CD8, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4 and 41BB. In some embodiments, for example, such antigens are CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4 and 41BB. In some embodiments, the first binding domain specifically binds such antigens.
In some embodiments, the first antigen is a tethered moiety to enhance tissue specificity. In some embodiments, for example, such antigens are CD103 (ITGAE), CD166, CD177, CXCR3, and S1000A8/9. In some embodiments, the first binding domain specifically binds such antigens.
In some embodiments, the first antigen is an agonist to enhance cd8kir+ Treg cell migration. In some embodiments, for example, such antigen is CCR7, CXCR3, or CXCR5. In some embodiments, the first binding domain specifically binds such antigens.
In some embodiments, the first antigen is selected from PD-1, ICOS, and CXCR3. In some embodiments, the first binding domain specifically binds such antigens.
In some embodiments, the first antigen is selected from CD3 or CD8. In some embodiments, the first antigen is selected from CD3, CD5, or CD8. In some embodiments, the first antigen is selected from a subunit of CD3 or CD8. In some embodiments, the first antigen is CD3 epsilon. In some embodiments, the first antigen is CD8 a.
The second binding domain of the binding agent specifically binds to an inhibitory KIR protein (killer cell immunoglobulin-like receptor protein). For example, the inhibitory KIR protein may be KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2, or KIR2DL3, or a combination thereof, such as a KIR2DL1/2/3 or KIR2DL1/2 protein, specifically binding. In some embodiments, the KIR protein is selected from KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2, or KIR2DL3, or a combination thereof, such as KIR2DL1/2/3 or KIR2DL1/2 protein. In some embodiments, the second binding domain is a KIR protein antagonist that blocks the interaction of KIR proteins with their binding partners.
The binding agent may be any suitable agent comprising binding domains for both antigens. In some embodiments, the binding agent is bispecific (i.e., has binding domains for two different antigens). In some embodiments, the binding agent is bivalent (i.e., has two binding domains). In some embodiments, the binding agent is tetravalent (i.e., has four binding domains).
The binding domain of the binding agent may be from an antibody or from a non-antibody form. In some embodiments, the binding domain is from an antibody or antigen-binding portion thereof (i.e., an antibody fragment). In some embodiments, the antibody fragment is a Fab, fab ', F (ab') 2, fv, scFv, or single domain antibody (also known as VHH, VNAR, sdAb or nanobody). In some embodiments, the binding domain is or is derived from an anticalin, an affibody, avimer, DARPin, or an adnectin.
In some embodiments, the binding agent is a bispecific antibody, diabody antibody, antibody Fc fusion protein, scFv1-ScFv2, scFv12-Fc-scFv22, igG-scFv, DVD-Ig, trifunctional antibody/tetravalent body tumor, diabody IgG, scFv2-Fc, tandAb, scFv-HSA-scFv, scFv-VHH, fab-scFv-Fc, fab-VHH-Fc, dAb-IgG, igG-VHH, tandem scFv-Fc, (scFv 1) 2 -Fc-(VHH) 2 BiTe, DART, cross-mab mab, anticalin, affibody, avimer, DARPin, adnectin, scFv-Fc, single arm tandem scFv-Fc or DART-Fc (see, e.g., FIGS. 2 and 3). In some embodiments, the IgG-scFv is an IgG (H) -scFv, a scFv- (H) IgG, an IgG (L) -scFv, a scFv- (L) IgG, a 2scFv-IgG, or an IgG-2scFv (as shown in fig. 1).
In some embodiments, the binding agent comprises a first binding domain comprising a heavy chain variable region and a light chain variable region. In some embodiments, the heavy and light chain variable regions of the first binding domain specifically bind to antigens expressed on cd8+kir+treg, such as CD3, CD8, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4, and 41BB. In some embodiments, the heavy and light chain variable regions of the first binding domain specifically bind to antigens expressed on cd8+kir+treg, such as CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4, and 41BB. In some embodiments, the heavy and light chain variable regions of the first binding domain specifically bind to subunits of an antigen expressed on cd8+kir+treg, such as CD3, CD8, CD40L, CD122, HLA-DR, OX-40, S1000A8/9 and 41BB/CD137. In some embodiments, the heavy and light chain variable regions of the first binding domain specifically bind to subunits of an antigen expressed on cd8+kir+treg, such as CD3, CD5, CD8, CD40L, CD122, HLA-DR, OX-40, S1000A8/9 and 41BB/CD137.
In some embodiments, the first antigen is selected from a functional agonist capable of activating cd8kir+ tregs. In some embodiments, for example, such antigens are CD3, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB, and HLA-DR. In some embodiments, for example, such antigens are CD3, CD5, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB, and HLA-DR. In some embodiments, the first binding domain has agonist activity when bound to such antigen.
In some embodiments, the first antigen is selected from a functional antagonist that reduces the functional inhibition of cd8kir+ Treg. In some embodiments, for example, such antigens are LAG-3/CD223, TIM-3, PD-1, S1000A8/9 and TLT2. In some embodiments, when binding such antigens, the first binding domain has antagonist activity (e.g., blocking activity).
In some embodiments, the first antigen is a tethered moiety to enhance the specificity of the binding agent for cd8kir+ tregs. In some embodiments, for example, such antigens are CD3, CD8, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4 and 41BB. In some embodiments, for example, such antigens are CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4 and 41BB. In some embodiments, the first binding domain specifically binds such antigens.
In some embodiments, the first antigen is a tethered moiety to enhance tissue specificity. In some embodiments, for example, such antigens are CD103 (ITGAE), CD166, CD177, CXCR3, and S1000A8/9. In some embodiments, the first binding domain specifically binds such antigens.
In some embodiments, the first antigen is an agonist to enhance cd8kir+ Treg cell migration. In some embodiments, for example, such antigen is CCR7, CXCR3, or CXCR5. In some embodiments, the first binding domain specifically binds such antigens.
In some embodiments, the first antigen is selected from PD-1, ICOS, and CXCR3. In some embodiments, the first binding domain specifically binds such antigens.
In some embodiments, the first antigen is selected from CD3 or CD8. In some embodiments, the first antigen is selected from CD3, CD5, or CD8. In some embodiments, the first antigen is selected from a subunit of CD3 or CD8. In some embodiments, the first antigen is CD3 epsilon. In some embodiments, the first antigen is CD8 a.
Antibodies for use in the binding domains described herein are well known in the art.
Such as those described in U.S. Pat. nos. 5,929,212;5,885,573; and 8,551,478 and International patent publication No. WO2018223004 describe antibodies to CD 3.
Antibodies to CD8 have been described, for example, in published U.S. patent application nos. 20190382488 and 20190071500 and international patent publications nos. WO2014164553 and WO 2017134306.
Antibodies to CD5 have been described, for example, in published U.S. patent application nos. 2018/0104308, 2011/0250203, and 2008/0254027.
Antibodies to CD27 have been described, for example, in published U.S. patent application nos. 20210009706, 20200247898 and 20200131272.
Antibodies to CD38 have been described, for example, in published U.S. patent application nos. 20200408765, 20200399391, 20090304710 and 20050158305.
Antibodies to CD39 have been described, for example, in published U.S. patent application nos. 20190062448, 20130273062 and 20100303828.
Antibodies to CD40L have been described, for example, in published U.S. patent application nos. 20190092868, 20100092482, 20030031668 and 20010018041.
Antibodies to CD45RA, CD45RB and CD45RO have been described, for example, in published, U.S. patent application nos. 20030232009 and 20020168362, and are available from commercial sources.
Antibodies to CD73 have been described, for example, in published U.S. patent application nos. 20200148781, 20200071404, 20190256598 and 20160145350.
Antibodies against CD103 (ITGAE) have been described, for example, in published U.S. patent application No. 20050266001.
Antibodies to CD122 have been described, for example, in published U.S. patent application nos. 20180362655 and 20110250213.
Antibodies to CD166 have been described, for example, in published U.S. patent application nos. 20160355587 and 20090269787.
Antibodies to CD177 have been described, for example, in published U.S. patent application No. 20190125832.
Antibodies to CCR7 have been described, for example, in published U.S. patent application nos. 20200216548, 20180237529 and 20150344580.
Antibodies to CXCR3 have been described, for example, in published U.S. patent application nos. 20190119391, 20190008955 and 20130251733.
Antibodies to CXCR5 have been described, for example, in published U.S. patent application nos. 20190169283, 20160053014 and 20130236476.
Antibodies against HLA-DR have been described, for example, in published U.S. patent application Nos. 20180355043 and 20190071503.
Antibodies to ICOS have been described, for example, in published U.S. patent application nos. 20160304610 and 20110243929.
Antibodies to LAG-3/CD223 have been described, for example, in published U.S. patent application nos. 20210009687, 20200277372, 20200071403, and 20190276538.
Antibodies to OX40 have been described, for example, in published U.S. patent application nos. 20140377284, 20140308276 and 20100196359.
Antibodies to PD-1 have been described, for example, in published U.S. patent application nos. 20190322749, 20190309069, 20170313774 and 20110171215.
Antibodies to S1000A8/9 have been described, for example, in published U.S. patent application Nos. 20180256710 and 20200023045.
Antibodies to TIM-3 have been described, for example, in published U.S. patent application nos. 20180072804, 20170306016 and 20150086574.
Antibodies to TLT-2 have been described, for example, in published U.S. patent application No. 20130216540.
For example, antibodies to 2B4 may be obtained from commercial suppliers.
Antibodies against 41BB have been described, for example, in published U.S. patent application nos. 20170198050 and 20200347144.
In some embodiments, the first binding domain specifically binds to CD3 epsilon and the heavy and light chain variable domains have the amino acid sequences shown below, respectively: SEQ ID NO. 1 and SEQ ID NO. 2; SEQ ID NO. 9 and SEQ ID NO. 10; 17 and 18; 25 and 26; 33 and 34; SEQ ID NO. 41 and SEQ ID NO. 34; SEQ ID NO. 45 and SEQ ID NO. 34; 49 and 50; SEQ ID NO. 57 and SEQ ID NO. 58; 65 and 66; or SEQ ID NO. 65 and SEQ ID NO. 166.
In some embodiments, the first binding domain specifically binds to CD3 epsilon and the heavy and light chain variable domains have the amino acid sequences shown below, respectively: SEQ ID NO. 1 and SEQ ID NO. 2; SEQ ID NO. 9 and SEQ ID NO. 10; 17 and 18; 25 and 26; 33 and 34; SEQ ID NO. 41 and SEQ ID NO. 34; SEQ ID NO. 45 and SEQ ID NO. 34; 49 and 50; SEQ ID NO. 57 and SEQ ID NO. 58; or SEQ ID NO. 65 and SEQ ID NO. 66; or SEQ ID NO. 65 and SEQ ID NO. 166; wherein the framework regions of the heavy and light chain variable regions are optionally substituted, deleted or inserted with 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acids, and wherein the CDRs of the heavy or light chain variable regions are unmodified.
In some embodiments, the first binding domain comprises an amino acid sequence according to any one or more of SEQ ID NOS: 1-72 and 166-169 (e.g., VH, VL, hCDR1, hCDR3, lCDR1, lCDR2 and/or lCDR 3).
In some embodiments, the first binding domain specifically binds to CD8 a and the heavy and light chain variable domains have the amino acid sequences shown below, respectively: SEQ ID NO. 73 and SEQ ID NO. 74; or SEQ ID NO. 81 and SEQ ID NO. 82; or the binding domain comprises a VHH chain having the amino acid sequence shown in SEQ ID NO. 89, SEQ ID NO. 93 or SEQ ID NO. 97.
In some embodiments, the first binding domain specifically binds to CD8 a and the heavy and light chain variable domains have the amino acid sequences shown below, respectively: SEQ ID NO. 73 and SEQ ID NO. 74; or SEQ ID NO. 81 and SEQ ID NO. 82; or the binding domain comprises a VHH chain having the amino acid sequence shown in SEQ ID NO. 89, SEQ ID NO. 93 or SEQ ID NO. 97; wherein the framework regions of the heavy and light chain variable regions or the VHH chain are optionally modified by 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions, and wherein the CDRs or VHH chains of the heavy or light chain variable regions are unmodified.
In some embodiments, the first binding domain comprises an amino acid sequence according to any one or more of SEQ ID NOS: 73-100 (e.g., VH, VL, hCDR, hCDR1, hCDR3, lCDR1, lCDR2 and/or lCDR 3).
In some embodiments, the first binding domain specifically binds CD3 epsilon and the heavy chain variable region has complementarity determining regions hCDR1, hCDR2, and hCDR3, light chain variable regions CDR1, CDR2, and CDR3, and the amino acid sequences of the heavy and light chain variable region CDRs are shown below, respectively: SEQ ID NO 3 to SEQ ID NO 8; 11 to 16; SEQ ID NO 19 through SEQ ID NO 24; 27 to 32; SEQ ID NO. 35 to SEQ ID NO. 40; 42 to 44 and 38 to 40; 46 to 48 and 38 to 40; SEQ ID NO. 51 to SEQ ID NO. 56; 59 to 64; or SEQ ID NO. 67 to SEQ ID NO. 72. In some embodiments, the first binding domain specifically binds CD3 ε and comprises light chain variable regions lCDR1, lCDR2 and lCDR3, which have the amino acid sequences shown in SEQ ID NOS 167, 168 and 169, respectively.
In some embodiments, the first binding domain specifically binds to CD8 a and the heavy chain variable region has complementarity determining regions hCDR1, hCDR2, and hCDR3, light chain variable regions CDR1, CDR2, and CDR3, and the amino acid sequences of the heavy and light chain variable region CDRs are shown below, respectively: 75 to 80; or SEQ ID NO 83 to SEQ ID NO 88; or the first binding domain comprises a VHH chain having the amino acid sequences hCDR1, hCDR2 and hCDR3, and VHH CDRs respectively as follows: SEQ ID NO 90 through SEQ ID NO 92; 94 to 96; or SEQ ID NO 98 to SEQ ID NO 100.
In some embodiments, the first binding domain specifically binds ICOS and the heavy and light chain variable regions have the amino acid sequences shown below, respectively: 170 and 171.
In some embodiments, the first binding domain specifically binds ICOS and the heavy and light chain variable regions have the amino acid sequences shown below, respectively: 170 and 171; wherein the framework regions of the heavy and light chain variable regions or the VHH chain are optionally modified by 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions, and wherein the CDRs of the heavy or light chain variable regions are unmodified.
In some embodiments, the first binding domain comprises an amino acid sequence according to any one or more of SEQ ID NOS 170-177 (e.g., VH, VL, hCDR, hCDR1, hCDR3, lCDR1, lCDR2 and/or lCDR 3).
In some embodiments, the first binding domain specifically binds PD-1 and the heavy and light chain variable domains have the amino acid sequences shown below, respectively: 178 and 179.
In some embodiments, the first binding domain specifically binds PD-1 and the heavy and light chain variable domains have the amino acid sequences shown below, respectively: 178 and 179; wherein the framework regions of the heavy and light chain variable regions or the VHH chain are optionally modified by 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions, and wherein the CDRs of the heavy or light chain variable regions are unmodified.
In some embodiments, the first binding domain comprises an amino acid sequence according to any one or more of SEQ ID NOS 178-185 (e.g., VH, VL, hCDR, hCDR1, hCDR3, lCDR1, lCDR2 and/or lCDR 3).
In some embodiments, the first binding domain specifically binds CXCR3 and the heavy and light chain variable domains have the amino acid sequences shown below, respectively: 186 and 187.
In some embodiments, the first binding domain specifically binds CXCR3 and the heavy and light chain variable domains have the amino acid sequences shown below, respectively: 186 and 187; wherein the framework regions of the heavy and light chain variable regions or the VHH chain are optionally modified by 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions, and wherein the CDRs of the heavy or light chain variable regions are unmodified.
In some embodiments, the first binding domain comprises an amino acid sequence according to any one or more of SEQ ID NOS 186-193 (e.g., VH, VL, hCDR, hCDR1, hCDR3, lCDR1, lCDR2 and/or lCDR 3).
In some embodiments, the first binding domain specifically binds CD5 and the heavy and light chain variable domains have the amino acid sequences shown below, respectively: 194 and 195.
In some embodiments, the first binding domain specifically binds CD5 and the heavy and light chain variable domains have the amino acid sequences shown below, respectively: 194 and 195; wherein the framework regions of the heavy and light chain variable regions or the VHH chain are optionally modified by 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions, and wherein the CDRs of the heavy or light chain variable regions are unmodified.
In some embodiments, the first binding domain comprises an amino acid sequence according to any one or more of SEQ ID NOs 194-201 (e.g., VH, VL, hCDR, hCDR1, hCDR3, lCDR1, lCDR2 and/or lCDR 3).
In some embodiments, the binding agent comprises a second binding domain comprising a heavy chain variable region and a light chain variable region. The second binding domain of the binding agent specifically binds to an inhibitory KIR protein (killer cell immunoglobulin-like receptor protein). The inhibitory KIR protein may be KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2 or KIR2DL3 or a combination thereof, such as a specific binding KIR2DL1/2/3 or KIR2DL1/2 protein.
Antibodies against inhibitory KIR proteins are well known in the art.
Antibodies to WO2018148223 have been described, for example, in U.S. patent No. 5,770,387 and international patent publication No. WO 2018148223.
Antibodies to KIR3DL2 have been described, for example, in published U.S. patent application nos. 20200199228 and 20150232556.
Antibodies to KIR2DL1, KIR2DL2, KIR2DL3, and combinations thereof have been described, for example, in U.S. patent nos. 10,668,180 and 10,253,095, international patent publication No. WO2006003179, published U.S. patent application nos. 20150290316 and 20130251711, and european patent No. 3072522.
In some embodiments, the second binding agent specifically binds KIR3DL1 and the heavy and light chain variable regions have the amino acid sequences shown below, respectively: 133 and 134; 141 and 142; or SEQ ID NO:149 and SEQ ID NO:150.
In some embodiments, the first binding domain specifically binds KIR3DL1 and the heavy and light chain variable domains have the amino acid sequences shown below, respectively: 133 and 134; 141 and 142; or SEQ ID NO. 149 and SEQ ID NO. 150; wherein the framework regions of the heavy and light chain variable regions or the VHH chain are optionally modified by 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions, and wherein the CDRs of the heavy or light chain variable regions are unmodified.
In some embodiments, the first binding domain specifically binds KIR3DL2 and the heavy and light chain variable domains have the amino acid sequences shown below, respectively: SEQ ID NO. 157 and SEQ ID NO. 158.
In some embodiments, the first binding domain specifically binds KIR3DL2 and the heavy and light chain variable domains have the amino acid sequences shown below, respectively: 157 and 158; wherein the framework regions of the heavy and light chain variable regions or the VHH chain are optionally modified by 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions, and wherein the CDRs of the heavy or light chain variable regions are unmodified.
In some embodiments, the first binding domain specifically binds KIR2DL1/2/3 and the heavy and light chain variable domains have the amino acid sequences shown below, respectively: SEQ ID NO. 101 and SEQ ID NO. 102; SEQ ID NO 109 and SEQ ID NO 110; 117 and 118; or SEQ ID NO. 125 and SEQ ID NO. 126.
In some embodiments, the first binding domain specifically binds KIR2DL1/2/3 and the heavy and light chain variable domains have the amino acid sequences shown below, respectively: SEQ ID NO. 101 and SEQ ID NO. 102; SEQ ID NO 109 and SEQ ID NO 110; 117 and 118; or SEQ ID NO. 125 and SEQ ID NO. 126; wherein the framework regions of the heavy and light chain variable regions or the VHH chain are optionally modified by 1 to 8, 1 to 6, 1 to 4 or 1 to 2 amino acid substitutions, deletions or insertions, and wherein the CDRs of the heavy or light chain variable regions are unmodified.
In some embodiments, the first binding domain specifically binds KIR3DL1 and the heavy chain variable region has Complementarity Determining Regions (CDRs) hCDR1, hCDR2, and hCDR3, the light chain variable region has amino acid sequences of CDR1, CDR2, and CDR3, and the CDRs of the heavy and light chain variable regions have amino acid sequences as shown below, respectively: SEQ ID NO. 135 to SEQ ID NO. 140; 143 to 148; or SEQ ID NO 151 through SEQ ID NO 156.
In some embodiments, the first binding domain specifically binds KIR3DL2 and the heavy chain variable region has complementarity determining regions hCDR1, hCDR2, and hCDR3, the light chain variable region has CDR1, CDR2, and CDR3, and the amino acid sequences of the CDRs of the heavy and light chain variable regions have the amino acid sequences shown below, respectively: 159 to 164.
In some embodiments, the first binding domain specifically binds KIR2DL1/2/3 and the heavy chain variable region has complementarity determining regions hCDR1, hCDR2, and hCDR3, the light chain variable region has amino acid sequences of CDR1, CDR2, and CDR3, and the heavy and light chain variable regions have amino acid sequences as shown below, respectively: 103 to 108; SEQ ID NO 111 through SEQ ID NO 116; 119 to 124; or SEQ ID NO 127 through SEQ ID NO 132.
Binding agent
The binding agent may be any suitable agent comprising at least a first binding domain and a second binding domain, wherein the first binding domain specifically binds to a first antigen selected from antigens expressed on cd8+kir+t regulatory cells (tregs) other than KIR proteins; and a second binding domain specifically binds to an inhibitory KIR protein, wherein the binding agent binds to cd8+ kir+ tregs.
In some embodiments, the binding agent is bispecific (i.e., has binding domains for two different antigens). In some embodiments, the binding agent is bivalent (i.e., has two binding domains). In some embodiments, the binding agent is tetravalent (i.e., has four binding domains). In some embodiments, the binding agent is trivalent, hexavalent or octavalent.
The binding domain of the binding agent may be from an antibody or from a non-antibody form. In some embodiments, the binding domain is from an antibody or antigen-binding portion thereof (i.e., an antigen-binding antibody fragment). In some embodiments, the antibody fragment is a Fab, fab ', F (ab') 2, fv, scFv, or single domain antibody (also known as VHH, VNAR, sdAb or nanobody). In some embodiments, the binding domain is from an anticalin, affibody, avimer, DARPin, adnectin, or receptor ectodomain Fc fusion protein.
In some embodiments, the binding agent is a bispecific antibody, diabody antibody, antibody Fc fusion protein, scFv1-ScFv2, scFv12-Fc-scFv22, igG-scFv, DVD-Ig, trifunctional antibody/tetravalent body tumor, diabody IgG, scFv2-Fc, tandAb, scFv-HSA-scFv, scFv-VHH, fab-scFv-Fc, fab-VHH-Fc, dAb-IgG, igG-VHH, tandem scFv-Fc, (scFv 1) 2 -Fc-(VHH) 2 BiTe, DART, cross mab mab, anticalin, affibody, avimer, DARPin, adnectin, scFv-Fc, single arm tandem scFv-Fc or DART-Fc. In some embodiments, the IgG-scFv is an IgG (H) -scFv, a scFv- (H) IgG, an IgG (L) -scFv, a scFv- (L) IgG, a 2scFv-IgG, or an IgG-2scFv (as shown in fig. 1).
As used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulins, i.e., molecules that contain an antigen binding site (antigen binding portion) that specifically binds to a target antigen. The term generally refers to antibodies consisting of two immunoglobulin heavy chain variable regions and two immunoglobulin light chain variable regions, including full length antibodies (having heavy and light chain constant regions) and antigen binding portions thereof; including, for example, intact monoclonal antibodies, fab ', F (ab') 2 Fv, disulfide-linked Fv, scFv, single domain antibodies (dAb), diabodies, multispecific antibodies, bispecific antibodies, specific antibodies, and single chain antibodies (see, e.g., huston et al, proc.Natl.Acad.Sci.U.S. A.,85,5879-5883 (1988) and Bird et al, science 242,423-426 (1988), which are incorporated herein by reference).
In antibodies, each heavy chain consists of a variable region (abbreviated VH) and a constant region. The heavy chain constant region may comprise three domains CH1, CH2 and CH3, and optionally a fourth domain CH4. Each of these domains is referred to as an "Fc domain". As used herein, when a binding agent comprises an Fc domain, it may include one or more Fc domains, or the entire Fc region, unless the context dictates otherwise. Each light chain consists of a variable region (abbreviated VL) and a constant region or constant domain. The light chain constant region is the CL domain. VH and VL regions can be further divided into hypervariable regions known as Complementarity Determining Regions (CDRs) and conserved regions known as Framework Regions (FR). Thus, each VH and VL region consists of three CDRs and four FRs, arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. This structure is well known to those skilled in the art.
As used herein, an "antigen-binding portion" of an antibody refers to a portion of an antibody described herein that has VH and VL sequences or heavy and light chain variable region CDRs. Examples of antibody binding moieties include Fab, fab ', F (ab') 2 Fv, disulfide-linked Fv, scFv, single domain antibodies (dabs), diabodies and single chain antibodies. The terms Fab, F (ab') 2 And Fv is referred to as follows: (i) Fab fragments, i.e., monovalent fragments consisting of the VL, VH, CL and CH1 domains (ii) F (ab') 2 Fragments, i.e., bivalent fragments comprising two Fab fragments linked at the hinge region by disulfide bonds and (iii) Fv fragments consisting of the VL and VH domains of an antibody. Although the two domains of the Fv fragment, namely VL and VH, are encoded by separate coding regions, it is also possible to use synthetic linkers, such as poly G4S amino acid sequences (as disclosed in SEQ ID NO: 165) '(G4S) n', where n=1 to 5), such that they can be made as single protein chains of VL and VH regions combined to form monovalent molecules, known as single chain Fv (ScFv). The term "antigen binding portion" of an antibody is also intended to include such single chain antibodies.
Other forms of single chain antibodies, such as "diabodies" are also included herein. Diabodies are bivalent bispecific antibodies in which VH and VL domains are expressed on a single polypeptide chain, but use a linker connecting the VH and VH domains that is too short to allow the two domains to bind on the same chain, forcing the VH and VL domains to pair with complementary domains of different chains (VL and VHF, respectively) and forming two antigen binding sites (see, e.g., holliger, R et al, (1993) proc. Natl. Acad. Sci. Usa 90:64446448; poljak, R. J et al, (1994) Structure 2:1121-1123).
The immunoglobulin constant region or Fc region is referred to as the heavy chain or light chain constant region. The human heavy and light chain constant region amino acid sequences are well known in the art. The constant region may be of any suitable type and may be selected from the immunoglobulin class IgA, igD, igE, igG and IgM. Several immunoglobulin classes can be further divided into isotypes, e.g. IgGl, igG2, igG3, igG4 or IgAl and IgA2. The heavy chain constant regions (Fc) corresponding to different classes of immunoglobulins may be α, δ, ε, γ and μ, respectively. The light chain may be one of kappa (or kappa) and lambda (or lambda).
In some embodiments, the binding agent lacks an FC region or domain thereof. In some embodiments, the binding agent has an integrated Fc region or Fc domain thereof. In some embodiments, the binding agent has an Fc region or Fc domain of an IgG1 isotype. In some embodiments, the binding agent has an Fc region or Fc domain of an IgG2 isotype. In some embodiments, the binding agent has an Fc region or Fc domain of an IgG3 isotype. In some embodiments, the binding agent has an Fc region or Fc domain of an IgG4 isotype. In some embodiments, the Fc domain may have a hybrid isotype comprising constant regions from two or more isotypes. In some embodiments, the Fc region or Fc domain may be an IgG1 or IgG4 constant region.
In some embodiments, the C-terminus of an Fc domain (e.g., heavy chain) may be the full C-terminus ending with the amino acid residue PGK. In some embodiments, the C-terminus of the Fc domain may also be a truncated C-terminus, wherein one or both C-terminal amino acid residues have been removed. In some embodiments, the C-terminus of the FC domain is truncated and terminates at the C-terminus of PG. In some embodiments, the binding agent comprising a heavy chain comprising a C-terminal CH3 domain comprises a C-terminal glycine-lysine dipeptide (G446 and K447, numbered according to the Kabat EU index). In some embodiments, the binding agent comprising a heavy chain comprising a C-terminal CH3 domain comprises a C-terminal glycine residue (G446, numbered according to the Kabat EU index).
The binding agents as described herein are multispecific, typically bispecific binding agents. In some embodiments, the binding agent is a multispecific antibody or antibody-like molecule, such as a bispecific antibody. Multispecific antibodies are monoclonal antibodies that have binding specificities for at least two different sites or antigens. The binding agents described herein generally have binding specificities for different antigens. Bispecific antibodies can be prepared as full length antibodies or antibody fragments. Bispecific and multispecific antibodies include the following: scFv1-ScFv2, scFv1 2 -Fc-scFv2 2 IgG-scFv, DVD-Ig, trifunctional antibody/tetravalent body tumor, two-in-one IgG, scFv 2 -Fc, tandAb, scFv-HSA-scFv, scFv-VHH, fab-scFv-Fc, fab-VHH-Fc, dAb-IgG, igG-VHH, tandem scFv-Fc, (scFv 1) 2 -Fc-(VHH) 2 scFv-Fc, single arm tandem scFv-Fc and DART-Fc. In some embodiments, the IgG-scFv is an IgG (H) -scFv, scFv- (H) IgG, igG (L) -scFv, svFc- (L) IgG, 2scFv-IgG, or IgG-2scFv (as shown in fig. 1).
Techniques for preparing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs with different specificities (see Milstein and Cuello, nature 305:537 (1983)), WO 93/08829 and Traunecker et al, EMBO J.10:3655 (1991)), and "knob" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be prepared by engineering electrostatic manipulation effects to antibody Fc heterodimer molecules (WO 2009/089004 A1); cross-monoclonal antibodies to two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan et al, science,229:81 (1985)); bispecific antibodies were generated using leucine zippers (see, e.g., kostelny et al, j. Immunol.,148 (5): 1547-1553 (1992)); using "diabody" techniques for the preparation of bispecific antibody fragments (see, e.g., hollinger et al, proc. Natl. Acad. Sci. USA,90:6444-6448 (1993)); and the use of single chain Fv (scFv) dimers (see, e.g., gruber et al, J.Immunol.,152:5368 (1994)); and the preparation of trispecific antibodies (as described, for example, in Tutt et al, J.Immunol.147:60 (1991)).
Also included herein are engineered antibodies having three or more functional antigen binding sites, including "octopus antibodies" (see, e.g., US2006/0025576 A1).
The binding agents (e.g., antibodies or antigen binding fragments) herein also include "double-acting FAb" or "DAF" that comprises antigen binding sites that bind to two different antigens (see, e.g., US2008/0069820 and Bostrom et al, 2009, science 323:1610-14). "Cross mab" antibodies are also included herein (see, e.g., WO 2009/080251, WO 2009/080252, WO2009/080253, WO2009/080254 and WO 2013/026833).
In some embodiments, the binding agent comprises a different antigen binding site fused to one or the other of the two subunits of the Fc domain; thus, two subunits of an Fc domain may be contained in two different polypeptide chains. Recombinant co-expression and subsequent dimerization of these polypeptides results in several possible combinations of the two polypeptides. In order to increase the yield and purity of bispecific molecules in recombinant production, it would therefore be advantageous to introduce modifications in the Fc domain of the binding agent that promote binding of the desired polypeptide.
Thus, in a particular aspect, a binding agent is involved comprising (a) at least a first binding domain, (b) a second binding domain, and (c) an Fc domain consisting of a first and a second subunit capable of stable binding, wherein the Fc domain comprises a modification that facilitates binding of the first and the second subunit of the Fc domain. The site of the most extensive protein-protein interaction between the two subunits of the Fc domain of human IgG is in the CH3 domain of the Fc structure. Thus, in one aspect, the modification is in the CH3 domain of the Fc domain.
In a particular aspect, the Fc modification is a so-called "mortar" modification, including a "mortar" modification of one of the two subunits of the Fc domain and a "mortar" modification of the other of the two subunits of the Fc domain. In one particular aspect, the first subunit of the Fc domain comprises amino acid substitutions S354C and T366W (EU numbering), and the second subunit of the Fc domain comprises amino acid substitutions Y349C, T S and Y407V (numbering according to the Kabat EU numbering).
The pestle and mortar technique is described, for example, in U.S. Pat. nos. 5,731,168;7,695,936; ridgway et al, prot Eng 9,617-621 (1996) and Carter, J Immunol Meth 248,7-15 (2001). Generally, the method includes introducing a protrusion ("slug") at the interface of the first polypeptide and a corresponding cavity ("socket") in the interface of the second polypeptide, such that the protrusion can be positioned in the cavity, thereby promoting heterodimer formation and hindering homodimer formation. The protrusions are constructed by replacing small amino acid side chains from the first polypeptide interface with larger side chains (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensation cavity of the same or similar size as the protuberance is created in the interface of the second polypeptide.
Thus, in some embodiments, in the CH3 domain of the Fc domain, the amino acid residues are replaced with amino acid residues having a larger side chain volume, thereby creating a protrusion within the CH3 domain that is positionable in the cavity within the CH3 domain of the second Fc domain, and in the CH3 domain of the second Fc domain, the amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby creating a cavity within the CH3 domain of the second Fc domain within which the protrusion within the CH3 domain of the first Fc domain is positionable. The protrusions and cavities may be prepared by altering the nucleic acid encoding the polypeptide, for example by site-specific mutagenesis or by peptide synthesis. In a particular embodiment, the threonine residue at position 366 is replaced with a tryptophan residue in the CH3 domain of the first Fc domain (T366W), and the tyrosine residue at position 407 is replaced with a valine residue in the CH3 domain of the second Fc domain (Y407V). In another embodiment, in the second Fc domain, the threonine residue at position 366 is additionally replaced with a serine residue (T366S) and the leucine residue at position 368 is replaced with an alanine residue (L368A).
In yet other aspects, in the first Fc domain, the serine residue at position 354 is additionally replaced with a cysteine residue (S354C), and in the second Fc domain, the tyrosine residue at position 349 is additionally replaced with a cysteine residue (Y349C). The introduction of these two cysteine residues results in the formation of disulfide bridges between the two Fc domains, thereby further stabilizing the dimer (Carter (2001), J Immunol Methods 248,7-15). In some embodiments, the first Fc domain comprises amino acid substitutions S354C and T366W (EU numbering), and the second Fc domain comprises amino acid substitutions Y349C, T S and Y407V (numbering according to the Kabat EU index).
In some embodiments, modifications that promote binding of the first and second Fc domains include modifications that mediate electrostatic manipulation effects, e.g., as described in PCT publication No. WO 2009/089004. Typically, the method involves replacing one or more amino acid residues at the interface of two Fc domains with charged amino acid residues, such that homodimer formation is electrostatically unfavorable, but heterodimer is electrostatically favorable.
In some embodiments, the binding agent comprises one or more scFv or "single chain variable fragment. scFv are fusion proteins of the heavy (VH) and light (VL) variable regions of antibodies, linked to short linker peptides of 10 to about 25 amino acids. The linker is typically glycine-rich for flexibility, serine or threonine-rich for solubility, and may link the N-terminus of VH to the C-terminus of VL, or vice versa. This protein retains the original antibody specificity, although the constant region is removed and a linker is introduced. scFv antibodies are described, for example, in Houston, j.s., methods in enzymol.203 (1991) 46-96. Methods of making scFv molecules and designing suitable peptide linkers are described, for example, in U.S. Pat. nos. 4,704,692; U.S. Pat. nos. 4,946,778; raag and Whitlow, FASEB 9:73-80 (1995), bird and Walker, TIBTECH,9:132-137 (1991).
The binding agent is scFv-Fcs, which has been described by Sokolowska-Wedzina et al mol. Cancer Res.15 (8): 1040-1050, 2017.
In some embodiments, the binding agent is a "dual specific T cell adaptor" or BiTE (see, e.g., WO2004/106381, WO2005/061547, WO2007/042261, and WO 2008/119567). This approach utilizes two antibody variable domains arranged on a single polypeptide. For example, a single polypeptide chain may comprise two single chain Fv (scFv) fragments, each fragment having a Variable Heavy (VH) and a Variable Light (VL) domain separated by a polypeptide linker of sufficient length to allow intramolecular binding between the two domains. The single polypeptide also comprises a polypeptide spacer sequence between the two scFv fragments. Each scFv recognizes different epitopes and these epitopes may be specific for different proteins, such that both proteins are bound by BiTE.
Since it is a single polypeptide, the bispecific T cell adaptors can be expressed using any prokaryotic or eukaryotic cell expression system known in the art, such as CHO cell lines. However, specific purification techniques (see, e.g., EP 1691833) may be necessary to separate monomeric bispecific T cell adaptors from other multimeric species that may have biological activity other than that expected by the monomers. In one exemplary purification scheme, a solution containing the secreted polypeptide is first subjected to metal affinity chromatography and the polypeptide is eluted with an imidazole concentration gradient. The eluate was further purified using anion exchange chromatography and the polypeptide was eluted using a sodium chloride concentration gradient. Finally, the eluate is subjected to size exclusion chromatography to separate monomers from the multimeric species. In some embodiments, the binding agent that is a bispecific antibody consists of a single polypeptide chain comprising two single chain FV fragments (scFV) that are fused to each other by a peptide linker.
Single domain antibodies are antibody fragments consisting of a single monomer variable antibody domain. Single domain antibodies may be derived from the variable domain of the antibody heavy chain of a camelid (e.g. nanobodies or VHH fragments). Furthermore, the term single domain antibody includes the self-owner heavy chain variable domain (aVH) or a VNAR fragment from shark (see, e.g., hasler et al, mol. Immunol.75:28-37,2016).
Techniques for producing single domain antibodies (DAB or VHH) are well known in the art, as disclosed, for example, in Cossins et al, 2006,Prot Express Purif 51:253-259 and Li et al, immunol. Lett.188:89-95,2017. The single domain antibodies may be obtained from camels, alpacas or llamas by standard immunization techniques. (see, e.g., muyledermans et al, TIBS26:230-235,2001; yau et al, J Immunol Methods 281:161-75,2003; and Maass et al, J Immunol Methods 324:324:13-25,2007). VHH may have strong antigen binding capacity and may interact with neoepitopes that are not accessible by conventional VH-VL pairs (see, e.g., muydermans et al, 2001). Alpaca serum IgG contains about 50% of camel-only heavy chain IgG antibodies (hcabs) (see, e.g., maass et al, 2007). Alpaca can be immunized with antigen and VHH that binds to and neutralizes target antigen can be isolated (see, e.g., maass et al, 2007). PCR primers that amplify alpaca VHH coding sequences have been identified and can be used to construct alpaca VHH-phage display libraries, which can be used to isolate antibody fragments by standard biological screening techniques well known in the art (see, e.g., maass et al, 2007).
In some embodiments, the binding agent is an IgG-scFV. IgG-scFv forms include IgG (H) -scFv, scFv- (H) IgG, igG (L) -scFv, svFc- (L) IgG, 2scFV-IgG, and IgG-2scFv. These and other bispecific antibody formats and methods of making the same are described, for example, in Brinkmann and Kontermann, MAbs 9 (2): 182-212 (2017); wang et al, antibodies,2019,8,43; dong et al 2011, MAbs 3:273-88; natsume et al, J.biochem.140 (3): 359-368,2006; cheal et al mol.cancer Ther.13 (7): 1803-1812,2014; and Bates and Power, antibodies,2019,8,28.
Igg-like double variable domain antibodies (DVD-Ig) have been described by Wu et al, 2007,Nat Biotechnol 25:1290-97; hasler et al, mol.Immunol.75:28-37,2016, WO 08/024188 and WO 07/024715. Trifunctional antibodies have been described by Chelius et al, MAbs 2 (3): 309-319, 2010. Two-in-one IgG has been described by Kontermann et al, drug Discovery Today 20 (7): 838-847, 2015. The Tanden antibody or TandAb has been described by Kontermann et al (supra). ScFv-HSA-scFv has been described by Kontermann et al (supra).
In some embodiments, the binding agent is a scaffold antigen binding protein, such as, for example, fibronectin and engineered ankyrin repeat protein (DARPin), which has been used as a surrogate scaffold for antigen binding domains, see, for example, gebauer and Skerra, engineered protein scaffolds as next-generation antibody therapeutics, curr Opin Chem Biol 13:245-255 (2009), and Stumpp et al, darpins: A new generation of protein therapeutics, drug Discovery Today 13:695-701 (2008). In some embodiments, the scaffold antigen binding protein is selected from the following: lipocalins (Anticalin) molecules of protein A origin, such as the Z domain of protein A (affibody), the A domain (Avimer/Maxibody), serum transferrin (transformant); designed ankyrin repeat protein (DARPin), fibronectin (AdNectin), C-lectin domain (Tetranectin); a variable domain of the neoantigen receptor beta lactamase (VNAR fragment), a human gamma crystallin or ubiquitin (Affilin molecule); kunitz-type domains of human protease inhibitors and micro-entities such as proteins from the knotin family, peptide aptamers and fibronectin.
Lipocalin is a family of extracellular proteins that transport small hydrophobic molecules such as steroids, bile, retinoids, and lipids. It has a rigid beta sheet secondary structure with multiple loops at the open end of the conical structure, which loops can be designed to bind different target antigens. The size of the Anticalin is between 160 and 180 amino acids and is derived from lipocalin. For more details, see Biochim Biophys Acta 1482:337-350 (2000), U.S. Pat. No. 7,250,297B1 and U.S. Pat. No. 3, 20070224633.
The designed ankyrin repeat protein (DARPin) is derived from ankyrin, a family of proteins that mediate the attachment of integral membrane proteins to the cytoskeleton. A single ankyrin repeat is a 33-residue motif, consisting of two alpha helices and one beta turn. Which can bind different target antigens by randomizing residues in the first alpha helix and beta turn of each repeat. Its binding interface can be increased by increasing the number of modules (a method of affinity maturation). For more details, see J.mol.biol.332,489-503 (2003), PNAS100 (4), 1700-1705 (2003) and J.mol.biol.369,1015-1028 (2007), and US20040132028A1.
Modification of Fc domains to alter effector function
In some embodiments, the Fc region or Fc domain does not substantially bind to at least one Fc receptor selected from the group consisting of: fcyRI (CD 64), fcyRIIA (CD 32 a), fcyRIIB (CD 32 b), fcyRIIIA (CD 16 a) and FcyRIIIB (CD 16 b). In some embodiments, the Fc region or Fc domain exhibits substantially no binding to any Fc receptor selected from the group consisting of: fcyRI (CD 64), fcyRIIA (CD 32 a), fcyRIIB (CD 32 b), fcyRIIIA (CD 16 a) and FcyRIIIB (CD 16 b). As used herein, "substantially unbound" refers to weak or no binding to a selected fcγ receptor. In some embodiments, "substantially not bind" refers to a decrease in binding affinity for fcγ receptor of at least 1000-fold (e.g., an increase in Kd). In some embodiments, the Fc receptor or region is Fc null. As used herein, "Fc free" refers to an Fc region or Fc domain that exhibits weak or no binding to any fcγ receptor. In some embodiments, the binding affinity of the Fc domain or region for the fcγ receptor is reduced (e.g., kd increased) by at least a factor of 1000.
In some embodiments, the Fc receptor has reduced or substantially no effector function activity. As used herein, "effector function activity" refers to antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent macrophage phagocytosis (ADCP), and/or complement-dependent cytotoxicity (CDC). In some embodiments, the Fc domain exhibits reduced ADCC, ADCP or CDC activity compared to the wild-type Fc domain. In some embodiments, the Fc domain exhibits reduced ADCC, ADCP and CDC as compared to the wild-type Fc domain. In some embodiments, the Fc domain exhibits substantially no effector function (i.e., the ability to stimulate ADCC, ADCP, or CDC). As used herein, "substantially null effector function" refers to a reduction in effector function activity by at least 1000-fold as compared to a wild-type Fc domain.
In some embodiments, the Fc domain has reduced or no ADCC activity. As used herein, reduced or no ADCC activity refers to at least a 10-fold, at least a 20-fold, at least a 30-fold, at least a 50-fold, at least a 100-fold, or at least a 500-fold reduction in ADCC activity of an Fc domain.
In some embodiments, the Fc domain has reduced or no CDC activity. As used herein, reduced or absent CDC activity refers to at least a 10-fold, at least a 20-fold, at least a 30-fold, at least a 50-fold, at least a 100-fold, or at least a 500-fold reduction in CDC activity of an Fc domain.
In vitro and/or in vivo cytotoxicity assays may be performed to confirm a reduction/depletion of ADCC and/or CDC activity. For example, an Fc receptor (FcR) binding assay may be performed to ensure that the antibody lacks fcγ receptors (and thus may lack ADCC activity). Primary cells mediating ADCC NK cells express fcyriii only, whereas monocytes express fcyri, fcyrii and fcyriii. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, annu. Rev. Immunol.9:457-492 (1991). Non-limiting examples of in vivo assays for assessing ADCC activity of a target molecule are described in U.S. Pat. No. 5,500,362 (see, e.g., hellstrom, I. Et al, proc. Nat 'l Acad. Sci. USA 83:7059-7063 (1986) and Hellstrom, I. Et al, proc. Nat' l Acad. Sci. USA 82:1499-1502 (1985)); U.S. Pat. No. 5,821,337 (see Bruggemann, M. Et al, J. Exp. Med.166:1351-1361 (1987)). Alternatively, non-radioactive assay methods (see, e.g., ACTI TM Non-radioactive cytotoxicity assays for flow cytometry (CellTechnology, inc.Mountain View, calif.); and CytoTox96 TM Non-radioactive cytotoxicity assay (Promega, madison, wis.). Useful effector cells for such assays include Peripheral Blood Mononuclear Cells (PBMC) and natural killer cells (NK). Alternatively or additionally, ADCC activity of the target molecule may be assessed in vivo, for example, in an animal model, as disclosed in Clynes et al, proc.Nat' l Acad.Sci.USA 95:652-656 (1998).
A C1q binding assay may also be performed to confirm that the antibody or Fc domain or Fc region is unable to bind C1q and therefore lacks CDC activity or has reduced CDC activity. See, e.g., C1q and C3C binding ELISA in WO 2006/029879 and WO 2005/100402. To assess complement activation, CDC assays can be performed (see, e.g., gazzano-Santoro et al, J.Immunol. Methods 202:163 (1996); cragg, M.S. et al, blood 101:1045-1052 (2003); and Cragg, M.S. and M.J. Glennie, blood 103:2738-2743 (2004)).
In some embodiments, the Fc domain has reduced or no ADCP activity. As used herein, reduced or no ADCP activity refers to at least a 10-fold, at least a 20-fold, at least a 30-fold, at least a 50-fold, at least a 100-fold, or at least a 500-fold reduction in ADCP activity of an Fc domain.
ADCP binding assays may also be performed to confirm that the antibodies or Fc domains or Fc regions lack ADCP activity or have reduced ADCP activity. See, for example, US20190079077 and US20190048078 and references disclosed therein.
Antibodies with reduced effector function activity include antibodies with substitutions of one or more of Fc region residues 238, 265, 269, 270, 297, 327 and 329 (see U.S. Pat. No. 6,737,056). Such Fc mutants include Fc mutants having substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including so-called "DANA" Fc mutants in which residues 265 and 297 are replaced with alanine (see U.S. Pat. No. 7,332,581). Certain antibody variants that have reduced binding to FcR are also known. ( See, for example, U.S. Pat. nos. 6,737,056; WO 2004/056312, and Shields et al J.biol.chem.9 (2): 6591-6604 (2001). )
In certain embodiments, the binding agent comprises an Fc domain or Fc region having one or more amino acid substitutions that reduce fcγr binding, e.g., substitutions at positions 234 and 235 of the Fc region (EU numbering of residues). In some embodiments, the substitutions are L234A and L235A (LALA). In some embodiments, the Fc domain further comprises D265A and/or P329G in an Fc region derived from a human IgG1-Fc region. In some embodiments, the substitutions are L234A, L235A and P329G (LALA-PG) in the Fc region derived from a human IgG1-Fc region. (see, e.g., WO 2012/130831). In some embodiments, the substitutions are L234A, L235A and D265A (LALA-DA) in the Fc region derived from a human IgG1-Fc region.
In some embodiments, the Fc region is altered, resulting in altered (i.e., or reduced) C1q binding and/or Complement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat. No. 6,194,551, WO 99/51642 and Idusogie et al, J.Immunol.164:4178-4184 (2000).
Modification of binding domains
In some embodiments, the binding domain may be modified by one or more conservative substitutions. For conservative amino acid substitutions, a given amino acid may be substituted by residues having similar physicochemical characteristics, e.g., one aliphatic residue for another (e.g., ile, val, leu or Ala for each other), or one polar residue for another (e.g., between Lys and Arg, between Glu and Asp, or between Gln and Asn). Other such conservative amino acid substitutions, for example, substitutions of the entire region with similar hydrophobic characteristics, are well known. Polypeptides comprising conservative amino acid substitutions may be tested in any of the assays described herein to confirm that the desired activity, e.g., antigen binding activity and specificity of the native or reference polypeptide, is retained.
For conservative substitutions, amino acid groupings can be made according to the similarity of their side chain properties (A.L. Lehninger, in Biochemistry, 2 nd edition, pp.73-75,Worth Publishers,New York (1975)): (1) nonpolar: ala (A), val (V), leu (L), ile (I), pro (P), phe (F), trp (W), met (M); (2) uncharged polarity: gly (G), ser (S), thr (T), cys (C), tyr (Y), asn (N), gln (Q); (3) acidity: asp (D), glu (E); and (4) alkaline: lys (K), arg (R), his (H).
Alternatively, for conservative substitutions, naturally occurring residues may be divided into groups based on common side chain properties: (1) hydrophobicity: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilicity: cys, ser, thr, asn, gln; (3) acidity: asp, glu; (4) alkaline: his, lys, arg; (5) residues affecting the chain direction: gly, pro; and (6) aromatic: trp, tyr, phe. Non-conservative substitutions will require the exchange of members of one or the other class.
Specific conservative substitutions include, for example: ala to Gly or to Ser; arg to Lys; asn to Gln or to His; asp to Glu; cys to Ser; gln to Asn; glu to Asp; gly to Ala or to Pro; his to Asn or to Gln; lie to Leu or to Val; leu to Ile or to Val; lys to Arg, to gin, or to Glu; met to Leu, to Tyr or to Ile; phe to Met, to Leu, or to Tyr; ser to Thr; thr to Ser; trp to Tyr; tyr to Trp; and/or Phe to Val, to Ile or to Leu.
In some embodiments, conservatively modified variants of the binding domain are preferably 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 more identical to the reference VH or VL sequence, where the VH and VL CDRs are unmodified. The degree of homology (percent identity) between a reference sequence and a modified sequence can be determined by comparing the two sequences, for example, using a freely available computer program on the world wide web (e.g., BLASTp or BLASTn with default settings) typically used for this purpose.
Modification of the native (or reference) amino acid sequence may be accomplished by any of a variety of techniques well known to those skilled in the art. For example, mutations can be introduced at specific sites by synthesizing oligonucleotides containing the desired mutant sequences, which are flanked by restriction sites that can be ligated to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes a variant with the desired amino acid insertion, substitution or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide nucleotide sequences with specific codon changes that vary according to the desired substitution, deletion or insertion. Techniques for making such changes are well established and include, for example, those described by Walder et al (Gene 42:133, 1986); bauer et al (Gene 37:73, 1985); craik (BioTechniques, january 1985,12-19); smith et al (Genetic Engineering: principles and Methods, plenum Press, 1981); and those disclosed in U.S. patent nos. 4,518,584 and 4,737,462, the entire contents of which are incorporated herein by reference.
Cd8+kir+t regulatory cells
Regulatory T cells are characterized by a cd8+kir+ phenotype and are typically MHC class I-restricted. In humans, cd8+kir+t regulatory cells express inhibitory KIR proteins. In some embodiments, KIR proteins expressed by cells may include one or more inhibitory KIR proteins, e.g., KIR2DL1, KIR2DL2, KIR2DL3, KIR2DL5, KIR3DL1, and KIR3DL2; and may specifically include one or more of KIR2DL2, KIR2DL3, and KIR3DL 1. In some embodiments, the CD8+KIR+T regulatory cells are not HLA E (Qa-1 b) restricted. (see, e.g., lohwasser et al International Immunology 13:321-327 (2001) and Sarantopoulos et al J.Clin. Invest.114 (9): 1218-1221 (2004), general explanation of mouse Qa-1b and human HLA E restrictions). In some embodiments, cd8+kir+t regulatory cells may also be characterized as cd44+, cd122+, rather than HLA E (Qa-1 b) restricted. In some embodiments, cd8+kir+t regulatory cells may also be characterized as CD28-. In some embodiments, cd8+kir+t-modulating cells may also be characterized as cd28-, cd44+ and cd122+. In some embodiments, cd8+kir+t regulatory cells may also be characterized as CD28-, cd44+ and cd122+, rather than HLA E (Qa-1 b) restricted.
In some embodiments, the cd8+kir+ tregs express the following antigens: CD3, CD8, PD-1, CD16, CD122, CD39, CXCR3, ICOS, CD103 and inhibitory KIR proteins.
In some embodiments, the cd8+kir+ tregs express one or more of the following antigens: CD3, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4 and 41BB. In some embodiments, the cd8+kir+ tregs express one or more of the following antigens: CD3, CD5, CD16, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, KLRB1, KLRG1, LAG-3/CD223, NKG2C, NKG2D, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4 and 41BB. In some embodiments, the cd8+kir+ tregs express one or more of the following antigens: CD39, KLRB1, KLRG1, NKG2C, NKG2D, CXCR and CD122.
Production of binding agent
In various embodiments, the binding agent may be produced by a cell line of human, mouse, or other animal origin. Recombinant DNA expression can be used to produce binding agents. This allows for the production of antibodies as well as a range of antigen binding portions and other binding agents (including fusion proteins) in the host species of choice. The production of antibodies, antigen binding portions thereof and other binding agents in bacteria, yeast, transgenic animals and eggs is also an alternative to cell-based production systems. The main advantage of transgenic animals is the potentially high yield of renewable resources.
As used herein, the term "nucleic acid" or "nucleic acid sequence", "polynucleotide sequence" or "nucleotide" refers to a polymer molecule comprising ribonucleic acid, deoxyribonucleic acid, or analog units thereof. The nucleic acid may be single-stranded or double-stranded. The single-stranded nucleic acid may be a single-stranded nucleic acid of denatured double-stranded DNA. In some embodiments, the nucleic acid may be a cDNA, e.g., a nucleic acid lacking introns.
Nucleic acid molecules encoding the amino acid sequences of antibodies, or antigen binding portions thereof, as well as other binding agents, can be prepared by various methods well known in the art. These methods include, but are not limited to, preparing synthetic nucleotide sequences encoding antibodies, antigen binding portions, or other binding agents. In addition, oligonucleotide-mediated (or site-directed) mutagenesis, PCR-mediated mutagenesis, and cassette mutagenesis can be used to prepare nucleotide sequences encoding antibodies or antigen binding portions, as well as other binding agents. As described herein, the nucleic acid sequence encoding at least an antibody, antigen binding portion thereof, binding agent or polypeptide thereof, may be recombined with the vector DNA according to conventional techniques, e.g., blunt-ended or staggered-ended ends for ligation, restriction endonuclease digestion to provide suitable ends, appropriate filling of cohesive ends, alkaline phosphatase treatment to avoid undesired ligation, and ligation with an appropriate ligase. Techniques for such manipulation are disclosed, for example, maniatis et al, molecular Cloning, lab. Manual (Cold Spring Harbor Lab. Press, N.Y., 1982and 1989), and Ausubel et al, current Protocols in Molecular Biology (John Wiley & Sons), 1987-1993, and may be used to construct nucleic acid sequences and vectors encoding antibodies or antigen-binding portions thereof or VH and/or VL polypeptides thereof. Where the binding agent comprises an antibody or antigen-binding portion thereof, in some embodiments, the VH polypeptide is encoded by the first nucleic acid. In some embodiments, the VL polypeptide is encoded by a second nucleic acid. In some embodiments, the VH and VL polypeptides are encoded by one nucleic acid.
A nucleic acid molecule (e.g., DNA) is said to be "capable of expressing" a polypeptide if it comprises nucleotide sequences that contain transcriptional and translational regulatory information, and such sequences are "operably linked" to the nucleotide sequence encoding the polypeptide. An operable linkage is one in which the regulatory DNA sequence and the expression DNA sequence (e.g., antibody or antigen binding portion thereof) are sought to be linked in a manner that allows expression of the polypeptide or antigen binding portion in recoverable amounts of the gene. The precise nature of the regulatory regions required for gene expression may vary from organism to organism and is well known in the art. See, e.g., sambrook et al, 1989; ausubel et al, 1987-1993.
Thus, expression of an antibody or antigen binding portion thereof or other binding agent described herein may occur in a prokaryotic cell or eukaryotic cell. Suitable hosts include bacterial or eukaryotic hosts, including yeast, insect, fungal, avian and mammalian cells, in vivo or in situ, or host cells of mammalian, insect, avian or yeast origin. The mammalian cells or tissues may be of human, primate, hamster, rabbit, rodent, bovine, porcine, ovine, equine, caprine, canine or feline origin, although any other mammalian cell may be used. Furthermore, in vivo synthesis of ubiquitin transmembrane polypeptide fusion proteins can be achieved by using e.g. a yeast ubiquitin hydrolase system. The fusion proteins so produced may be processed in vivo or purified and processed in vitro, allowing the synthesis of antibodies or antigen binding portions thereof having specific amino terminal sequences as described herein. Furthermore, problems associated with retention of the initial codon-derived methionine residue in yeast (or bacterial) direct expression can be avoided. (see, e.g., sabin et al, 7 Bio/technology.705 (1989); miller et al, 7 Bio/technology.698 (1989)). When the yeast is grown in a glucose-rich medium, any of a series of yeast gene expression systems comprising a promoter and termination elements from an active expression gene encoding a high-yield glycolytic enzyme can be used to obtain the recombinant antibody or antigen-binding portion thereof or other binding agent. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, the promoter and terminator signals of phosphoglycerate kinase gene may be utilized.
The production of antibodies or antigen binding portions thereof and other binding agents in insects can be accomplished, for example, by infecting an insect host with baculovirus engineered to express polypeptides by methods well known to those of ordinary skill in the art. See Ausubel et al, 1987-1993.
In some embodiments, the introduced nucleic acid sequence (encoding an antibody or antigen binding portion thereof or polypeptide thereof or other binding agent) is incorporated into a plasmid or viral vector capable of autonomous replication in a recipient host cell. Any of a variety of carriers may be used for this purpose and are well known and available to those of ordinary skill in the art. See, e.g., ausubel et al, 1987-1993. Important factors in selecting a particular plasmid or viral vector include: the recipient cells containing the vector can be readily identified and selected from those that do not; the number of vector copies required in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
Exemplary prokaryotic vectors known in the art include plasmids, such as those capable of replication in E.coli. Other gene expression elements and other binding agents that may be used to express antibodies or antigen binding portions thereof encoding DNA include, but are not limited to, (a) viral transcription promoters and their enhancer elements, such as the SV40 early enhancer (Okayama et al, 3mol. Cell. Biol.280 (1983)), the Rous sarcoma virus LTR (Gorman et al, 79pnas 6777 (1982)) and the moloney mouse leukemia virus LTR (gronschel et al, 41cell 885 (1985)); (b) Splice and polyadenylation sites such as those derived from the late region of SV40 (Okayarea et al, 1983) and (c) polyadenylation sites such as in SV40 (Okayama et al, 1983). Immunoglobulin encoding DNA genes can be expressed as described by Liu et al, supra, and Weidle et al, 51Gene 21 (1987), using the SV40 early promoter and its enhancer, the mouse immunoglobulin H chain promoter enhancer, SV40 late region mRNA splicing, rabbit S-globulin intervening sequences, immunoglobulin and rabbit S-globulin polyadenylation sites, and SV40 polyadenylation elements as expression elements.
For nucleotide sequences encoding immunoglobulins, the transcriptional promoter may be, for example, human cytomegalovirus, and the promoter enhancers may be cytomegalovirus and mouse/human immunoglobulins.
In some embodiments, for expression of the DNA coding region in a rodent cell, the transcriptional promoter may be a viral LTR sequence, the transcriptional promoter enhancer may be one or both of a mouse immunoglobulin heavy chain enhancer and a viral LTR enhancer, and polyadenylation and transcription termination regions. In other embodiments, DNA sequences encoding other proteins are combined with the above-described expression elements to achieve expression of the proteins in mammalian cells.
Each coding region or gene fusion is assembled in or inserted into an expression vector. The recipient cell capable of expressing the variable region or antigen-binding portion thereof is then transfected with the nucleotide encoding the antibody or antibody polypeptide or antigen-binding portion thereof alone or cotransfected with the polynucleotide encoding the VH and VL chain encoding regions. Transfected recipient cells are cultured under conditions that allow expression of the bound coding region, and the expressed antibody chain or intact antibody or antigen binding portion is recovered from the culture.
In some embodiments, nucleic acids comprising a coding region encoding an antibody or antigen-binding portion thereof are assembled in an isolated expression vector and then used to cotransfect a recipient host cell. Each vector may contain one or more selectable genes. For example, in some embodiments, two selectable genes are used, the first being a selectable gene designed for selection in bacterial systems and the second being a selectable gene designed for selection in eukaryotic systems, wherein each vector has a set of coding regions. Vectors generated by this strategy first direct the production of nucleotide sequences in bacterial systems and allow for amplification. The DNA vectors so produced and amplified in a bacterial host are then used to co-transfect eukaryotic cells and allow selection of co-transfected cells carrying the desired transfected nucleic acids (e.g., encoding antibody heavy and light chains). Non-limiting examples of selectable genes for bacterial systems are genes conferring resistance to ampicillin and genes conferring resistance to chloramphenicol. Alternative genes for eukaryotic transfectants include the xanthine-guanine phosphoribosyl transferase gene (designated gpt) and the phosphotransferase gene from Tn5 (designated neo). Alternatively, the fusion nucleotide sequences encoding the VH and VL chains may be assembled on the same expression vector.
For transfection of expression vectors and production of antibodies or antigen binding portions thereof or other binding agents, the recipient cell line may be a chinese hamster ovary cell line (e.g., DG 44) or myeloma cells. Myeloma cells can synthesize, assemble and secrete immunoglobulins encoded by transfected immunoglobulin genes and have mechanisms for immunoglobulin glycosylation. For example, in some embodiments, the recipient cell is an Ig-producing recombinant myeloma cell SP2/0.SP2/0 cells produce only immunoglobulins encoded by the transfected gene. Myeloma cells can be grown in culture or in the abdominal cavity of mice, where secreted immunoglobulins can be obtained from the ascites fluid.
The expression vector or other binding agent encoding the antibody or antigen-binding portion thereof may be introduced into a suitable host cell by any of a number of suitable means, including biochemical means such as transformation, transfection, protoplast fusion, calcium phosphate precipitation, application of polycations such as Diethylaminoethyl (DEAE) dextran, and mechanical means such as electroporation, direct microinjection, and microinjection bombardment. Johnston et al, 240Science 1538 (1988), as known to those of ordinary skill in the art.
Yeast has certain advantages over bacteria in the production of immunoglobulin heavy and light chains. Yeast undergo post-translational peptide modifications, including glycosylation. There are many recombinant DNA strategies that utilize strong promoter sequences and high copy number plasmids, which can be used to produce the desired protein in yeast. Yeast recognizes the leader sequence of the cloned mammalian gene product and secretes a polypeptide (i.e., a pre-polypeptide) carrying the leader sequence. See, e.g., hitzman et al, 11th Intl.Conf.Yeast,Genetics&Molec.Biol (montalier, france, 1982).
Yeast gene expression systems can routinely assess the level of production, secretion and stability of antibodies, assembled antibodies, and antigen-binding portions thereof. When yeast are grown in glucose-rich media, a variety of yeast gene expression systems can be utilized that integrate promoter and termination elements from active expression genes encoding high-volume produced glycolytic enzymes. Known glycolytic genes can also provide very efficient transcriptional control signals. For example, the promoter and terminator signals of phosphoglycerate kinase (PGK) gene may be utilized. Another example is the translational elongation factor 1 alpha promoter. A variety of methods can be used to assess the optimal expression plasmid for immunoglobulin expression in yeast. See IIDNA Cloning 45 (Glover et al, IRL Press, 1985) and, for example, U.S. patent publication No. US2006/0270045 A1.
Bacterial strains can also be used as hosts for the production of antibody molecules or antigen-binding portions thereof or other binding agents described herein. Coli K12 strains such as E.coli W3110, bacillus species, E.coli such as Salmonella typhimurium (Salmonella typhimurium) or Serratia marcescens (Serratia marcescens), and various Pseudomonas species may be used. Plasmid vectors containing replicon and control sequences derived from species compatible with the host cells are used in conjunction with these bacterial hosts. The vector carries a replication site and a specific gene capable of providing phenotypic selection in transformed cells. Various methods can be employed to evaluate expression plasmids for the production of antibodies and antigen-binding portions thereof in bacteria (see Glover,1985; ausubel,1987,1993; sambrook,1989; colligan, 1992-1996).
The host mammalian cells may be cultured in vitro or in vivo. Mammalian cells provide post-translational modifications of immunoglobulin molecules, including removal of leader peptides, folding and assembly of VH and VL chains, glycosylation of antibody molecules, and secretion of functional antibodies and/or antigen-binding portions thereof.
In addition to the above-described lymphoid-derived cells, mammalian cells that can be used as hosts for the production of antibody proteins include fibroblasts-derived cells, such as Vero or CHO-K1 cells. Exemplary eukaryotic cells that may be used to express an immunoglobulin polypeptide include, but are not limited to, COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, CHO-K1 and DG44 cells; PERC6 TM Cells (Crucell); and NSO cells. In some embodiments of the present invention, in some embodiments,the particular eukaryotic host cell is selected based on its ability to make the desired post-translational modification of the heavy and/or light chain. For example, in some embodiments, CHO cells produce polypeptides having a higher level of sialylation than the same polypeptide produced in 293 cells.
In some embodiments, one or more antibodies or antigen binding portions thereof or other binding agents may be produced according to any suitable method in an animal that has been engineered or transfected with one or more nucleic acid molecules encoding the polypeptide.
In some embodiments, the antibody or antigen binding portion thereof is produced in a cell-free system. Non-limiting exemplary cell-free systems have been described, for example, sitaraman et al, methods mol. Biol.498:229-44 (2009); spirin, trends Biotechnol.22:538-45 (2004); and Endo et al, biotechnol. Adv.21:695-713 (2003).
A number of vector systems are available for expression of VH and VL chains in mammalian cells (see Glover, 1985). Various methods can be employed to obtain intact antibodies. As described above, VH and VL chains, and optionally related constant regions, can be co-expressed in the same cell to achieve intracellular binding and ligation of VH and VL chains into an intact tetrameric H2L2 antibody or antigen-binding portion thereof. Co-expression can occur by using the same or different plasmids in the same host. The nucleic acid encoding the VH and VL chains, or antigen binding portions thereof, may be placed into the same plasmid and then transfected into cells, thereby directly selecting cells expressing both chains. Alternatively, the cells may be transfected first with a plasmid encoding one strand (e.g., the VL chain), and then the resulting cell line transfected with a VH chain plasmid containing a second selectable marker. Cell lines that produce antibodies, antigen-binding portions thereof, or other binding agents by either route can be transfected with plasmids encoding peptides, VH, VL, or other copies of VH plus VL chains, along with other selectable markers, to produce cell lines with enhanced properties, such as higher yields of assembled antibodies or antigen-binding portions thereof, or enhanced stability of transfected cell lines.
In addition, plants have become an alternative expression system for convenient, safe and economical production of recombinant antibodies, which is based on large-scale cultivation of microorganisms or animal cells. The antibody or antigen binding portion may be expressed in plant cell culture or in conventionally grown plants. Expression in plants may be systemic, restricted to subcellular plastids, or to seeds (endosperm). See, for example, U.S. patent publication No. 2003/0167531; U.S. patent No. 6,080,560; U.S. patent No. 6,512,162; and WO 0129242. Several plant-derived antibodies have entered advanced stages of development, including clinical trials (see, e.g., biolex, n.c.).
For an intact antibody, the variable regions (VH and VL) of the antibody are typically linked to at least a portion of an immunoglobulin constant region (Fc), typically a constant region of a human immunoglobulin. The human constant region DNA sequence may be isolated from a variety of human cells according to well known procedures, such as immortalized B cells (see, e.g., WO 87/02671; incorporated herein by reference in its entirety). Antibodies may comprise both light and heavy chain constant regions. The heavy chain constant region may comprise CH1, hinge, CH2, CH3 and sometimes CH4 regions. In some embodiments, the CH2 domain may be deleted or omitted.
Alternatively, the techniques described for producing single chain antibodies (see, e.g., U.S. Pat. No. 4,946,778;Bird,Science 242:423-42 (1988); huston et al, proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); ward et al, nature 334:544-54 (1989); the entire contents of which are incorporated herein by reference) may be adapted to produce single chain antibodies that specifically bind to a desired antigen. Single chain antibodies are formed by linking the heavy and light chain variable regions of the Fv region via an amino acid bridge, thereby producing a single chain polypeptide. Techniques for assembling functional Fv fragments in E.coli can also be used (see, e.g., skerra et al, science242:1038-1041 (1988); the entire contents of which are incorporated herein by reference). The method of preparing the other binders is as described above.
The intact (e.g., intact) antibodies, dimers thereof, individual light and heavy chains, or antigen-binding portions thereof, can be recovered and purified by known techniques, such as immunoadsorption or immunoaffinity chromatography, chromatographic methods such as HPLC (high performance liquid chromatography), ammonium sulfate precipitation, gel electrophoresis, or any combination of these. See generally, scens, protein Purification (Springer-Verlag, n.y., 1982). Substantially pure antibodies or antigen binding portions thereof having at least about 90% to 95% homology are advantageous, as are antibodies or antigen binding portions having 98% to 99% or more homology, particularly for pharmaceutical use. Once purified, the whole antibody or antigen binding portion thereof may be used in therapy or for developing and performing assay procedures, immunofluorescent staining, and the like, as desired, in part or in homogeneity. See generally vols.i & II immunol.meth. (profkovits & Pernis, acad.press, NY,1979and 1981).
Pharmaceutical preparation
In some aspects, the binding agent relates to a composition comprising an active ingredient (i.e., comprising a binding agent described herein or a nucleic acid encoding an antibody or antigen-binding portion thereof or other binding agent described herein). In some embodiments, the composition is a pharmaceutical composition. As used herein, the term "pharmaceutical composition" refers to a combination of an active agent and a pharmaceutically acceptable carrier for use in the pharmaceutical industry. The term "pharmaceutically acceptable" refers herein to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
The preparation of pharmacological compositions containing the active ingredient dissolved or dispersed therein is well known in the art and need not be limited based on any particular formulation. Typically, such compositions are prepared as injectable liquid solutions or suspensions; however, solid forms or suspensions suitable for rehydration in a liquid prior to use may also be prepared. The formulation may also be emulsified or presented as a liposome composition. The antibody or antigen-binding portion thereof or other binding agent may be admixed with excipients that are pharmaceutically acceptable and compatible with the active ingredients and in amounts suitable for use in the methods of treatment described herein. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol and the like, and combinations thereof. In addition, if desired, the pharmaceutical compositions may contain minor amounts of auxiliary substances, such as wetting or emulsifying agents, pH buffering agents, and the like, which enhance or maintain the effectiveness of the active ingredient (e.g., an antibody or antigen-binding portion thereof, or other binding agent). The pharmaceutical compositions described herein may include pharmaceutically acceptable salts of the components thereof. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of the polypeptide) with inorganic acids such as hydrochloric or phosphoric acids, or with organic acids such as acetic, tartaric, mandelic, and the like. Salts with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides, and organic bases such as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. Physiologically tolerable carriers are well known in the art. Exemplary liquid carriers are sterile aqueous solutions containing the active ingredient (e.g., antibody and/or antigen binding portion thereof or other binding agent) and water, and may contain buffers such as sodium phosphate at physiological pH, physiological saline, or both, such as phosphate buffered saline. Still further, the aqueous carrier may contain more than one buffer salt, as well as salts such as sodium and potassium chloride, dextrose, polyethylene glycol, and other solutes. The liquid composition may also comprise a liquid phase in addition to water. Examples of such other liquid phases are glycerol, vegetable oils such as cottonseed oil and water-oil emulsions. The amount of active agent effective in treating a particular disorder or condition will depend on the nature of the disorder or condition and can be determined by standard clinical techniques.
In some embodiments, a pharmaceutical composition comprising an antibody or antigen-binding portion thereof or other binding agent or nucleic acid encoding an antibody or antibody-binding portion thereof or antigen thereof or other binding agent as described herein may be a lyophilizate.
In some embodiments, a syringe comprising a therapeutically effective amount of a binding agent or pharmaceutical composition described herein is provided.
Treatment of inflammatory and autoimmune diseases
In some aspects, the binding agents described herein are useful in one or more methods comprising administering to a subject having an inflammatory disease a binding agent or pharmaceutical composition described herein. In some aspects, the binding agents described herein are useful in one or more methods comprising administering to a subject suffering from or having an immune response to an autoantigen or to an antigen that elicits or results in the onset of an autoimmune disorder a binding agent or pharmaceutical composition described herein (which may be collectively referred to herein as an autoimmune disease). In some embodiments, the subject is in need of treatment for an autoimmune disease. In some embodiments, methods of treating an autoimmune disease are provided, comprising administering to a subject in need thereof any of the binding agents described herein or the pharmaceutical compositions described herein in an amount effective to reduce the number or activity of pathogenic immune cells in the subject, thereby ameliorating a symptom of an autoimmune disease. In some embodiments, methods for inhibiting an immune response mediated by pathogenic immune cells are provided, comprising contacting cd8+ kir+ T regulatory cells (tregs) with an amount of any binding agent described herein or a pharmaceutical composition described herein effective to activate or stimulate cd8+ kir+ tregs (activated tregs), thereby reducing the number or activity of pathogenic immune cells. In some embodiments, methods for inhibiting an immune response to an autoantigen are provided, comprising administering to a subject in need thereof any of the binding agents described herein or the pharmaceutical compositions described herein in an amount effective to activate or stimulate cd8+kir+ tregs, thereby reducing the number or activity of pathogenic immune cells that respond to an autoantigen. In some embodiments, methods are provided for preventing an immune response to an autoantigen that causes or results in the onset of an autoimmune disease, comprising administering any of the binding agents described herein or the pharmaceutical compositions described herein to a subject in need thereof in an amount effective to activate or stimulate cd8+kir+ tregs, thereby reducing the number or activity of pathogenic immune cells or the titer of autoantibodies in the subject to respond to the autoantigen. In some embodiments, methods are provided for inhibiting an immune response to an autoantigen that causes or results in the onset of an autoimmune disease, comprising administering any of the binding agents described herein or the pharmaceutical compositions described herein to a subject in need thereof in an amount effective to activate or stimulate cd8+kir+ tregs, thereby reducing the number or activity of pathogenic immune cells or the titer of autoantibodies in the subject to respond to the autoantigen. In some embodiments, there is provided a method for inhibiting a response to an antigen or autoantigen that causes or results in the onset of an autoimmune disorder, comprising administering to a subject in need thereof any of the binding agents described herein or the pharmaceutical compositions described herein in an amount effective to activate or stimulate cd8+kir+ tregs, thereby reducing or inhibiting an immune response to the antigen or autoantigen in the subject.
As used herein, the term "autoantigen" refers to an antigen (e.g., a cell surface protein or other antigen) that is generally recognized as self by the immune system (e.g., a healthy human immune system). In autoimmune diseases or conditions, antigens or autoantigens become targets for humoral or cell-mediated immune responses, or trigger an excessive immune response (collectively, "autoimmune diseases"). As used herein, the terms "activate or stimulate" cd8+kir+ Treg, or activated cd8+kir+ Treg, refer to an increase in regulatory T cell function of these cells, such as the ability to suppress an immune response, particularly to an autoantigen, or to suppress an immune response to an autoantigen that causes or leads to the onset of an autoimmune disease. Activation or stimulation of cd8+ kir+ tregs may include removal of the inhibitory effect on these cells to restore cd8+ KIR-tregs (e.g., restore balance of the immune system or restore balanced immune activity in a subject or in a subject in need of treatment prior to onset of disease). Activation or stimulation of cd8+ kir+ tregs may also include removal of cd4+ cells, B cells, or other cells that mediate an immune response, such as removal of such cells by depletion, e.g., cell lysis.
As used herein, pathogenic immune cells refer to immune cells that react with or induce a response to self-antigens. Examples of such pathogenic immune cells include autoreactive cd4+ T cells, autoantibody-producing B cells, dendritic cells presenting autoantigens, and cells presenting other autoantigens, as is well known in the art.
In some embodiments, the cd8+kir+ tregs are contacted with the binding agent in vivo. In some embodiments, the cd8+kir+ tregs are contacted ex vivo with a binding agent. The activated cd8+kir+ tregs may then be administered to a subject in need thereof in an effective amount.
In some embodiments, activated cd8+kir+ tregs exert an inhibitory effect on pathogenic immune cells such as autoreactive cd4+ T cells, autoantibody-producing B cells, dendritic cells presenting autoantigens, and cells presenting other autoantigens. In some embodiments, activated cd8+kir+ tregs exert an inhibitory effect on pathogenic immune cells such as autoreactive cd4+ T cells, autoantibody-producing B cells, and dendritic cells presenting autoantigens. In some embodiments, activated cd8+kir+ tregs deplete pathogenic immune cells, such as autoreactive cd4+ T cells, autoantibody-producing B cells, and autoantigen-presenting dendritic cells. In some embodiments, the activated cd8+kir+ tregs modulate pathogenic effects of pathogenic immune cells and reduce the titer of autoantibodies in the subject. In some embodiments, the activated cd8+kir+ tregs reduce the titer of autoantibodies in the subject.
In some embodiments, the binding agent is selected from any binding agent described herein that has reduced or substantially no effector function activity in each case. In some embodiments, the reduced effector function activity is reduced or no ADCC, ADCP or CDC effector function activity. In some embodiments, substantially no effector function activity means substantially no ADCC, ADCP and CDC effector function activity. In some embodiments, the binding agent lacks an Fc domain or Fc region and has reduced effector function or substantially no effector function. In some embodiments, the binding agent has an Fc domain or Fc region, has reduced effector function or substantially no effector function due to amino acid substitutions in the Fc domain or Fc region. In some embodiments, the binding agent has an Fc domain or Fc region with reduced effector function or substantially no effector function due to amino acid substitutions (e.g., fc no substitutions) in the Fc domain or Fc region. In some embodiments, the binding agent lacks an Fc domain or Fc region, or has an Fc domain or Fc region with reduced binding to one or more fcγ receptors, or is an Fc domain-free. In some embodiments, the binding agent lacks an Fc domain or Fc region. In some embodiments, the binding agent has an Fc domain or Fc region with reduced binding to one or more fcγ receptors, or is an Fc domain-free. In some embodiments, the binding agent has an Fc domain or Fc region that has reduced binding to one or more fcγ receptors due to amino acid substitutions in the Fc domain or Fc region.
Without being bound by any particular theory, the reduction or absence of effector function activity of the binding agent may limit the interaction of the binding agent with other cell types (i.e., non-cd8+kir+treg) and/or limit the depletion of cd8+kir+treg bound by the binding agent.
In some embodiments, the subject in need of treatment has an autoimmune disease. In some embodiments, the subject in need of treatment suffers from an autoimmune disease, such as, for example, autoimmune hepatitis, addison's disease, alopecia areata, albert's syndrome, ankylosing spondylitis, antiphospholipid syndrome, arthritis, ascariasis, aspergillosis atopic allergy, atopic dermatitis, atopic rhinitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune myositis, behcet's disease, bird feeder's Lung (Bird-Fancier's Lung), bronchial asthma, kaplan syndrome, cardiomyopathy, celiac disease, chagas's disease, chronic glomerulonephritis, chronic graft versus host disease, cogan's syndrome, condensed collectin disease, CREST syndrome, crohn's disease, cryoglobulinemia, cushing's syndrome, dermatomyositis, discoid lupus, dress ier syndrome, eaton-Lambert syndrome, encephalomyelitis, endocrinopathies, erythema, evan's syndrome, feltey's syndrome, fibromyalgia, french's cyclotis (Fuch's cyclotis), gastric atrophy, gastrointestinal allergies, giant cell arteritis, glomerulonephritis, goodpasture's syndrome, graft versus host disease, graves disease, guillain-Barre disease (syndrome), hashimoto thyroiditis, hemolytic anemia, allergic purpura, hyperviscosity syndrome, idiopathic adrenal atrophy, idiopathic pulmonary fibrosis, idiopathic thrombocytopenic purpura, igA nephropathy, inflammatory bowel disease (syndrome), insulin dependent diabetes mellitus (IDDM or type I), juvenile arthritis, juvenile idiopathic arthritis, juvenile diabetes (type I), lambert-Eaton syndrome laminitis, lichen planus, lupus hepatitis, lupus nephritis, lymphopenia, macroglobulinemia, meniere's disease, mixed connective tissue disease, unknown monoclonal immunoglobulin disease, multiple sclerosis, myasthenia gravis, myocarditis, pemphigus/pemphigoid, pernicious anemia, POEMS syndrome, polyadenylic syndrome, polyarteritis nodosa, polymyositis, senile dementia, primary aγ -globulinemia, primary biliary cirrhosis/cholangitis, psoriasis, psoriatic arthritis, raynaud's phenomenon, rette's syndrome, rheumatic fever, rheumatoid arthritis, sang Pute's syndrome, schmidt's syndrome, scleroderma/systemic sclerosis, shulman's syndrome, sjogren's syndrome, sympathological ophthalmology, systemic lupus erythematosus, large arteritis, temporal arteritis, thyroiditis, thrombocytopenia, thyroid toxicity, toxic epidermolysis, type B insulin resistance, type I diabetes, ulcerative colitis, uveitis, waldenstrom's and wegener's granulomatosis.
In some embodiments, the autoimmune disease is autoimmune hepatitis, celiac disease, crohn's disease, juvenile idiopathic arthritis, inflammatory Bowel Disease (IBD), insulin dependent diabetes mellitus (IDDM or type 1 diabetes), lupus nephritis, myasthenia gravis, myocarditis, multiple Sclerosis (MS), pemphigus/pemphigoid, primary biliary cirrhosis/cholangitis, rheumatoid Arthritis (RA), scleroderma/systemic sclerosis, sjogren's syndrome (SjS), systemic Lupus Erythematosus (SLE), or ulcerative colitis.
In some embodiments, the autoimmune disease is selected from autoimmune hepatitis, celiac disease, crohn's disease, inflammatory Bowel Disease (IBD), insulin dependent diabetes mellitus (IDDM or type 1 diabetes), multiple Sclerosis (MS), rheumatoid Arthritis (RA), systemic Lupus Erythematosus (SLE), or ulcerative colitis.
The methods described herein comprise administering a therapeutically effective amount of a binding agent to a subject having an autoimmune disease. As used herein, the phrases "therapeutically effective amount," "effective amount," and "effective dose" may refer to an amount of a binding agent as described herein that provides therapeutic benefit in the treatment, management, prevention of recurrence, delay of onset, or prevention of an autoimmune disease, e.g., provides a statistically significant reduction in at least one symptom, sign, or marker of an autoimmune disease. Determination of therapeutically effective amounts is well within the ability of those skilled in the art. In general, a therapeutically effective amount can vary with the subject's medical history, age, condition and sex, the severity and type of the subject's medical condition, and the administration of other pharmaceutically active agents.
It is contemplated that the methods herein reduce symptoms, pathology, disease progression, or onset of a disease in a subject. As used herein, "subject" refers to a human or animal. Typically, the animal is a vertebrate, such as a primate, rodent, livestock or hunting animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus monkeys. Rodents include mice, rats, woodchuck, ferrets, rabbits, and hamsters. Domestic animals and hunting animals include cows, horses, pigs, deer, bison, buffalo, felines (e.g., domestic cats), canines (e.g., dogs, foxes, wolves), birds (e.g., chickens, emus, ostrich) and fish (e.g., trout, catfish, and salmon). In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms "patient," "individual," and "subject" are used interchangeably herein.
Preferably, the subject is a mammal. The mammal may be a human, non-human primate, mouse, rat, canine, feline, equine, or dairy cow, but is not limited to these examples. For example, mammals other than humans can be advantageously used as subjects representing animal models of, for example, various autoimmune diseases. In addition, the methods described herein may be used to treat domestic animals and/or pets. The subject may be male or female. In certain embodiments, the subject is a human.
The subject may be a human previously diagnosed with or determined to have an autoimmune disease and in need of treatment, but need not have received treatment for an autoimmune disease. Alternatively, the subject may be a human not previously diagnosed as having an autoimmune disease in need of treatment. The subject may be a subject that exhibits one or more conditions associated with an autoimmune disease or one or more complications without exhibiting a risk factor. A "subject in need of treatment" for an autoimmune disease may be a human suffering from or diagnosed with the disease. In some embodiments, a subject is in need of treatment if the subject is at risk of developing an immune response to an antigen or autoantigen that may lead to or cause the onset of an autoimmune disease. In other embodiments, a subject "at risk of developing an autoimmune disease" refers to a subject diagnosed as at risk of developing the disease or condition.
As used herein, the term "treatment" or "improvement," when used in reference to a disease, disorder, or medical condition (e.g., autoimmune disease), refers to the treatment of a condition, wherein the purpose is to reverse, alleviate, ameliorate, inhibit, slow or stop the development or severity of symptoms or the condition. The term "treating" includes reducing or alleviating at least one adverse reaction or symptom of a condition or disease. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the condition progresses less or ceases. That is, "treatment" may include not only improvement of symptoms or markers, but also cessation or at least slowing of progression or worsening of symptoms that would be expected without treatment. Beneficial or desired clinical results include, but are not limited to, reducing one or more symptoms, reducing the onset of disease in a subject, reducing one or more symptoms, reducing the extent of a deficit, stabilizing (i.e., not worsening) an autoimmune disease state, or preventing, delaying or slowing the onset or progression of an autoimmune disease. As used herein, the term "administering" refers to contacting a binding agent described herein or a nucleic acid encoding a binding agent described herein (e.g., by administration to a subject) by a method or pathway that results in binding of the binding agent to cd8+kir+ tregs. Similarly, pharmaceutical compositions comprising the binding agents described herein or nucleic acids encoding the binding agents disclosed herein may be administered by any suitable route, thereby producing an effective treatment for a subject.
The dosage range of the binding agent depends on efficacy and includes an amount large enough to produce the desired effect, e.g., to reduce one or more symptoms, to reduce the onset of disease in the subject, to reduce one or more symptoms, to reduce the extent of a defect, to stabilize (i.e., not worsen) an autoimmune disease state, or to slow the onset or progression of an autoimmune disease. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage will vary with the age, condition and sex of the subject and can be determined by one skilled in the art. In the event of any complications, the individual physician may also adjust the dosage. In some embodiments, the dosage ranges from about 0.01mg/kg body weight to about 20mg/kg body weight. In some embodiments, the dosage ranges from about 0.5mg/kg body weight to about 15mg/kg body weight. In some embodiments, the dosage ranges from about 0.5mg/kg body weight to about 5mg/kg body weight. Alternatively, a dose range may be titrated to maintain serum levels between 1 μg/mL and 1000 μg/mL.
In some embodiments, the subject receives a single dose of any of the binding agents described herein, e.g., for treating an acute autoimmune disease or condition. In some embodiments, the subject receives a single dose of any of the binding agents described herein, e.g., for preventing an immune response that may result in an autoimmune disease or condition. In some embodiments, the subject receives repeated doses of any of the binding agents described herein, e.g., for the treatment of a chronic autoimmune disease or condition. In some embodiments, the subject receives repeated doses of any of the binding agents described herein, e.g., for preventing an immune response that may result in an autoimmune disease or condition. In some embodiments, the dose is administered weekly, biweekly, tricyclically, monthly, bi-monthly, or 6 months for weeks, months, or years. The duration of treatment depends on the clinical progress of the subject and the response to the treatment.
In some embodiments, the dose may be administered intravenously. In some embodiments, intravenous administration may be an infusion that occurs over a period of about 10 minutes to about 4 hours. In some embodiments, intravenous administration may be an infusion that occurs over a period of about 30 minutes to about 90 minutes. In some embodiments, the dose may be administered subcutaneously.
Pharmaceutical compositions containing any of the binding agents described herein may be administered in unit doses. When referring to a pharmaceutical composition, the term "unit dose" refers to physically discrete units suitable as unitary dosages for subjects, each unit containing a predetermined quantity of active material (e.g., a binding agent), calculated to produce the desired therapeutic effect in association with the desired physiologically acceptable diluent (i.e., carrier or vehicle).
In some embodiments, administration of any of the binding agents described herein can result in improved therapeutic results, such as reduced systemic inflammatory cytokines, reduced pathology in the tissue affected by the disease, reduced frequency and/or severity of episodes, reduced self-reporting of symptoms associated with the disease, reduction and/or prevention of one or more symptoms, delay or slowing of the onset or progression of autoimmune disease.
In some embodiments, the binding agent or pharmaceutical composition of any of the binding agents described herein is administered with an immunotherapy. As used herein, "immunotherapy" refers to a therapeutic strategy aimed at modulating the subject's autoimmune system. Examples of immunotherapies include, but are not limited to, antibodies, such as checkpoint inhibitors and modulators, and immunosuppressants, such as cyclosporine, cyclosporine a, mycophenolate mofetil, sirolimus, tacrolimus, etanercept, prednisone, azathioprine, methotrexate, cyclophosphamide, prednisone, aminocaproic acid, chloroquine, hydroxychloroquine, hydrocortisone, dexamethasone, chlorambucil, DHEA, danazol, bromocriptine, meloxicam, infliximab, abamectin, beracemide, and adalimumab.
Treatment of graft complications
In some embodiments, methods of treating graft complications associated with graft versus host disease or GVHD are provided. In some embodiments, methods for reducing complications of GVHD in a subject are provided, comprising administering to a subject who has received a transplant a binding agent or pharmaceutical composition described herein to inhibit the immune response of the host to the transplant. In some embodiments, methods for reducing complications of GVHD in a subject are provided, comprising administering a binding agent or pharmaceutical composition described herein to a subject receiving a transplant to inhibit an immune response associated with the transplant. In some embodiments, methods of reducing GVHD are provided, comprising administering a binding agent or pharmaceutical composition described herein to a subject who has received a transplant and is experiencing GVHD, thereby reducing symptoms of GVHD. In some embodiments, methods of inhibiting GVHD are provided, comprising administering a binding agent or pharmaceutical composition described herein to a subject who has received a transplant and is experiencing GVHD, thereby inhibiting or reducing GVHD. In some embodiments, methods of inhibiting or reducing GVHD are provided comprising contacting a binding agent or pharmaceutical composition described herein with cd8+kir+t regulatory cells (tregs) from a subject who has received a transplant, thereby activating the cd8+kir+t regulatory cells (tregs). In some embodiments, methods of inhibiting or reducing GVHD are provided, comprising contacting a binding agent or pharmaceutical composition described herein with cd8+kir+t regulatory cells (tregs) from a subject who has received a transplant, thereby depleting the transplanted cd8+kir+t regulatory cells (tregs). In some embodiments, methods of inhibiting or reducing GVHD are provided comprising contacting a binding agent or pharmaceutical composition described herein with cd8+kir+t regulatory cells (Treg) from a subject who has received a transplant, thereby depleting the cd8+kir+t regulatory cells (Treg).
In some embodiments, the subject has received a hematopoietic stem cell graft, umbilical cord blood stem cell graft, an induced pluripotent stem cell-derived progenitor or differentiated cell graft, a bone marrow graft, or a solid organ graft. The graft is typically an allograft. In some embodiments, the subject has received a hematopoietic stem cell transplant, umbilical cord blood stem cell transplant, an induced pluripotent stem cell-derived progenitor or differentiated cell transplant, a bone marrow transplant, or a solid organ transplant, and is experiencing GVHD. In some embodiments, the subject has received a hematopoietic stem cell graft, umbilical cord blood stem cell graft, an induced pluripotent stem cell-derived progenitor or differentiated cell graft, a bone marrow graft, or a solid organ graft, and is at risk of GVHD. In some embodiments, the subject has received a hematopoietic stem cell transplant and is experiencing GVHD. In some embodiments, the subject has received a hematopoietic stem cell transplant and is at risk of GVHD. In some embodiments, the subject has received an umbilical cord blood stem cell graft and is experiencing GVHD. In some embodiments, the subject has received an umbilical cord blood stem cell graft and is at risk of GVHD. In some embodiments, the subject has received an induced pluripotent stem cell-derived progenitor or differentiated cell graft and is experiencing GVHD. In some embodiments, the subject has received an induced pluripotent stem cell-derived progenitor or differentiated cell graft and is at risk of GVHD. In some embodiments, the subject has received a bone marrow transplant and is experiencing GVHD. In some embodiments, the subject has received a bone marrow transplant and is at risk of GVHD. In some embodiments, the subject has received a solid organ transplant and is experiencing GVHD. In some embodiments, the subject has received a solid organ transplant and is at risk of GVHD.
In some embodiments, GVHD is inhibited or reduced by depleting pathogenic immune cells, such as CD 4T cells. In some embodiments, GVHD is inhibited or reduced by depleting pathogenic immune cells from the graft, such as cd8+kir+ tregs. In some embodiments, the cd8+kir+ tregs are contacted with the binding agent in vivo. In some embodiments, the cd8+kir+ tregs are contacted ex vivo with a binding agent. The activated cd8+kir+ tregs are then administered to a subject in need thereof in an effective amount.
In some embodiments, the binding agent is selected from any binding agent described herein that has reduced or substantially no effector function activity in each case. In some embodiments, the reduced effector function activity is reduced or no ADCC, ADCP or CDC effector function activity. In some embodiments, substantially no effector function activity means substantially no ADCC, ADCP and CDC effector function activity. In some embodiments, the binding agent lacks an Fc domain or Fc region and has reduced effector function or substantially no effector function. In some embodiments, the binding agent has an Fc domain or Fc region with reduced or substantially no effector function due to amino acid substitutions in the Fc domain or Fc region. In some embodiments, the binding agent has an Fc domain or Fc region with reduced effector function or substantially no effector function due to amino acid substitutions (e.g., fc no substitutions) in the Fc domain or Fc region. In some embodiments, the binding agent lacks an Fc domain or Fc region, or has an Fc domain or Fc region with reduced binding to one or more fcγ receptors. In some embodiments, the binding agent lacks an Fc domain or Fc region. In some embodiments, the binding agent has an Fc domain or Fc region with reduced binding to one or more fcγ receptors, or is an Fc domain-free. In some embodiments, the binding agent has an Fc domain or Fc region that has reduced binding to one or more fcγ receptors due to amino acid substitutions in the Fc domain or Fc region.
In some embodiments, the binding agent is selected from any binding agent described herein that has effector function activity comprising at least ADCC in each case. In some embodiments, the effector function activity is a combination of ADCC and ADCP and/or CDC effector function activity. In some embodiments, having effector function activity means having ADCC, ADCP and CDC effector function activity. In various embodiments, such binding agents have an Fc domain or have an Fc domain that binds to one or more fcγ receptors.
Without being bound by any particular theory, a reduction or absence of effector function activity of the binding agent may limit the interaction of the binding agent with other cell types (i.e., non-cd8+kir+ tregs) and/or limit the depletion of cd8+kir+ tregs. Rather, the presence of effector function activity of the binding agent is believed to bias the immune response towards depletion of cd8+kir+treg.
In some embodiments, the graft is a stem cell graft, a bone marrow graft, or a solid organ graft. As used herein, the term "transplant" refers to an organ, tissue, or cell that is transplanted from one subject to a different subject, or within the same subject (e.g., to a different region of the subject). Organs such as the liver, kidney, heart or lung or other body parts, such as bone or bone matrix, such as bone marrow, tissues, such as skin, cornea, intestine, endocrine glands or stem cells or various types, or hematopoietic cells, including hematopoietic stem and progenitor cells, cord blood stem cells and induced pluripotent stem cell-derived progenitor cells or differentiated cells, are examples of transplants. In some embodiments, the solid organ transplant is a liver, kidney, lung, pancreas, and/or heart transplant. The term transplant includes grafts. Inhibition may be allograft (or xenograft) or xenograft. The term "allograft" refers to a graft between members of a species in which two genes are not identical. The term "xenograft" refers to a graft between members of different species.
In some embodiments, the transplant is a bone marrow transplant. In some embodiments, the transplantation is hematopoietic stem cell transplantation. In some embodiments, the transplantation is cord blood stem cell transplantation. In some embodiments, the transplantation is an induced pluripotent stem cell-derived progenitor cell or differentiated cell transplantation.
In some embodiments, the subject has graft versus host disease or GVHD. In some embodiments, the subject is at risk of GVHD.
The methods described herein comprise administering a therapeutically effective amount of a binding agent to a subject receiving a transplant. As used herein, the phrases "therapeutically effective amount", "amount effective", "effective amount" and "effective dose" may refer to the amount of binding agent described herein that provides therapeutic benefit in managing graft-related GVHD. In some embodiments, the therapeutic benefit is to delay or prevent the onset of GVHD. In some embodiments, the therapeutic benefit is a statistically significant reduction in at least one symptom, sign, or marker of GVHD. Determination of therapeutically effective amounts is well within the ability of those skilled in the art. In general, a therapeutically effective amount can vary with the subject's medical history, age, condition and sex, the severity and type of the subject's medical condition, and the administration of other pharmaceutically active agents.
It is contemplated that the methods herein reduce, or prevent symptoms of GVHD in a subject receiving transplantation. As used herein, "subject" refers to a human or animal. Typically, the animal is a vertebrate, such as a primate, rodent, livestock or hunting animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus monkeys. Rodents include mice, rats, woodchuck, ferrets, rabbits, and hamsters. Domestic animals and hunting animals include cows, horses, pigs, deer, bison, buffalo, felines (e.g., domestic cats), canines (e.g., dogs, foxes, wolves), birds (e.g., chickens, emus, ostrich) and fish (e.g., trout, catfish, and salmon). In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms "patient," "individual," and "subject" are used interchangeably herein.
Preferably, the subject is a mammal. The mammal may be a human, non-human primate, mouse, rat, canine, feline, equine, or dairy cow, but is not limited to these examples. For example, mammals other than humans can be advantageously used as subjects representing animal models of, for example, various autoimmune diseases. In addition, the methods described herein may be used to treat domestic animals and/or pets. The subject may be male or female. In certain embodiments, the subject is a human.
The subject may be a human receiving the graft. Alternatively, the subject may be a person who has received the graft. The subject may be a subject who will receive the graft and is at risk of GVHD. The subject may be a subject who has received a graft and is at risk of GVHD. The subject may be a subject who has received an implant and has GVHD.
As used herein, the term "treatment" or "improvement," when used in reference to a disease, condition, or medical condition (e.g., GVHD), refers to the treatment of a condition in which the aim is to reverse, alleviate, ameliorate, inhibit, slow or stop the progression or severity of GVHD symptoms or conditions. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition or disease. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, if the disease progression is reduced or stopped, the treatment is "effective". That is, "treatment" may include not only amelioration of symptoms or markers, but also cessation or at least slowing of progression or worsening of the symptoms that would be expected without treatment. Beneficial or desired clinical results include, but are not limited to, reduction of one or more symptoms associated with GVHD.
As used herein, the term "administering" refers to contacting a binding agent described herein or a nucleic acid encoding a binding agent described herein (e.g., by administration to a subject) by a method or pathway that results in binding of the binding agent to cd8+kir+ tregs. Similarly, pharmaceutical compositions comprising the binding agents described herein or nucleic acids encoding the binding agents disclosed herein may be administered by any suitable route, thereby producing an effective treatment for a subject.
The dosage range of the binding agent depends on potency and includes amounts sufficient to produce the desired effect, e.g., to reduce one or more symptoms of GVHD or to reduce or prevent GVHD. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage will vary with the age, condition and sex of the subject and can be determined by one skilled in the art. In the event of any complications, the individual physician may also adjust the dosage. In some embodiments, the dosage ranges from about 0.01mg/kg body weight to about 20mg/kg body weight. In some embodiments, the dosage ranges from about 0.5mg/kg body weight to about 15mg/kg body weight. In some embodiments, the dosage ranges from about 0.5mg/kg body weight to about 5mg/kg body weight. Alternatively, a dose range may be titrated to maintain serum levels between 1 μg/mL and 1000 μg/mL.
In some embodiments, the subject receives a single dose of any of the binding agents described herein, e.g., for treating acute GVHD after transplantation. In some embodiments, the subject receives repeated doses of any of the binding agents described herein, e.g., for the treatment of chronic GVHD. In some embodiments, the dose is administered weekly, biweekly, tricyclically, monthly, bi-monthly, or 6 months for weeks, months, or years. The duration of treatment depends on the clinical progress of the subject and the response to the treatment.
In some embodiments, the dose may be administered intravenously. In some embodiments, intravenous administration may be an infusion that occurs over a period of about 10 minutes to about 4 hours. In some embodiments, intravenous administration may be an infusion that occurs over a period of about 30 minutes to about 90 minutes. In some embodiments, the dose may be administered subcutaneously.
Pharmaceutical compositions containing any of the binding agents described herein may be administered in unit doses. When referring to a pharmaceutical composition, the term "unit dose" refers to physically discrete units suitable as unitary dosages for subjects, each unit containing a predetermined quantity of active material (e.g., a binding agent), calculated to produce the desired therapeutic effect in association with the desired physiologically acceptable diluent (i.e., carrier or vehicle).
In some embodiments, administration of any of the binding agents described herein can result in improved therapeutic outcomes, such as reduction of systemic inflammatory cytokines, reduction of pathology in the tissues affected by the disease, reduction of self-reporting of symptoms associated with an immune response associated with adverse effects on host tissues, improvement or prolongation of graft implantation, alleviation of one or more symptoms, and/or prevention, delay or slowing of the occurrence or progression of graft rejection, or prolongation of graft implantation by reducing the use of broad-spectrum immunosuppressants (e.g., corticosteroids).
In some embodiments, the binding agent or pharmaceutical composition of any of the binding agents described herein is administered with an immunosuppressant such as a corticosteroid.
Treatment of infectious diseases
In some aspects, the binding agents described herein can be used in one or more methods comprising administering the binding agents or pharmaceutical compositions described herein to a subject having an infection. In some embodiments, the subject is in need of treatment for the infection. In some embodiments, a method of treating an infection is provided by administering any of the binding agents or pharmaceutical compositions described herein to a subject in need thereof in an amount effective to activate or stimulate cd8+kir+ tregs, thereby ameliorating symptoms of the infection. In some embodiments, the method comprises stimulating an immune response against an infectious agent by contacting cd8+kir+t regulatory cells (tregs) with any binding agent or pharmaceutical composition described herein in an amount effective to activate or stimulate cd8+kir-tregs (activated tregs). In some embodiments, the method comprises depleting the infected cells by contacting cd8+kir+t regulatory cells (tregs) with an amount of any binding agent or pharmaceutical composition described herein effective to activate or stimulate cd8+kir-tregs (activated tregs). In some embodiments, the method comprises stimulating an immune response against the infected cells by contacting cd8+kir+t regulatory cells (tregs) with any binding agent or pharmaceutical composition described herein in an amount effective to activate or stimulate cd8+kir-tregs (activated tregs).
In some embodiments of the methods of treating an infection, cd8+ kir+ tregs are contacted with any of the binding agents described herein in vivo. In some embodiments, the cd8+kir+ Treg is contacted ex vivo with any binding agent described herein. The activated cd8+ kir+ tregs are then administered to a subject in need thereof in an effective amount. In some embodiments, the immune response comprises immunosuppressive immunocytopenia. As used herein, immunosuppressive immune cells include CD4 tregs and tolerogenic DCs. In some embodiments, the number of infected cells is reduced in the subject.
In some embodiments, the binding agent is selected from any binding agent described herein that has reduced or substantially no effector function activity in each case. In some embodiments, the reduced effector function activity is reduced or no ADCC, ADCP or CDC effector function activity. In some embodiments, substantially no effector function activity means substantially no ADCC, ADCP and CDC effector function activity. In some embodiments, the binding agent lacks an Fc domain or Fc region and has reduced effector function or substantially no effector function. In some embodiments, the binding agent has an Fc domain or Fc region with reduced or substantially no effector function due to amino acid substitutions in the Fc domain or Fc region. In some embodiments, the binding agent has an Fc domain or Fc region with reduced effector function or substantially no effector function due to amino acid substitutions (e.g., fc no substitutions) in the Fc domain or Fc region. In some embodiments, the binding agent lacks an Fc domain or Fc region, or has an Fc domain or Fc region with reduced binding to one or more fcγ receptors. In some embodiments, the binding agent lacks an Fc domain or Fc region. In some embodiments, the binding agent has an Fc domain or Fc region with reduced binding to one or more fcγ receptors, or is an Fc domain-free. In some embodiments, the binding agent has an Fc domain or Fc region that has reduced binding to one or more fcγ receptors due to amino acid substitutions in the Fc domain or Fc region.
Without being bound by any particular theory, a reduction or absence of effector function activity of the binding agent may limit the interaction of the binding agent with other cell types (i.e., non-cd8+kir+ tregs) and/or limit the depletion of cd8+kir+ tregs.
In some embodiments, the subject is in need of treatment for an infectious disease or is infected with an infectious agent, such as, for example, a bacterial disease, a systemic fungal disease, a rickettsia disease, a parasitic disease, or a viral disease. In some embodiments, the infection is a bacterial disease such as diphtheria, pertussis, occult bacteremia, urinary tract infection, gastroenteritis, cellulitis, epilepsy, tracheitis, adenoid hypertrophy, post pharyngeal abscess, impetigo, eczema, pneumonia, endocarditis, septic arthritis, pneumococcal pneumonia, peritonitis, bacteremia, meningitis, acute suppurative meningitis, urethritis, cervicitis, prostatitis, pharyngitis, salpingitis, epididymitis, gonorrhea, syphilis, listeriosis, anthrax, canola, salmonellosis, typhoid fever, dysentery, conjunctivitis, sinusitis, brucellosis, cholera, plague, tetanus, necrotic enteritis, actinomycosis, mixed anaerobic infection, syphilis, recurrent fever, leptospirosis, lyme disease, murine bite fever, tuberculosis, including tuberculosis, lymphadenitis, leprosy, chlamydia, trachitis or peritonitis; systemic mycoses such as histoplasmosis, coccidiosis, explosive mycosis, sporosis, cryptococcosis, systemic candidiasis, aspergillosis, mucormycosis, myxoma or chromomycosis; rickettsia diseases such as typhus, rocky mountain spotted fever, rickettsia, eastern tick-borne rickettsia, Q fever or barbita disease; parasitic diseases such as malaria, babesia, african sleeping disease, chagas disease, leishmaniasis, dur-dur fever, toxoplasmosis, meningoepithymosis, keratitis, dieamebias, giardiasis, cryptosporidiosis, isospora, cyclosporin, microsporicosis, ascariasis, whipworm infection, hookworm infection, nematode infection, ocular larva migration, trichinosis, guinea worm, lymphangiosis, schistosomiasis, river blindness, canine heartworm infection, schistosomiasis, swimmer pruritus, eastern pulmonary influenza, eastern liver influenza, fasciolopsis, taeniasis, echinococcosis and metacercaria; the composition can be used for treating viral diseases, such as measles, acute sclerosing panencephalitis, common cold, mumps, rubella, rosea, fifth disease, varicella, coronavirus infection, covid19 disease, respiratory syncytial virus infection, membranous laryngitis, bronchitis, infectious mononucleosis, poliomyelitis, herpes, hand-foot-and-mouth disease, epidemic chest pain, genital herpes, genital warts, aseptic meningitis, myocarditis, epstein-barr virus infection, epstein-barr virus pericarditis, gastroenteritis, hepatitis a virus infection, hepatitis b virus infection, hepatitis c virus infection, HIV infection, human Papilloma Virus (HPV) infection, lei Yizeng syndrome, kawasaki syndrome, influenza, bronchitis, viral "walking" pneumonia, acute febrile respiratory disease, acute pharyngeal fever, epidemic keratoconjunctivitis, herpes simplex virus type 1 (HSV-1) infection, herpes simplex virus type 2 (HSV-2) infection, herpes zoster, cytomegalovirus, leukosis, progressive polyneuropathy, schrssis, schmitt-kun, szebra-disease, szebra-lear's disease, szechwan-type encephalitis, szebra's disease, szechwan-heat syndrome, lymphocytic fever, tsuk's disease, shak's disease, shabber's disease, sham's disease, shabber's disease, shaggy's disease, and lymphoheat disease, shaggy's disease, and lymphokawa disease.
In some embodiments, the infection is a viral infection. In some embodiments, the infection is a viral infection, such as an HIV infection, a hepatitis A virus infection, a hepatitis B virus infection, a hepatitis C virus infection, an Epstein-Barr virus infection, a coronavirus infection, such as a SARS-COV2 infection (Covid-19), and an influenza virus infection (influenza). In some embodiments, the infection is caused by an infectious agent, such as coronavirus, diphtheria virus, ebola virus, influenza virus, HIV, human Papilloma Virus (HPV), hepatitis a virus, hepatitis b virus, hepatitis c virus, measles virus, respiratory syncytial virus, rotavirus, and herpes virus.
The methods described herein comprise administering a therapeutically effective amount of a binding agent to a subject having an infection or having cells infected with an infectious agent. As used herein, the phrases "therapeutically effective amount," "effective amount," or "effective dose" may refer to the amount of any binding agent or pharmaceutical composition described herein that provides therapeutic benefit in treating, managing, or preventing recurrence of an infectious disease, e.g., provides a statistically significant reduction in at least one symptom, sign, or marker of an infection. Determination of therapeutically effective amounts is well within the ability of those skilled in the art. In general, a therapeutically effective amount can vary with the subject's medical history, age, condition and sex, the severity and type of the subject's medical condition, and the administration of other pharmaceutically active agents.
As used herein, "subject" refers to a human or animal. Typically, the animal is a vertebrate, such as a primate, rodent, livestock or hunting animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus monkeys. Rodents include mice, rats, woodchuck, ferrets, rabbits, and hamsters. Domestic animals and hunting animals include cows, horses, pigs, deer, bison, buffalo, felines (e.g., domestic cats), canines (e.g., dogs, foxes, wolves), birds (e.g., chickens, emus, ostrich) and fish (e.g., trout, catfish, and salmon). In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms "patient," "individual," and "subject" are used interchangeably herein.
Preferably, the subject is a mammal. The mammal may be a human, non-human primate, mouse, rat, canine, feline, equine, or dairy cow, but is not limited to these examples. For example, mammals other than humans may be advantageously used as subjects representing animal models of, for example, various infections. In addition, the methods described herein may be used to treat domestic animals and/or pets. The subject may be male or female. In certain embodiments, the subject is a human.
The subject may be a person who has been previously diagnosed with or determined to have an infection and is in need of treatment, but does not have to have received treatment for the infection. Alternatively, the subject may be a human who has not been previously diagnosed as infected, but in need of treatment. The subject may be a subject that exhibits one or more risk factors for one or more diseases or one or more complications associated with the infection, or may be a subject that does not exhibit such a risk factor. An infected "subject in need of treatment" may be an infected person or a person diagnosed with an infection. In other embodiments, a subject "at risk of infection" refers to a subject diagnosed as at risk of infection.
As used herein, the term "treatment" or "improvement," when used in reference to a disease, condition, or medical condition (e.g., infection), refers to the treatment of a condition in which the aim is to reverse, alleviate, ameliorate, inhibit, slow or stop the progression or severity of symptoms or conditions. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition or disease. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, if the disease progression is reduced or stopped, the treatment is "effective". That is, "treatment" may include not only amelioration of symptoms or markers, but also cessation or at least slowing of progression or worsening of the symptoms that would be expected without treatment. Beneficial or desired clinical results include, but are not limited to, reduction of infected cells, reduction of one or more symptoms, reduction of the extent of a defect, stabilization of the state of the infection (i.e., not worsening), or delay or slowing of the infection or progression of the infection compared to that expected without treatment. As used herein, the term "administering" refers to providing a binding agent described herein or a nucleic acid encoding a binding agent described herein into contact (e.g., by administration to a subject) by a method or route that results in binding of the binding agent to cd8+kir+tregs. Similarly, pharmaceutical compositions comprising the binding agents described herein or nucleic acids encoding the binding agents disclosed herein may be administered by any suitable route, thereby producing an effective treatment for a subject.
The dosage range of the binding agent depends on the efficacy and includes an amount sufficient to produce the desired effect, e.g., to stimulate an immune response against the infected cells, to reduce the number of infected cells, or to slow or prevent the progression of the infection. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage will vary with the age, condition and sex of the subject and can be determined by one skilled in the art. In the event of any complications, the individual physician may also adjust the dosage. In some embodiments, the dosage ranges from about 0.01mg/kg body weight to about 20mg/kg body weight. In some embodiments, the dosage ranges from about 0.01mg/kg body weight to about 10mg/kg body weight. In some embodiments, the dosage ranges from about 0.1mg/kg body weight to about 10mg/kg body weight. In some embodiments, the dosage ranges from about 0.5mg/kg body weight to about 10mg/kg body weight. In some embodiments, the dosage ranges from about 0.5mg/kg body weight to about 5mg/kg body weight. Alternatively, a dose range may be titrated to maintain serum levels between 1 μg/mL and 1000 μg/mL.
In some embodiments, the subject receives a single dose of any of the binding agents or pharmaceutical compositions described herein, e.g., for treating an acute infection. In some embodiments, the subject receives a single dose of any of the binding agents or pharmaceutical compositions described herein, e.g., for preventing an immune response to an infection that may result in an autoimmune disease or condition. In some embodiments, the subject receives repeated doses of any of the binding agents or pharmaceutical compositions described herein, e.g., for treating chronic infection. In some embodiments, the dose is administered weekly, biweekly, tricyclically, monthly, bi-monthly, or 6 months for weeks, months, or years. The duration of treatment depends on the clinical progress of the subject and the response to the treatment.
In some embodiments, the dose may be administered intravenously. In some embodiments, intravenous administration may be an infusion that occurs over a period of about 10 minutes to about 4 hours. In some embodiments, intravenous administration may be an infusion that occurs over a period of about 30 minutes to about 90 minutes. In some embodiments, the dose may be administered subcutaneously.
In some embodiments, a total of about 2 to about 10 doses are administered to the subject. In some embodiments, a total of 4 doses are administered. In some embodiments, a total of 5 doses are administered. In some embodiments, a total of 6 doses are administered. In some embodiments, a total of 7 doses are administered. In some embodiments, a total of 8 doses are administered. In some embodiments, a total of 9 doses are administered. In some embodiments, a total of 10 doses are administered. In some embodiments, a total of more than 10 doses are administered.
Pharmaceutical compositions containing any of the binding agents described herein may be administered in unit doses. When referring to a pharmaceutical composition, the term "unit dose" refers to physically discrete units suitable as unitary dosages for subjects, each unit containing a predetermined quantity of active material (e.g., a binding agent), calculated to produce the desired therapeutic effect in association with the desired physiologically acceptable diluent (i.e., carrier or vehicle).
In some embodiments, administration of any of the binding agents described herein can result in improved therapeutic outcomes, such as improved clinical symptoms, or reduced viral load, or pathogen elimination or reduction, or reduction of infected cells.
In some embodiments, the binding agent or pharmaceutical composition of any of the embodiments described herein is administered with an infectious disease controlling agent, such as an antibacterial, antifungal, or antiviral agent. The antibacterial agent may be, for example, an lactam antibiotic such as penicillin G, penicillin V, cloxacillin, dicloxacillin, methicillin, nafcillin, oxacillin, ampicillin, amoxicillin, bazacillin, azlocillin, carbenicillin, mezlocillin, piperacillin or ticarcillin; aminoglycosides such as amikacin, gentamicin, kanamycin, neomycin, netilmicin, streptomycin or tobramycin; macrolides such as azithromycin, clarithromycin, erythromycin, lincomycin or clindamycin; tetracyclines, such as demerits, doxycyclines, minocycline, oxytetracycline, or tetracyclines; quinolones, such as cinrofloxacin or nalidixic acid; fluoroquinolones such as ciprofloxacin, enoxacin, glafloxacin, levofloxacin, lomefloxacin, norfloxacin, ofloxacin, sparfloxacin or tefloxacin; polypeptides, such as bacitracin, colistin or polymyxin B; sulfonamides, such as sulfoisoxazole, sulfamethoxazole, sulfadiazine, sulfamethylthiadiazole, or sulfacetamide; and other antibacterial agents such as trimethoprim, sulfamethoxazole, chloramphenicol, vancomycin, metronidazole, quinupristin, dapoxetine, rifampin, spectinomycin, or nitrofurantoin; and antiviral agents such as, for example, general antiviral agents such as iodate, vitamin a, trifluoperazine, acyclovir, famciclovir, penciclovir, valacyclovir, ganciclovir, foscarnet sodium, ribavirin, amantadine, rimantadine, or cidofovir; an antisense oligonucleotide; an immunoglobulin; an interferon; and other drugs such as zidovudine, didanosine, zalcitabine, stavudine, lamivudine, nevirapine, delavirdine, saquinavir, oseltamivir and peramivir, ritonavir, indinavir or nelfinavir.
Treatment of cancer
In some aspects, a binding agent or pharmaceutical composition as described herein may be used in a method of treating cancer comprising administering to a subject in need thereof a binding agent or pharmaceutical composition as described herein. In some embodiments, there is provided a method of treating cancer comprising administering to a subject in need thereof any of the binding agents described herein or any of the pharmaceutical compositions described herein, wherein the binding agent has substantially no effector function activity, in an amount effective to activate or stimulate cd8+kir+ tregs and thereby ameliorate symptoms of cancer. In some embodiments, methods of stimulating an immune response against an antigen associated with cancer (cancer antigen; e.g., an antigen expressed on cancer cells) are provided, comprising contacting cd8+ kir+ T regulatory cells (tregs) with an amount of any binding agent described herein or any pharmaceutical composition described herein effective to activate or stimulate cd8+ kir+ tregs (activated tregs), thereby increasing the immune response to the cancer antigen, wherein the binding agent has substantially no effector function activity.
In some embodiments, the binding agent is selected from any binding agent described herein that has reduced or substantially no effector function activity in each case. In some embodiments, the reduced effector function activity is reduced or no ADCC, ADCP or CDC effector function activity. In some embodiments, substantially no effector function activity means substantially no ADCC, ADCP and CDC effector function activity. In some embodiments, the binding agent lacks an Fc domain or Fc region and has reduced effector function or substantially no effector function. In some embodiments, the binding agent has an Fc domain or Fc region with reduced or substantially no effector function due to amino acid substitutions in the Fc domain or Fc region. In some embodiments, the binding agent has an Fc domain or Fc region with reduced effector function or substantially no effector function due to amino acid substitutions (e.g., fc no substitutions) in the Fc domain or Fc region. In some embodiments, the binding agent lacks an Fc domain or Fc region, or has an Fc domain or Fc region with reduced binding to one or more fcγ receptors. In some embodiments, the binding agent lacks an Fc domain or Fc region. In some embodiments, the binding agent has an Fc domain or Fc region with reduced binding to one or more fcγ receptors, or is an Fc domain-free. In some embodiments, the binding agent has an Fc domain or Fc region that has reduced binding to one or more fcγ receptors due to amino acid substitutions in the Fc domain or Fc region.
In some embodiments, there is provided a method of treating cancer comprising administering to a subject in need thereof any of the binding agents described herein or any of the pharmaceutical compositions described herein, wherein the binding agent has effector function activity including at least ADCC, in an amount effective to deplete cd8+kir+ tregs and thereby ameliorate symptoms of cancer. In some embodiments, a method of stimulating an immune response against cancer is provided, comprising contacting cd8+kir+t regulatory cells (tregs) with an amount of any binding agent described herein, or any pharmaceutical composition described herein, effective to deplete cd8+kir+tregs, thereby increasing the immune response against cancer, wherein the binding agent has effector function activity including at least ADCC. In some embodiments, a method of stimulating an immune response against an antigen associated with cancer (cancer antigen) is provided, comprising contacting cd8+kir+t regulatory cells (tregs) with an amount of any binding agent described herein or any pharmaceutical composition described herein effective to deplete cd8+kir+tregs, thereby increasing the immune response to the cancer antigen, wherein the binding agent has effector function activity including at least ADCC.
In some embodiments, the binding agent is selected from any of the binding agents described herein, in each case having effector function activity comprising at least ADCC. In some embodiments, the effector function activity is a combination of ADCC and ADCP and/or CDC effector function activity. In some embodiments, having effector function activity means having ADCC, ADCP and CDC effector function activity. In various embodiments, such binding agents have an Fc domain or have an Fc domain that binds one or more fcγ receptors.
In some embodiments, wherein the cd8+ kir+ Treg is contacted with the binding agent in vivo. In some embodiments, the cd8+kir+ tregs are contacted ex vivo with a binding agent. In some embodiments, the activated cd8+kir+ tregs are administered to a subject in need thereof in an effective amount.
Without being bound by any particular theory, a reduction or absence of effector function activity of the binding agent may limit the interaction of the binding agent with other cell types (i.e., non-cd8+kir+ tregs) and/or limit the depletion of cd8+kir+ tregs. In contrast, the presence of effector function activity of the binding agent is believed to bias the immune response towards depletion of cd8+kir+ tregs.
In some embodiments, the increased immune response includes cancer cytopenia or immunosuppressive immune cell depletion. By immunosuppressive immune cells is meant herein, for example, tumor-associated macrophages, cd4+ tregs and/or tolerogenic Dendritic Cells (DCs). In some embodiments, the number of cancer cells in the subject is reduced. In some embodiments, the number of immunosuppressive immune cells in the subject is reduced.
The terms "cancer" and "malignant tumor" refer to uncontrolled cell growth that interferes with the normal function of body organs and systems. Cancers or malignant tumors may be primary or metastatic, i.e. they have become invasive, with tumor growth implanted in tissues distant from the original tumor site. "tumor" refers to uncontrolled growth of cells that interfere with normal function of body organs and systems. As used herein, the term cancer includes malignant tumors and tumors, unless the context indicates otherwise. A subject with cancer is a subject having objectively measurable cancer cells in the subject. This definition includes benign tumors and malignant tumors, as well as potentially dormant tumors and micrometastases. Cancer migrates from its original location and implants other vital organs, eventually possibly leading to death of the subject by deterioration of the function of the affected organ. Hematological malignancies (hematopoietic cancers), such as leukemia and lymphoma, are capable of competing for normal hematopoietic compartments in a subject, resulting in hematopoietic failure (in the form of anemia, thrombocytopenia, and neutropenia), ultimately leading to death.
Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, myeloma, and leukemia. More specific examples of such cancers include, but are not limited to, basal cell carcinoma, biliary tract carcinoma, bladder carcinoma, bone carcinoma, brain and CNS cancers, breast cancer (e.g., triple negative breast cancer), peritoneal cancer, cervical cancer; bile duct cancer, choriocarcinoma, chondrosarcoma, colorectal and rectal cancer (colorectal cancer), connective tissue cancer, digestive system cancer, endometrial cancer, esophageal cancer, eye cancer, head and neck cancer, gastric cancer (including gastrointestinal cancer and gastric cancer), glioblastoma (GBM), liver cancer (hepatic carcinoma), liver cancer (hepatoma), intraepithelial tumors, renal cancer (e.g., clear cell renal cancer or non-clear cell renal cancer), laryngeal cancer, leukemia, liver cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma and lung squamous cell carcinoma), lymphomas including hodgkin and non-hodgkin's lymphoma, melanoma, mesothelioma, myeloma, neuroblastoma, oral cancer (e.g., lip, tongue, rhabdomyosarcoma, respiratory system cancer, salivary gland carcinoma, sarcoma, skin cancer, squamous cell carcinoma, testicular cancer, thyroid cancer, uterine or endometrial cancer, uterine heavy cancer, urinary system cancer, vulval cancer); and other carcinomas and sarcomas, as well as B-cell lymphomas (low grade/follicular non-Huo Jishi gold lymphomas (NHL), small lymphocytic lymphomas (SL) NHL, medium grade/follicular NHL, medium grade diffuse NHL, high grade immunoblastic NHL, high grade lymphocytic NHL, high grade small non-cutting cell NHL, giant tumor NHL, mantle cell lymphomas, AIDS-related lymphomas and Waldenstrom's macroglobulinemia), chronic Lymphocytic Leukemia (CLL), acute Lymphocytic Leukemia (ALL), hairy cell leukemia, chronic myelogenous leukemia and post-transplant lymphoproliferative disease (PTLD), as well as abnormal vascular proliferation and myeloma associated with mole-type hamartoma, oedema (e.g., oedema associated with brain tumors), and Meigs syndrome, such as multiple myeloma.
In some embodiments, the cancer is selected from solid tumors, including, but not limited to, hepatocellular carcinoma, lung cancer such as small cell lung cancer and large cell lung cancer, colorectal cancer, esophageal cancer, cervical cancer, ovarian cancer, renal cell carcinoma, prostate cancer, and bladder cancer.
The methods described herein comprise administering to a subject having cancer or malignancy a therapeutically effective amount of a binding agent or pharmaceutical composition described herein. As used herein, the phrases "therapeutically effective amount," "effective amount," or "effective dose" may refer to an amount of a binding agent or pharmaceutical composition described herein that provides therapeutic benefit in the treatment, management, or prevention of a cancer or malignancy, e.g., an amount of statistically significant reduction in at least one symptom, sign, or marker of cancer, tumor, or malignancy. Determination of therapeutically effective amounts is well within the ability of those skilled in the art. In general, a therapeutically effective amount can vary with the subject's medical history, age, condition and sex, the severity and type of the subject's medical condition, and the administration of other pharmaceutically active agents.
In some embodiments, the methods described herein reduce tumor size or tumor burden in a subject and/or reduce metastasis in a subject. In various embodiments, the tumor size in the subject is reduced by about 25-50%, about 40-70%, or about 50-90% or more. In various embodiments, the method reduces tumor size by 10%, 20%, 30% or more. In various embodiments, the method reduces tumor size by 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.
As used herein, "subject" refers to a human or animal. Typically, the animal is a vertebrate, such as a primate, rodent, livestock or hunting animal. Primates include chimpanzees, cynomolgus monkeys, spider monkeys, and macaques, e.g., rhesus monkeys. Rodents include mice, rats, woodchuck, ferrets, rabbits, and hamsters. Domestic animals and hunting animals include cows, horses, pigs, deer, bison, buffalo, felines (e.g., domestic cats), canines (e.g., dogs, foxes, wolves), birds (e.g., chickens, emus, ostrich) and fish (e.g., trout, catfish, and salmon). In certain embodiments, the subject is a mammal, e.g., a primate, e.g., a human. The terms "patient," "individual," and "subject" are used interchangeably herein.
Preferably, the subject is a mammal. The mammal may be a human, non-human primate, mouse, rat, canine, feline, equine, or dairy cow, but is not limited to these examples. For example, mammals other than humans may be advantageously used as subjects representing animal models of, for example, various infections. In addition, the methods described herein may be used to treat domestic animals and/or pets. The subject may be male or female. In certain embodiments, the subject is a human.
The subject may be a person who has been previously diagnosed with or determined to have cancer and is in need of treatment, but need not have received cancer treatment. Alternatively, the subject may be a subject who has not been previously diagnosed as having cancer in need of treatment. The subject may be a subject that exhibits one or more risk factors for a condition or one or more complications associated with cancer, or a subject that does not exhibit a risk factor. A "subject in need of treatment" for a particular cancer may be a subject suffering from or diagnosed with the disease. In other embodiments, a subject "at risk of developing" refers to a subject diagnosed as at risk of developing.
As used herein, the terms "treatment", "treatment" or "improvement", when used in reference to a disease, condition or medical condition (e.g., cancer), refer to the treatment of a condition, wherein the purpose is to reverse, alleviate, ameliorate, inhibit, slow or stop the progression or severity of symptoms or conditions. The term "treating" includes reducing or alleviating at least one adverse effect or symptom of a condition or disease. Treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, if the disease progression is reduced or stopped, the treatment is "effective". That is, "treatment" may include not only amelioration of symptoms or markers, but also cessation or at least slowing of progression or worsening of the symptoms that would be expected without treatment. Beneficial or desired clinical results include, but are not limited to, reduction of infected cells, reduction of one or more symptoms, reduction of the extent of a defect, stabilization of the state of the infection (i.e., not worsening), or delay or slowing of the infection or progression of the infection compared to that expected without treatment. As used herein, the term "administering" refers to providing a binding agent described herein or a nucleic acid encoding a binding agent described herein into contact (e.g., by administration to a subject) by a method or route that results in binding of the binding agent to cd8+kir+tregs. Similarly, pharmaceutical compositions comprising the binding agents described herein or nucleic acids encoding the binding agents disclosed herein may be administered by any suitable route, thereby producing an effective treatment for a subject.
The dosage range of the binding agent depends on the efficacy and includes amounts sufficient to produce the desired effect, e.g., slowing tumor growth or shrinking tumor size. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage will vary with the age, condition and sex of the subject and can be determined by one skilled in the art. In the event of any complications, the individual physician may also adjust the dosage. In some embodiments, the dosage ranges from about 0.01mg/kg body weight to about 20mg/kg body weight. In some embodiments, the dosage ranges from about 0.01mg/kg body weight to about 10mg/kg body weight. In some embodiments, the dosage ranges from about 0.1mg/kg body weight to about 10mg/kg body weight. In some embodiments, the dosage ranges from about 0.5mg/kg body weight to about 10mg/kg body weight. In some embodiments, the dosage ranges from about 0.5mg/kg body weight to about 5mg/kg body weight. Alternatively, a dose range may be titrated to maintain serum levels between 1 μg/mL and 1000 μg/mL.
The above dosage may be repeatedly administered. In a preferred embodiment, the above doses are administered weekly, biweekly, tricyclically or monthly, weekly or monthly. The duration of treatment depends on the clinical progress of the subject and the response to the treatment.
In some embodiments, the dose may be administered intravenously. In some embodiments, intravenous administration may be an infusion that occurs over a period of about 10 minutes to about 4 hours. In some embodiments, intravenous administration may be an infusion that occurs over a period of about 30 minutes to about 90 minutes. In some embodiments, the dose may be administered subcutaneously.
Pharmaceutical compositions comprising the binding agents may be administered in unit doses. When referring to a pharmaceutical composition, the term "unit dose" refers to physically discrete units suitable as unitary dosages for subjects, each unit containing a predetermined quantity of active material (e.g., a binding agent), calculated to produce the desired therapeutic effect in association with the desired physiologically acceptable diluent (i.e., carrier or vehicle).
In some embodiments, administration of any of the binding agents described herein can result in an improved therapeutic outcome as an objective response selected from stable disease, partial response, or complete response as determined by standard medical criteria for the cancer being treated. In some embodiments, the improved treatment results in a decrease in tumor burden. In some embodiments, the improved treatment outcome is progression free survival or disease free survival.
In some embodiments, the binding agent or pharmaceutical composition of any of the embodiments described herein is administered with an immunotherapy or chemotherapy. As used herein, "immunotherapy" refers to a therapeutic strategy intended to induce or enhance the subject's autoimmune system to combat cancer or malignancy. Examples of immunotherapy include, but are not limited to, antibodies such as checkpoint inhibitors. In some embodiments, the chemotherapy is, for example, an alkylating agent, such as, for example, a nitrogen mustard, such as cyclophosphamide, ifosfamide, qu Luolin amine, or chlorambucil; nitrosoureas such as carmustine (BCNU) and lomustine (CCNU); alkyl sulfonates such as busulfan and trosoxion; triazenes such as dacarbazine; platinum-containing compounds such as cisplatin and carboplatin; plant alkaloids such as vinca alkaloids, e.g. vincristine, vinblastine, vindesine and vinorelbine; taxanes such as paclitaxel and docetaxel; DNA topoisomerase inhibitors; epipodophyllotoxins such as etoposide, teniposide, topotecan, 9-aminocamptothecin, camptothecin and krestin; mitomycin such as mitomycin C; antimetabolites, such as antimetates, e.g., DHFR inhibitors, e.g., methotrexate and trimetrexate; IMP dehydrogenase inhibitors such as mycophenolic acid, thiazolecarboxamide nucleosides, ribavirin and EICAR; ribonucleotide reductase inhibitors such as hydroxyurea and deferoxamine; and pyrimidine analogs such as uracil analogs, e.g., 5-fluorouracil, fluorouridine, deoxyfluorouridine, and Ratitrexed; cytosine analogues such as cytarabine (ara C), cytosine arabinoside, and fludarabine; purine analogs such as mercaptopurine and thioguanine; hormone therapies such as receptor antagonists, e.g., antiestrogens, e.g., tamoxifen, raloxifene, and megestrol; LHRH agonists such as goserelin and leuprorelin acetate; antiandrogens such as flutamide and bicalutamide; retinoids/deltaids: vitamin D3 analogs such as EB 1089, CB 1093, and KH 1060; photodynamic therapy such as verteporfm (BPD-MA), phthalocyanine, photosensitizer Pc4, methoxy hypocrellin a and (2 BA-2-DMHA); cytokines such as interferon alpha and interferon gamma; tumor necrosis factor; and other inhibitors such as isopropanylation inhibitors such as lovastatin; dopaminergic neurotoxins, such as l-methyl-4-phenylpyridinium ion; cell cycle inhibitors such as staurosporine; actinomycins such as actinomycin D and dactinomycin; bleomycins such as bleomycin A2, bleomycin B2 and percomycin; anthracyclines such as daunorubicin, doxorubicin (doxorubicin), idarubicin, epirubicin, pirarubicin, zolobicin, and mitoxantrone; MDR inhibitors such as verapamil and Ca2+ ATPase inhibitors such as thapsigargin.
Exemplary embodiments
The following embodiments further illustrate the invention, which should not be construed as limiting.
1. A binding agent, comprising:
a first binding domain that specifically binds a first antigen selected from antigens expressed on cd8+kir+t regulatory cells (tregs) other than KIR proteins; and
a second binding domain that specifically binds an inhibitory KIR protein, wherein the binding agent binds cd8+kir+treg.
2. The binding agent according to the preceding embodiment, wherein the first antigen is selected from the group consisting of: CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4 and 41BB.
3. The binding agent of any one of the preceding embodiments, wherein the first antigen is selected from the group consisting of: CD3, CD8, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4 and 41BB.
4. The binding domain according to any one of the preceding embodiments, wherein the first antigen is selected from the group of antigens consisting of:
CD3, CD5, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB and HLA-DR;
lag-3/CD223, TIM-3, PD-1, S1000A8/9 and TLT2;
CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4 and 41BB;
CD103 (ITGAE), CD166, CD177, CXCR3 and S1000A8/9;
ccr7, CXCR3 and CXCR5;
pd-1, ICOS and CXCR3;
cd3, CD5 and CD8; and
cd3 and CD8.
5. The binding domain according to any one of the preceding embodiments, wherein the first antigen is selected from the group of antigens consisting of:
CD3, CD27, CD38, CD39, CD40L, CD45RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB and HLA-DR;
lag-3/CD223, TIM-3, PD-1, S1000A8/9 and TLT2;
CD3, CD8, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4 and 41BB;
CD103 (ITGAE), CD166, CD177, CXCR3 and S1000A8/9;
ccr7, CXCR3 and CXCR5; and
cd3 and CD8.
6. The binding agent according to any one of the preceding embodiments, wherein the binding agent is a bispecific antibody, diabody antibody, antibody Fc fusion protein, scFv1-scFv2, scFv1 2 -Fc-scFv2 2 IgG-scFv, DVD-Ig, trifunctional antibody/tetravalent body tumor, two-in-one IgG, scFv 2 -Fc, tandAb, scFv-HSA-scFv, scFv-VHH, fab-scFv-Fc, fab-VHH-Fc, dAb-IgG, igG-VHH, tandem scFv-Fc, (scFv 1) 2 -Fc-(VHH) 2 BiTe, DART, cross mab, scFv-Fc, single arm tandem scFv-Fc, DART-Fc, anticalin, affibody, avimer, DARPin or adnectin.
7. The binding agent according to any one of the preceding embodiments, wherein either the first binding domain or the second binding domain is selected from an antibody or antigen binding portion thereof and the other binding domain is an antibody fragment.
8. Root of Chinese characterThe binding agent of embodiment 7, wherein the antigen binding portion is Fab, fab ', F (ab') 2 Fv, scFv or single domain antibodies, such as VHH, VNAR, sdAb or nanobodies.
9. The binding agent of any one of the preceding embodiments, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region.
10. The binding agent of any one of the preceding embodiments, wherein the second binding domain comprises a heavy chain variable region and a light chain variable region.
11. The binding agent according to any one of the preceding embodiments, wherein the first binding domain specifically binds CD3 or a subunit of CD3, optionally CD3 epsilon.
12. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL amino acid sequences being selected from the group consisting of amino acid sequence pairs shown in seq id nos:
a. SEQ ID NO. 1 and SEQ ID NO. 2, respectively;
b. SEQ ID NO 9 and SEQ ID NO 10, respectively;
c. SEQ ID NO. 17 and SEQ ID NO. 18, respectively;
d. SEQ ID NO. 25 and SEQ ID NO. 26, respectively;
e. SEQ ID NO 33 and SEQ ID NO 34, respectively;
f. SEQ ID NO 41 and SEQ ID NO 34, respectively;
g. SEQ ID NO 45 and SEQ ID NO 34, respectively;
h. SEQ ID NO. 49 and SEQ ID NO. 50, respectively;
i. SEQ ID NO 57 and SEQ ID NO 58, respectively;
j. SEQ ID NO. 65 and SEQ ID NO. 66, respectively; and
k. SEQ ID NO. 65 and SEQ ID NO. 166, respectively.
13. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 1 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 2.
14. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 9 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 10.
15. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 17 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 18.
16. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 25 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 26.
17. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 33 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 34.
18. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 41 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 34.
19. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 45 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 34.
20. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 49 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 50.
21. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 57 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 58.
22. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 65 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 66.
23. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 65 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 166.
24. The binding agent of embodiment 11, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region comprising hCDR1, hCDR1 and hCDR3, respectively, and CDR1, CDR2 and CDR3, respectively, the CDRs having amino acid sequences selected from the group of amino acid sequences shown in seq id nos:
a. SEQ ID NO 3 to SEQ ID NO 8, respectively;
b. SEQ ID NO. 11 to SEQ ID NO. 16, respectively;
c. SEQ ID NO 19 through SEQ ID NO 24, respectively;
d. SEQ ID NO 27 through SEQ ID NO 32, respectively;
e. SEQ ID NO 35 to SEQ ID NO 40, respectively;
f. SEQ ID NO. 42 to SEQ ID NO. 44 and SEQ ID NO. 38 to SEQ ID NO. 40, respectively;
g. 46 to 48 and 38 to 40, respectively;
h. SEQ ID NO. 51 to SEQ ID NO. 56, respectively;
i. 59 to 64, respectively;
j. SEQ ID NO 67 to SEQ ID NO 72, respectively; and
k. SEQ ID NOS 67-69 and 167-169, respectively.
25. The binding agent of any one of embodiments 1 to 10, wherein the first binding domain specifically binds CD8 or a subunit of CD8, optionally CD8 a.
26. The binding agent of embodiment 25, wherein the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having amino acid sequences selected from the group consisting of amino acid sequence pairs shown in seq id nos:
a. SEQ ID NO 73 and SEQ ID NO 74, respectively; and
b. SEQ ID NO. 81 and SEQ ID NO. 82, respectively;
or the first binding domain comprises a VHH chain having an amino acid sequence selected from the group of amino acid sequences shown in seq id no:
c.SEQ ID NO:89；
d, SEQ ID NO. 93; and
e.SEQ ID NO:97。
27. the binding agent of embodiment 25, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 73 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 74.
28. The binding agent of embodiment 25, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 81 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 82.
29. The binding agent of embodiment 25, wherein the first binding domain comprises a VHH chain having the amino acid sequence shown in SEQ ID No. 89.
30. The binding agent of embodiment 25, wherein the first binding domain comprises a VHH chain having the amino acid sequence shown in SEQ ID No. 93.
31. The binding agent of embodiment 25, wherein the first binding domain comprises a VHH chain having the amino acid sequence shown in SEQ ID No. 97.
32. The binding agent of embodiment 25, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region comprising hCDR1, hCDR1 and hCDR3, respectively, and CDR1, CDR2 and CDR3, respectively, the CDRs having amino acid sequences selected from the group of amino acid sequences shown in seq id nos:
a. 75 to 80 respectively; or (b)
b. 83 to 88 respectively;
or the first binding domain comprises a VHH chain having hCDR1, hCDR2 and hCDR3, the VHH CDR having an amino acid sequence selected from the group of amino acid sequences shown in the group consisting of seq id nos:
c. SEQ ID NO 90 to SEQ ID NO 92, respectively;
d. 94 to 96 respectively; and
e. 98 to 100, respectively.
33. The binding agent of any one of embodiments 1 to 10, wherein the first binding domain specifically binds ICOS.
34. The binding agent of embodiment 33, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 170 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 171.
35. The binding agent of embodiment 33, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region comprising hCDR1, hCDR1 and hCDR3 amino acid sequences according to SEQ ID NOs 172, 173 and 174, respectively, and cdr1, cdr2 and cdr3 amino acid sequences according to SEQ ID NOs 175, 176 and 177, respectively.
36. The binding agent of any one of embodiments 1 to 10, wherein the first binding domain specifically binds PD-1.
37. The binding agent of embodiment 36, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 178 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 179.
The binding agent according to embodiment 36, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region comprising hCDR1, hCDR1 and hCDR3 amino acid sequences according to SEQ ID NOs 180, 181 and 182 respectively, and ilcdr 1, lCDR2 and lCDR3 amino acid sequences according to SEQ ID NOs 183, 184 and 185 respectively.
39. The binding agent of any one of embodiments 1 to 10, wherein the first binding domain specifically binds CXCR3.
40. The binding agent of embodiment 39, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 186 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 187.
41. The binding agent of embodiment 39, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region comprising hCDR1, hCDR1 and hCDR3 amino acid sequences according to SEQ ID NOs 188, 189 and 190, respectively, and cdr1, cdr2 and cdr3 amino acid sequences according to SEQ ID NOs 191, 192 and 193, respectively.
42. The binding agent of any one of embodiments 1 to 10, wherein the first binding domain specifically binds CD5.
43. The binding agent of embodiment 42, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 194 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 195.
44. The binding agent of embodiment 42, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region comprising hCDR1, hCDR1 and hCDR3 amino acid sequences according to SEQ ID NOs 196, 197 and 198, respectively, and cdr1, cdr2 and cdr3 amino acid sequences according to SEQ ID NOs 199, 200 and 201, respectively.
45. The binding agent of any one of the preceding embodiments, wherein the inhibitory KIR protein is selected from KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2, and KIR2DL3, or a combination thereof.
46. The binding agent of embodiment 45, wherein the second binding agent specifically binds KIR2DL1/2/3 or KIR2DL1/2.
47. The binding agent of any one of the preceding embodiments, wherein the second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL having amino acid sequences selected from the group of amino acid sequence pairs shown in:
a. SEQ ID NO. 101 and SEQ ID NO. 102, respectively;
b. SEQ ID NO 109 and SEQ ID NO 110, respectively;
c. SEQ ID NO 117 and SEQ ID NO 118, respectively;
d. SEQ ID NO. 125 and SEQ ID NO. 126, respectively;
e. SEQ ID NO 133 and SEQ ID NO 134, respectively;
f. 141 and 142, respectively;
g. 149 and 150, respectively; and
h. SEQ ID NO. 157 and SEQ ID NO. 158, respectively.
48. The binding agent of any one of the preceding embodiments, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 101 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 102.
49. The binding agent of any one of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 109 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 110.
50. The binding agent of any one of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 117 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 118.
51. The binding agent of any one of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 125 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 126.
52. The binding agent of any one of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 133 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 134.
53. The binding agent of any one of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 141 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 142.
54. The binding agent of any one of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 149 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 150.
55. The binding agent of any one of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region (VH) having the amino acid sequence set forth in SEQ ID No. 157 and a light chain variable region (VL) having the amino acid sequence set forth in SEQ ID No. 158.
56. A binding agent according to any one of claims 1 to 47, wherein the second binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable region comprising hCDR1, hCDR1 and hCDR3, respectively, and CDR1, CDR2 and CDR3, respectively, the CDRs having amino acid sequences selected from the group of amino acid sequences shown in seq id nos:
a. 103 to 108 respectively;
b. SEQ ID NO 111 through SEQ ID NO 116, respectively;
c. 119 to 124 respectively;
d. 127 to 132 respectively;
e. SEQ ID NO. 135 to SEQ ID NO. 140, respectively;
f. SEQ ID NO 143 to SEQ ID NO 148, respectively;
g. 151 to 156 respectively; and
h. 159 to 164, respectively.
57. The binding agent of any one of the preceding embodiments, wherein the binding agent does not comprise an Fc domain.
58. The binding agent of any one of embodiments 1 to 56, further comprising an Fc domain.
59. The binding agent of embodiment 58, wherein the Fc domain is selected from the group consisting of IgG1 and IgG4 Fc domains.
60. The binding agent of embodiment 59, wherein the binding agent has substantially no effector function activity.
61. The binding agent of any one of embodiments 58 to 60, wherein the Fc domain is an IgG1 Fc domain.
62. The binding agent of any one of embodiments 58 to 61, wherein the Fc domain is an IgG1 Fc null.
63. The binding agent according to any one of the preceding embodiments, wherein the binding agent is divalent or tetravalent.
64. The binding agent of any one of the preceding embodiments, wherein the binding agent is bispecific.
65. A pharmaceutical composition comprising the binding agent of any one of embodiments 1 to 64 and a pharmaceutically acceptable carrier.
66. A nucleic acid encoding the binding agent of any one of embodiments 1 to 64.
67. A vector comprising the nucleic acid of embodiment 66.
68. A cell line comprising the vector of embodiment 67.
69. A method of treating an autoimmune disease comprising administering to a subject in need thereof the binding agent of any one of embodiments 1-64 or the pharmaceutical composition of embodiment 65 in an amount effective to reduce the number or activity of pathogenic immune cells and thereby ameliorate a symptom of an autoimmune disease.
70. A method of inhibiting an immune response mediated by pathogenic immune cells comprising contacting cd8+kir+t regulatory cells (tregs) with an amount of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 effective to activate or stimulate cd8+kir+tregs (activated tregs), thereby reducing the number or activity of pathogenic immune cells.
71. A method of inhibiting an immune response to an antigen (such as an autoantigen), comprising administering to a subject in need thereof the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 in an amount effective to activate or stimulate cd8+ kir+ tregs, thereby reducing the number or activity of pathogenic immune cells that are responsive to the antigen or autoantigen.
72. The method of embodiment 70, wherein the cd8+ kir+ Treg is contacted with the binding agent in vivo.
73. The method of embodiment 70, wherein the cd8+ kir+ Treg is contacted with the binding agent ex vivo.
74. The method of embodiment 73, wherein the activated cd8+ kir+ tregs are administered to a subject in need thereof in an effective amount.
75. The method of any one of embodiments 69 to 74 wherein the autoimmune cell is an autoreactive cd4+ T cell, an autoantibody producing B cell, or a dendritic cell presenting an autoantigen.
76. The method of any one of embodiments 69 to 74 wherein the pathogenic immune cell is a self antigen presenting cell.
77. The method of embodiment 71, wherein the titer of autoantibodies in the subject is decreased.
78. The method of any one of embodiments 69 and 74-77, wherein the subject has an autoimmune disease selected from the group consisting of: celiac disease, crohn's disease, juvenile idiopathic arthritis, inflammatory Bowel Disease (IBD), insulin dependent diabetes mellitus (IDDM or type 1 diabetes), lupus nephritis, myasthenia gravis, myocarditis, multiple Sclerosis (MS), pemphigus/pemphigoid, rheumatoid Arthritis (RA), scleroderma/systemic sclerosis, sjogren's syndrome (SjS), systemic Lupus Erythematosus (SLE), and ulcerative colitis.
79. The method of embodiment 78, wherein the autoimmune disease is selected from the group consisting of: celiac disease, crohn's disease, inflammatory Bowel Disease (IBD), insulin dependent diabetes mellitus (IDDM or type 1 diabetes), lupus nephritis, multiple Sclerosis (MS), rheumatoid Arthritis (RA), scleroderma/systemic sclerosis, sjogren's syndrome (SjS), systemic Lupus Erythematosus (SLE) and ulcerative colitis.
80. The method of any one of embodiments 69-79, wherein the binding agent specifically binds to an inhibitory KIR protein on CD8 and cd8+ kir+ tregs.
81. The method of any one of embodiments 69-79, wherein the binding agent specifically binds to an inhibitory KIR protein on CD3 and cd8+ kir+ tregs.
82. The method of any one of embodiments 69-79, wherein the binding agent specifically binds to an inhibitory KIR protein on CD5 and cd8+ kir+ tregs.
83. The method of any one of embodiments 69-79, wherein the binding agent specifically binds to an inhibitory KIR protein on PD-1 and cd8+ kir+ tregs.
84. The method of any one of embodiments 69-79, wherein the binding agent specifically binds to inhibitory KIR proteins on ICOS and cd8+ kir+ tregs.
85. The method of any one of embodiments 69-79, wherein the binding agent specifically binds to an inhibitory KIR protein on CXCR3 and cd8+ kir+ tregs. 86. The method of any one of embodiments 69-85, wherein the cd8+ kir+ tregs are MHC class I-restricted.
87. The method of any one of embodiments 69-86, wherein the cd8+ kir+ tregs are not MHC HLA E (Qa-1 b) restricted.
88. The method of any one of embodiments 69 to 87, further comprising administering to the subject an immunosuppressant.
89. The method of any one of embodiments 69-88, wherein administering the binding agent to the subject results in improved therapeutic outcome in the subject.
90. The method of embodiment 89, wherein the improved therapeutic result is a reduced frequency and severity of disease onset, a reduced systemic inflammatory cytokine, or a reduced self-reporting of symptoms associated with the autoimmune disease.
91. The method of any one of embodiments 69 to 90 wherein the binding agent is administered intravenously.
92. The method of any one of embodiments 69 to 91 wherein the binding agent is administered subcutaneously.
93. The method of any one of embodiments 69 to 92 wherein the binding agent is administered at a dose of about 0.01mg/kg to about 20 mg/kg.
94. The method of any one of embodiments 69 to 93 wherein the binding agent has substantially no effector function activity.
95. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for treating an autoimmune disease in a subject by activating or stimulating cd8+kir+ tregs.
96. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for reducing the immune response of pathogenic immune cells by activating or stimulating cd8+kir+ tregs.
97. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for reducing autoantibody titers in a subject by activating or stimulating cd8+kir+ tregs.
98. A method of treating cancer comprising administering to a subject in need thereof the binding agent of any one of embodiments 1-64 or the pharmaceutical composition of embodiment 65, wherein the binding agent has substantially no effector function activity, in an amount effective to activate or stimulate cd8+kir+ tregs and thereby ameliorate symptoms of the cancer.
99. A method of stimulating an immune response against an antigen associated with cancer (cancer antigen), comprising contacting cd8+kir+t regulatory cells (tregs) with the binding agent according to any one of embodiments 1 to 64 or the pharmaceutical composition according to embodiment 65 in an amount effective to activate or stimulate cd8+kir+tregs (activated tregs), thereby increasing the immune response to the cancer antigen, wherein the binding agent has substantially no effector function activity.
100. A method of treating cancer comprising administering to a subject in need thereof a binding agent according to any one of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65, wherein the binding agent has effector function activity comprising at least ADCC, in an amount effective to deplete cd8+kir+ tregs and thereby ameliorate a symptom of the cancer.
101. A method of stimulating an immune response against an antigen associated with cancer (cancer antigen), comprising contacting cd8+kir+t regulatory cells (tregs) with the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 in an amount effective to deplete cd8+kir+tregs, thereby increasing the immune response to the cancer antigen, wherein the binding agent has effector function activity including at least ADCC.
102. The method of embodiment 99 or embodiment 101, wherein the cd8+ kir+ Treg is contacted with the binding agent in vivo.
103. The method of embodiment 99, wherein the cd8+ kir+ Treg is contacted with the binding agent ex vivo.
104. The method of embodiment 103, wherein the activated cd8+ kir+ Treg is administered to a subject in need thereof in an effective amount.
105. The method of any one of embodiments 99 and 101-104, wherein the increased immune response comprises cancer cytopenia or immunosuppressive immune cell depletion.
106. The method of any one of embodiments 98 to 105, wherein the cancer cells in the subject are reduced.
107. The method according to any one of embodiments 98 to 106, wherein the cancer is selected from the group consisting of: carcinoma, lymphoma, blastoma, sarcoma, myeloma, and leukemia.
108. The method according to any one of embodiments 98 to 107, wherein the cancer is selected from the group consisting of: solid tumors such as breast cancer, cervical cancer, ovarian cancer, lung cancer, CRC (and other bowel cancers), skin cancer, esophageal cancer, adenocarcinoma, bladder cancer, and prostate cancer; lymphoma.
109. The method of any one of embodiments 98-108, wherein the binding agent specifically binds to an inhibitory KIR protein on CD8 and cd8+ kir+ tregs.
110. The method of any one of embodiments 98-108, wherein the binding agent specifically binds to an inhibitory KIR protein on CD3 and cd8+ kir+ tregs.
111. The method of any one of embodiments 98-108, wherein the binding agent specifically binds to an inhibitory KIR protein on CD5 and cd8+ kir+ tregs.
112. The method of any one of embodiments 98-108, wherein the binding agent specifically binds to an inhibitory KIR protein on PD-1 and cd8+ kir+ tregs.
113. The method of any one of embodiments 98-108, wherein the binding agent specifically binds to inhibitory KIR proteins on ICOS and cd8+ kir+ tregs.
114. The method of any one of embodiments 98-108, wherein the binding agent specifically binds to an inhibitory KIR protein on CXCR3 and cd8+ kir+ tregs.
115. The method of any one of embodiments 98-114, wherein the cd8+kir+ Treg is MHC class I-restricted.
116. The method of any one of embodiments 98-115, wherein the cd8+kir+ Treg is not MHC HLA E (Qa-1 b) restricted.
117. The method of any one of embodiments 98 to 116, further comprising administering to the subject a chemotherapeutic agent or an immunotherapy, such as an immune checkpoint inhibitor.
118. The method of any one of embodiments 98 to 117, wherein administration of the binding agent to the subject results in improved therapeutic outcome in the subject.
119. The method of embodiment 118, wherein the improved therapeutic result is a partial response or a complete response.
120. The method of embodiment 118, wherein the improved therapeutic result is remission.
121. The method of any one of embodiments 98 to 120, wherein the binding agent is administered intravenously.
122. The method of any one of embodiments 98 to 120, wherein the binding agent is administered subcutaneously.
123. The method of any one of embodiments 98 to 122, wherein the binding agent is administered at a dose of about 0.01mg/kg to about 20 mg/kg.
124. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for treating cancer in a subject by activating or stimulating cd8+kir+ tregs, wherein the binding agent has substantially no effector function activity.
125. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for reducing immunosuppression of immunosuppressive immune cells by activating or stimulating cd8+kir+ tregs, wherein the binding agent has substantially no effector function activity.
126. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for reducing tumor burden in a subject by activating or stimulating cd8+kir+ tregs, wherein the binding agent has substantially no effector function activity.
127. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for treating cancer in a subject by depleting cd8+kir+ Treg, wherein the binding agent has effector function activity comprising at least ADCC.
128. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for depleting cd8+kir+ tregs, wherein said binding agent has effector function activity including at least ADCC.
129. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for reducing tumor burden in a subject, wherein the binding agent has effector function activity comprising at least ADCC.
130. A method of treating an infection comprising administering to a subject in need thereof the binding agent of any one of embodiments 1-64 or the pharmaceutical composition of embodiment 65 in an amount effective to activate or stimulate cd8+kir+ tregs and thereby ameliorate symptoms of the infection.
131. A method of stimulating an immune response against an infected cell caused by an infection comprising contacting a cd8+kir+t regulatory cell (Treg) with the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 in an amount effective to activate or stimulate a cd8+kir+treg (activated Treg), thereby generating the immune response against the infected cell.
132. The method of embodiment 131, wherein the cd8+ kir+ Treg is contacted with the binding agent in vivo.
133. The method of embodiment 131, wherein the cd8+ kir+ Treg is contacted with the binding agent ex vivo.
134. The method of embodiment 133, wherein the activated cd8+ kir+ tregs are administered to a subject in need thereof in an effective amount.
135. The method of any one of embodiments 131 to 134, wherein the immune response comprises an infectious cytopenia or an immunosuppressive immunocytopenia selected from the group consisting of: cd4+ T regulatory cells and tolerogenic DCs.
136. The method of embodiment 135, wherein the number of infected cells in the subject is reduced.
137. The method according to any one of embodiments 130 to 136, wherein the infection is selected from the group consisting of a bacterial disease, a systemic fungal disease, a rickettsiosis, a parasitic disease, and a viral disease.
138. The method of embodiment 137, wherein the infection is selected from the group consisting of: HIV infection, hepatitis C Virus (HCV) infection, human Papilloma Virus (HPV) infection, epstein Barr Virus (EBV) infection, coronavirus infection such as SARS-COV2 infection (Covid-19), cytomegalovirus (CMV) infection, and influenza virus infection.
139. The method of any one of embodiments 130-138, wherein the binding agent specifically binds to an inhibitory KIR protein on CD8 and cd8+ kir+ tregs.
140. The method of any one of embodiments 130-138, wherein the binding agent specifically binds to an inhibitory KIR protein on CD3 and cd8+ kir+ tregs.
141. The method of any one of embodiments 130-138, wherein the binding agent specifically binds to an inhibitory KIR protein on CD5 and cd8+ kir+ tregs.
142. The method of any one of embodiments 130-138, wherein the binding agent specifically binds to an inhibitory KIR protein on PD-1 and cd8+ kir+ tregs.
143. The method of any one of embodiments 130-138, wherein the binding agent specifically binds to inhibitory KIR proteins on ICOS and cd8+ kir+ tregs.
144. The method of any one of embodiments 130-138, wherein the binding agent specifically binds to an inhibitory KIR protein on CXCR3 and cd8+ kir+ tregs.
145. The method of any one of embodiments 130-144, wherein the cd8+kir+ Treg is MHC class I-restricted.
146. The method of any one of embodiments 130-145, wherein the cd8+kir+ Treg is not MHC HLA E (Qa-1 b) restricted.
147. The method of any one of embodiments 130-146, further comprising administering to the subject an antimicrobial or antiviral agent.
148. The method of any one of embodiments 130 to 147, wherein administering the binding agent to the subject results in improved therapeutic outcome in the subject.
149. The method of embodiment 148, wherein the improved therapeutic result is a reduction in infection or infected cells.
150. The method of any one of embodiments 130 to 149, wherein the binding agent is administered intravenously.
151. The method of any one of embodiments 130 to 149, wherein the binding agent is administered subcutaneously.
152. The method of any one of embodiments 130 to 151, wherein the binding agent is administered at a dose of about 0.01mg/kg to about 20 mg/kg.
153. The method of any one of embodiments 130 to 149, wherein the binding agent has substantially no effector function activity.
154. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for treating an infection in a subject by activating or stimulating cd8+kir+ tregs.
155. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for stimulating an immune response by activating or stimulating cd8+kir+ tregs and thereby inhibiting immunosuppressive immune cells.
156. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for reducing infection or infected cells in a subject by activating or stimulating cd8+kir+ tregs.
157. A method of reducing or preventing the onset of Graft Versus Host Disease (GVHD) after transplantation comprising administering to a subject in need thereof the binding agent of any one of embodiments 1-64 or the pharmaceutical composition of embodiment 65 in an amount effective to activate or stimulate cd8+kir+ Treg and thereby reduce or ameliorate at least one symptom of GVHD, wherein the binding agent has substantially no effector function activity.
158. A method of treating a subject receiving a graft, comprising contacting cd8+ kir+ T regulatory cells (tregs) with the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 in an amount effective to activate or stimulate cd8+ kir+ tregs (activated tregs), thereby reducing or inhibiting GVHD, wherein the binding agent has substantially no effector function activity.
159. A method of treating a subject receiving a graft, comprising administering to a subject in need thereof a binding agent according to any one of embodiments 1 to 64 or a pharmaceutical composition according to embodiment 65, wherein the binding agent has effector function activity comprising at least ADCC, in an amount effective to deplete cd8+kir+ tregs and thereby ameliorate symptoms of GVHD.
160. A method of inhibiting GVHD against a graft comprising contacting cd8+kir+t regulatory cells (tregs) with the binding agent according to any one of embodiments 1 to 64 or the pharmaceutical composition according to embodiment 65 in an amount effective to deplete cd8+kir+tregs, thereby reducing GVHD or symptoms thereof, wherein the binding agent has effector function activity including at least ADCC.
161. The method of any one of embodiments 158 or 160, wherein cd8+ kir+ tregs are contacted with said binding agent in vivo.
162. The method of embodiment 158, wherein the cd8+ kir+ Treg is contacted with the binding agent ex vivo.
163. The method of embodiment 162, wherein the activated cd8+ kir+ Treg is administered to a subject in need thereof in an effective amount.
164. The method of any one of embodiments 157 and 160-163, wherein reducing or reducing GVHD comprises reducing cd4+ T cell activity in GVHD.
165. The method according to any one of embodiments 157 to 164, wherein the graft is selected from the group consisting of: organ grafts, hematopoietic stem cell grafts, cord blood stem cell grafts, induced pluripotent stem cell-derived progenitor or differentiated cell grafts, and bone marrow grafts.
166. The method of any one of embodiments 165, wherein the graft is a hematopoietic stem cell graft, an umbilical cord blood stem cell graft, an induced pluripotent stem cell-derived progenitor cell, or a differentiated cell graft, or a bone marrow graft.
167. The method of any one of embodiments 157 to 166, wherein the graft is allogeneic.
168. The method of any one of embodiments 157-167, wherein the binding agent specifically binds to an inhibitory KIR protein on CD8 and cd8+ kir+ tregs.
169. The method of any one of embodiments 157-167, wherein the binding agent specifically binds to an inhibitory KIR protein on CD3 and cd8+ kir+ tregs.
170. The method of any one of embodiments 157-167, wherein the binding agent specifically binds to an inhibitory KIR protein on CD5 and cd8+ kir+ tregs.
171. The method of any one of embodiments 157-167, wherein the binding agent specifically binds to an inhibitory KIR protein on PD-1 and cd8+ kir+ tregs.
172. The method of any one of embodiments 157-167, wherein the binding agent specifically binds to inhibitory KIR proteins on ICOS and cd8+ kir+ tregs.
173. The method of any one of embodiments 157-167, wherein the binding agent specifically binds to an inhibitory KIR protein on CXCR3 and cd8+ kir+ tregs.
174. The method of any one of embodiments 157-173, wherein the cd8+ kir+ tregs are MHC class I-restricted.
175. The method of any one of embodiments 157-174, wherein the cd8+kir+ Treg is not MHC HLA E (Qa-1 b) restricted.
176. The method of any one of embodiments 157-175, further comprising administering an immunosuppressant to the subject.
177. The method of any one of embodiments 157 to 176, wherein administering the binding agent to the subject results in improved therapeutic outcome in the subject.
178. The method of embodiment 177, wherein the improved treatment results in reduced symptoms associated with GVHD, reduced systemic inflammatory cytokines, reduced pathology in tissues affected by GVHD, reduced self-reporting of symptoms associated with an immune response associated with an adverse effect on host tissue, improved or prolonged implantation of the graft, reduced one or more symptoms, and/or prevention, prolonged or slowed development or progression of graft rejection, or prolonged implantation of the graft with reduced use of a broad-spectrum immunosuppressive agent (e.g., a corticosteroid).
179. The method of any one of embodiments 157 to 178, wherein the binding agent is administered intravenously.
180. The method of any one of embodiments 157 to 178, wherein the binding agent is administered subcutaneously.
181. The use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for treating graft-related GVHD in a subject, wherein the binding agent has substantially no effector function activity.
182. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for treating graft-related GVHD in a subject by activating or stimulating cd8+kir+ tregs, wherein the binding agent has substantially no effector function activity.
183. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for reducing graft-related GVHD by activating or stimulating cd8+kir+ tregs, wherein the binding agent has substantially no effector function activity.
184. The use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for reducing GVHD of a graft, wherein the binding agent has substantially no effector function activity.
185. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for treating graft-related GVHD in a subject by depleting cd8+kir+ Treg, wherein the binding agent has effector function activity comprising at least ADCC.
186. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for depleting cd8+kir+ tregs, wherein said binding agent has effector function activity including at least ADCC.
187. Use of the binding agent of any one of embodiments 1 to 64 or the pharmaceutical composition of embodiment 65 for depleting cd8+kir+ tregs in a subject who has received an implant to reduce GVHD, wherein the binding agent has effector function activity including at least ADCC.
188. The use of the binding agent of any one of embodiments 1 to 64 or of embodiment 65 in any of the methods of embodiments 69 to 94, 98 to 123, 130 to 153 and 157 to 180.
189. Use of the binding agent of any one of embodiments 1 to 64 or according to embodiment 65 in the manufacture of a medicament for use in any of the methods of embodiments 69 to 94, 98 to 123, 130 to 153 and 157 to 180.
The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the precise form disclosed. Although specific implementations of, and examples for, the disclosure are described herein for illustrative purposes, various equivalent modifications are possible within the scope of the disclosure, as those skilled in the relevant art will recognize. The teachings of the present disclosure provided herein may be suitably applied to other processes or methods. The various embodiments described herein may be combined to provide further embodiments. Aspects of the disclosure can be modified, if necessary, to employ the compositions, functions and concepts of the above-described references and applications to provide yet another embodiment of the disclosure. These and other changes can be made to the disclosure in light of the detailed description.
Certain elements of any of the foregoing embodiments may be combined or substituted for elements of other embodiments. Moreover, while advantages associated with certain embodiments of the disclosure have been described in the context of those embodiments, other embodiments may also exhibit such advantages, and not all embodiments must exhibit such advantages to fall within the scope of the disclosure.
All patents and other publications identified are expressly incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that might be used in connection with the present application. These publications are provided solely for their disclosure prior to the filing date of the present application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior application or for any other reason. All statements as to the date or content of these documents is based on the information available to the applicant and does not constitute an admission as to the correctness of the dates or contents of these documents.
Examples
Example 1: monospecific and bispecific molecules activate cd8+ kir+ Treg cells in autoimmune disorders And testing for cytotoxicity against pathogenic immune cells
To test for functional recovery of cd8kir+ Treg cell activation and cytotoxicity-mediated elimination of pathogenic immune cells (e.g., autoreactive CD 4T cells) by a panel of monospecific and bispecific molecules (including inhibitory KIR blockers), primary cd8+ kir+ T cells are incubated with increasing concentrations of CD3 agonist antibodies that will mimic peptide/MHC binding of the CD8-kir+ T cell receptor in the presence of increasing concentrations of inhibitory KIR blocking molecules. It is expected that blocking of inhibitory KIR signaling will decrease the activation threshold of TCR involvement required for activation of CD8-kir+ Treg cells, resulting in the specificity and increased activation state of CD8-kir+ T cells. Increased activation of cd8+kir+ Treg cells will be demonstrated by increased secretion of certain cytokines (e.g., ifnγ, IL-10, tnfα, IL-35, or subunits thereof, etc.), increased activation-related expression markers (e.g., CD69, CD25, CD62L, CD44, CD 45), and increased proliferation.
Monospecific and bispecific molecules will be examined for their potency by inducing a functional outcome mediated by cd8kir+ Treg cells, and subsequently gluten re-stimulated CD 4T cells will be tested using peripheral blood mononuclear cell derived cd8kir+ Treg cells from celiac patients. Increased secretion by certain cytokines (e.g., ifnγ, IL-10, tnfα, and/or IL-35, or subunits thereof, etc.), increased expression of markers (e.g., CD69, CD25, CD62L, CD44, and/or CD 45), decreased inhibitory molecules (LAG-3, TIM-3, and/or PD-1), increased proliferation, increased inhibition of autoreactive cd4+ T cells and other pathogenic immune cells, such as autoantibody-producing B cells, autoantigen-presenting dendritic cells, and autoantipcs.
Example 2: monospecific and bispecific molecules activate and attenuate cd8+kir+ Treg cells in infection models Cytotoxicity test
A panel of monospecific and bispecific molecules (including inhibitory KIR blockers) will be tested for functional recovery of cd8kir+ Treg cell activation and cytotoxicity-mediated direct and indirect elimination of pathogen-infected cells. Human CMV specific T cells (Cello) will be incubated with an increased dose of a virus-derived dominant epitope (pp 65) that binds to MHC class I molecule HLA-A2. The peptide will be loaded onto lymphoma cell line T2 expressing HLA-A2. To test for specific inhibitory KIR molecule function, an MHC I-deficient lymphoma T1 cell line or K562 cell line will be transfected with a cognate MHC class I molecule (e.g., HLA-C2 when KIR2DL1 is blocked). To confirm that KIR blockade specifically and effectively reconstitutes the elimination of pathogen-infected cells, activation, cytotoxicity, cytokine production and proliferation of CD8 Treg cells will be examined. The extent of target cell elimination and apoptosis stained and propagated using annexin V will also be determined. As a control we will use, for example, an unrelated influenza hemagglutinin peptide (negative control), CD3 antibody bead activation (positive control) or MHC deficient lymphoma T1 cell line or parental K562 cells (negative control). To detect activation with greater sensitivity, we will transfect Jurkat cells with SHP1/2 or NFAT reporter to show enhanced activation upon inhibitory KIR blockade and/or agonist binding, thereby prioritizing use of the molecule.
Example 3: activation and cytotoxicity of single bispecific molecules against cd8+kir+ Treg cells in cancer models Testing
Cd8kir+ Treg cells will be tested against a set of tumor cell lines with high antigen loading in the presence of a set of KIR bispecific molecules to determine whether KIR blockade improves CD8 KIR-Treg cell activation and target cell killing. KIR bispecific molecules will be tested alone or in combination with other KIR bispecific molecules and in combination with other immune checkpoint inhibitors. For example, studies will test for increased doses of anti-CD 3 agonist antibodies in co-culture with tumor cell lines with increased doses of KIR molecules, including, for example: a549 (NSCLC), H1229 (NSCLC), a375 (melanoma), SK-Mel 3 (melanoma), caki-1 (RCC) and/or 786-O (RCC). The dependence of the haplotype on the response will be determined using, for example, primary NY-ESO-1 specific T cells (Cello) with NY-ESO1 peptide pulse T2 (HLA-A 2 restricted cell line) and/or HLA-A 2K 562. To assess whether HLA-expressing subpopulations of tumor cell targets are necessary for optimal response (e.g., HLA-B binds KIR3DL1 or KIR2DL1/2/3 binds HLA-C), the relevant HLA molecules will be overexpressed in K562 or T1 cells and pulsed with the relevant dominant epitopes (https:// anti bodies. Cancer. Gov/detail/MajorHistocompatibilityComplex ClassICPeptide 1).
Example 4: single bispecific molecule activation and cytotoxicity of CD8+KIR+ Treg cells in GVHD transplantation model Testing of sex
Following hematopoietic stem cell transplantation and other transplantation procedures, serious and life threatening complications may occur, donor-derived cells recognize the heterologous host tissue as foreign and become activated, destroying the healthy cells of the recipient, known as Graft Versus Host Disease (GVHD). Homoreactive graft versus host disease results in graft-related morbidity in up to 50% of the recipients of the graft, accounting for about 20% of deaths after the graft. KIR blockade of cd8+ kir+ tregs can reduce the severity of graft versus host disease in cases where transplanted cells destroy healthy tissue and recognize it as foreign. To test the effect of KIR blockade on graft versus host disease severity, a well-characterized GVHD model will be used in which human immune cells are injected into NOD/SCID/gamma chain (NSG) deficient mice, and then multiple organ acute pathology resulting from human cell activation and destruction of mouse tissues is observed. During the 30-45 day study, monospecific and bispecific molecules blocking KIR will be injected every 72 hours, and endpoint analysis will include histopathological analysis of serum pro-inflammatory cytokines, expression of activation markers for human T cells, disease scores (including survival and body weight), and intestinal tissue inflammation and epithelial cell killing. The present study will support the utility of KIR blocking as a method of reducing the severity of graft versus host disease while preserving a transplanted graft and determine the effect of KIR blocking on systemic disease that may affect multiple organs and tissues.
Example 5: ly49 blockade increases the activity of CD8+Ly49+T regulatory cells
The effect of Ly49 blocking cd8+ly49+ tregs was demonstrated in vitro. Briefly, cells were isolated from the spleen and lymph nodes of C57BL/6 mice on day 10 after EAE induction using standard MOG peptide protocols (see Saligrama et al, nature572:481-487 (2019)). Isolation of CD4+ T cells, CD8+CD28-regulatory T cells and CD8+CD28+ using magnetic separation and in the absence of blocking antibody (control) or F (ab') blocking antibody LY49C/l 2 Fragments (clone 5e6; fc portion of the lacking antibody) (anti-LY 49) were stimulated with CD3/CD28 and cultured with CD 4T cells 1:1.
Referring to fig. 4A-4D, cd8+ Treg activation (fig. 4A), immunosuppressive cytokine production (fig. 4B), cytolytic activity (granzyme B) (fig. 4C), and increased production of CD 4T cell anti-inflammatory IL-10 cytokines (fig. 4D) were statistically significant in the presence of Ly49 block. Similar results were observed with cells stimulated with CD3/CD28 in the presence of full length Ly49C/I blocking antibody (clone 5E 6) (data not shown). These results demonstrate that cd8+ly49+ T cells show increased activation by Ly49/KIR blockade.
Supernatants from cells (above) were collected 48 hours after the start of co-culture and analyzed for various analytes (cytokines) using the Bioplex assay.
The results of this analysis demonstrate that Ly49 blocker inhibits the following pro-inflammatory cytokines in mice samples treated with MOG and inhibitory peptides compared to mice treated with MOG peptide alone: RANTES, IL-6, IL-18, GM-CSF, tnfα and ifnγ (data not shown). In addition, IL-2 and IL-15 levels were reduced in the mice samples treated with MOG and inhibitory peptides compared to mice treated with MOG peptides (data not shown). The levels of anti-inflammatory cytokines IL-22 and MCP-3 were reduced in the samples of mice treated with MOG and inhibitory peptides compared to mice treated with MOG peptides (data not shown).
The effect of Ly49 blockade was evaluated in vivo in the mouse EAE model. Briefly, EAE was induced in C57BL/6 mice using standard MOG injection protocols (see Saligrama et al, nature 572:481-487 (2019)). Mice were administered MOG alone or in admixture with "surrogate peptide" ("SP") or F (ab') 2 Fragments (clone 5E6; lack of Fc portion of antibodies) (anti-LY 49 or "LY49 blocking") were administered in combination (FIG. 5). Ly49 blocking delayed disease onset and reduced disease severity (fig. 6), suggesting that mobilization of cd8+ tregs upon autoimmune triggering helps control the disease.
Example 6: in celiac disease patients, CD8 KIR+ T cells have a greater size than CD8T cells negative for KIR expression Cell lysis potential
Peripheral Blood Mononuclear Cells (PBMCs) were obtained from celiac disease patients and healthy donors. PBMCs were enriched for cd8+ T cells, and several surface markers, including a mixture of CD8 and pan-inhibitory KIR response peptides, were then stained and sorted to obtain cd8+ kir+ T cells and cd8+ KIR-cells. After sorting, PBMCs were stimulated with gluten peptides. Six days after stimulation, intracellular granzyme, perforin and ifnγ levels of CD8 Treg cells were assessed.
PBMCs of celiac patients had a higher percentage of cd8+kir+ Treg cells (fig. 7A). Kir+cd8+ T cells had a higher percentage of perforins, intracellular ifnγ, and granzyme B than cd8+kir-T cells (fig. 7B and 7C). These results indicate that celiac patients carry cd8+kir+ T cells with greater cytolytic potential than KIR negative cd8+ T cells.
Example 7: celiac patients have more kir+cd8+ T cells and more ICOS than healthy control group Expression on kir+ccd8+ T cells
PBMCs from celiac disease patients (six) or healthy donors were analyzed by flow cytometry and cd8+ T cells were gated. Celiac disease patients had PBMCs with more cd3+/pan kir+ T cells than PBMCs of healthy donors (fig. 8A). Celiac disease patients had PBMCs with more cd3+/pan kir+/icos+ cells than PBMCs of healthy donors (fig. 8B). These results indicate that celiac patients have more ICOS expression on kir+cd8+ T cells.
Example 8: gluten re-stimulation increases granzyme B levels, degranulation of cd8+kir+treg and cd4+ T cells Loss of
To determine the effect of gluten re-stimulation on cd8+kir+t cells, PMBC from celiac patients were stimulated with gluten peptides for 12 days to enrich for CD 4-reactive cells and cd8+ Treg cells in the presence of IL-7 and IL-15. CD8 tregs and CD4T cells were then selected and combined with autologous APC 1:1 without peptide, influenza peptide or gluten peptide. After 48 hours, the cells were analyzed by flow cytometry.
Restimulation with gluten peptide increased degranulation as measured by CD107 (fig. 9A, left panel) and granzyme B levels (fig. 9A, right panel) compared to restimulation of control influenza peptide or unstimulated cells. (598 refers to PBMC from patient 598). Re-stimulation of gluten also resulted in a reduced percentage of activated cd4+ cells, whereas re-stimulation of influenza peptide was absent (fig. 9B). These results indicate that the antigen response of cd8+kir+ tregs is specific, consistent with the Treg source of celiac patients, and rapid and sustained. These results indicate that cd8+ Treg cells are upregulated, cd4+ T cell activation is downregulated, and pathogenic cd4+ T lymphocytes are eliminated.
Example 9: KIR blockade increases granzyme B content and degranulation of cd8+ T cells
CD8+CD16+ T cells were selected from 3 patients diagnosed with celiac disease and cultured with CD4+ T cells and 1 μg/ml of anti-CD 3 agonist antibody (clone OKT 3) 1:1 in the presence or absence of 100 μg/ml of KIR2DL1/2/3 and KIR3DL1 antagonist antibody (50 μg each). After 48 hours, cd8+ Treg cells were analyzed using flow cytometry. KIR blockade ("KIR blockade") increased intracellular granzyme B levels (fig. 10A) and degranulation (CD 107) (fig. 10B).
In another experiment, the effect of KIR blockade was assessed in vitro using PBMCs from celiac patients. Briefly, cd8+ tregs were enriched from PBMCs of celiac patients and cultured with autologous CD 4T cells pulsed with a prolamin peptide mixture and antigen presenting cells and analyzed using flow cytometry. Following restimulation, cells administered with anti-inhibitory KIR antibodies (anti-KIR 2DL1/2/3, anti-KIR 3DL1, or a mixture of anti-KIR 2DL1/2/3 and KIR3 DLl) exhibited induction of cytolytic activity of cd8+ T cells, decreased activation of cd4+ T cells, and increased cd4+ T death (fig. 11). These results were consistently observed in several patient samples for monospecific and bispecific KIR blockade.
Example 10: KIR blockade reduces cd4+ T cell activation
CD8+CD16+ T cells were selected from 3 patients diagnosed with celiac disease and cultured with CD4+ T cells and 1 μg/ml of anti-CD 3 agonist antibody (clone OKT 3) 1:1 in the presence or absence of 100 μg/ml of KIR2DL1/2/3 and KIR3DL1 antagonist antibody (50 μg each). After 48 hours, cd8+ Treg cells were analyzed by flow cytometry. KIR blockade reduced activation and proliferation of cd4+ T cells (CD 69) in samples of all three patients (fig. 12).
Example 11: selection of the relevance of KIR proteins to cd8+ Treg cell HLA ligand expression in celiac disease
Celiac patient PBMC were stained with antibodies to KIR2DL1/2/3 and KIR3DL 1. After gating on CD 8T cells, the percentage of positive cells for KIR ligands and HLA haplotypes was determined. (HLA and KIR typing were performed in cooperation with Scisco Genetics). CD 8T cells from KIR expressing patients were as follows: in peripheral blood, 3/3 expresses KIR2DL and 2/3 expresses KIR3DL. The HLA ligands of the KIR are selected to be overexpressed in samples of celiac disease patients. 9/10 patients have at least one copy of HLA-C07:01 and 10/10 patients have at least one copy of HLA-B08:01:01.
Example 12: characterization of bispecific molecules co-binding CD8 and KIR2DL
Cross-mAb was prepared using Fab binding to KIR2L1/2/3 (prepared from IPH2102 IgG1r mAb (parent antibody VH and VL sequences, SEQ ID NOS: 101 and 102, respectively)) and scFv binding to CD8 alpha (prepared from Mb1b IgG1r mAb (parent antibody VH and VL sequences, SEQ ID NOS: 81 and 82, respectively). Fab and scFv are linked to IgG1 hinge-CH 2-CH3, wherein the CH3 domain is designed to contain a "knob" mutation to enhance proper binding of the two heterodimeric heavy chains. The "knob" heavy chain contains the mutations S354C and T366W. The "mortar" heavy chain contains mutations Y349C, T366S, L368A and Y407V.
Co-binding of KIR2L1/2/3-CD8 alpha cross-MAb to KIR2DL1 or KIR2DL3 and CD8 alpha was tested by biolayer interferometry using an Octet instrument. For co-binding studies, cross-MAb was captured onto AHC (anti-human Fc) biosensors using a 2-fold dilution of 0.3125 μg/ml to 20 μg/ml. Analytes (KIR 2DL1, KIR2DL3 and CD8 a) were kept constant at 100 nM. The analyte co-binding after capture is analyzed in two ways: first, KIR2DL1 or KIR2DL3, then binds directly to CD 8a, or CD 8a, then binds directly to KIRDL1 or KIR2DL 4. KIR2DL1, KIR2DL3 and CD 8a are tagged with hexahistidine peptides. The cross Mab is capable of co-binding to the target KIR2DL1 or KIR2DL3 and CD8 a.
Cross-MAb affinity for KIR2DL1, KIR2DL3 and CD8 alpha ligands was measured using an Octet instrument and compared to anti-CD 8 alpha and anti-KIR 2DL1/L2/L3 parent antibodies. For kinetic analysis, a loading concentration of 1.25 μg/ml was used to capture the cross-MAb onto the AHC (anti-human Fc) biosensor. The concentration of each analyte (KIR 2DL1, KIR2DL3 and CD8 a) ranged from 6.25nM to 200nM. The binding of the analytes after capture was first analyzed for KIR2DL1, KIR2DL3 or CD8 a binding, and then dissociation of each analyte was analyzed independently. This ensures that ka (binding rate), kD (dissociation rate) and kD values can be obtained and compared directly to the parent antibody. Kinetic analysis showed that the cross Mab retains affinity for the targets KIR2DL1, KIR2DL3 and CD8 a.
The parent antibodies were also analyzed for affinity against KIR2DL1/L2/L3 IPH2102 IgG1r and against CD 8. Alpha. Mb1b IgG1r mAb. For kinetic analysis, parent antibodies were individually captured onto an AHC (anti-human Fc) biosensor using a loading concentration of 1.25 μg/ml. For the IPH2102 IgG1r mAb, the KIR2DL1 or KIR2DL3 analyte ranged from 6.25nM to 200nM. The binding of the analytes after capture is first analyzed for KIR2DL1 or KIR2DL3 binding, and then dissociation of each analyte is independently analyzed. Similarly, for Mb1b IgG1rmAb, CD 8. Alpha. Analyte ranged from 6.25nM to 200nM. Analyte binding after capture was first analyzed for binding and then for dissociation of CD8 a.
Table 1: affinity comparison between parent antibody and Cross Mab
/>
Example 13: PBMC sample analysis of patients diagnosed with other autoimmune diseases
PBMCs from patients with lupus, ulcerative colitis, crohn's disease, multiple sclerosis and type 1 diabetes were analyzed using flow cytometry and biocomplex assays. Cd8+kir+ Treg cells were identified in these patient samples using a mixture of antibodies against the inhibitory KIR surface receptor sub-populations KIR2DL1/2/3 and KIR3DL1 (fig. 13). Tregs were found to express CXCR3, CD39 and other cell surface markers, consistent with cd8+ tregs from celiac disease patients (data not shown). Cd8+ tregs were found to produce soluble analytes related to cd8+ Treg cell function, including ifnγ and IL-22 (data not shown).
Example 14: phenotypic and functional characterization of CD8+KIR+ Treg cells
PBMCs from celiac disease patients and HLA DQ2.5 haplotypes were analyzed using flow cytometry and biocomplex assays to detect soluble analytes in the supernatant. Cd8+kir+ Treg cells were identified in these patient samples and found to express the surface markers CD39, KLRG1, NKG2D, NKG2C, KLRB, CXCR3 and CD122 (fig. 14).
When cultured with autologous CD 4T cells and antigen presenting cells pulsed with prolamin peptides under optimized conditions, cd8+kir+ tregs produce soluble analytes related to cd8+ Treg cell function, including the cytolytic markers granzyme B, perforin and CD107a, the intracellular anti-inflammatory cytokines IL-10, ifnγ and tnfα, and secreted cytokines (see, e.g., fig. 15). Stimulation of isolated cd8+kir+ Treg cells with increasing doses of anti-CD 3 antibodies produced many cytokines and chemokines, including RANTES and tnfβ, in a dose-dependent manner, which titrated with the intensity of the TCR signal delivered (fig. 16). When co-cultured with CD4+ T cells re-stimulated with prolamin, CD8+ KIR+ Treg cells controlled activation of CD4+ T lymphocytes and reduced the production of pro-inflammatory cytokines by CD4+ cells (FIGS. 17-19). The cd4+ T cell activation markers showed that the addition of cd8+ kir+ Treg cells during prolamin restimulation specifically reduced prolamin-reactive cd4+ T lymphocyte activation (fig. 18). Anti-inflammatory cytokines produced by CD 4T cells also increased (fig. 19), and some of the inflammatory cytokines and chemokines detected in the co-culture supernatant were down-regulated. These data indicate that cd8+kir+ tregs isolated from PBMCs derived from celiac patients are able to survive, amplify and function when cultured under optimal conditions, and thus functional defects in celiac patients appear to be reversible.
In addition, after repeated antigen exposure, an enriched recall (recovery) of cd8+ Treg cell responses and prevalence was observed, indicating that it has potential long term and disease modifying effects (fig. 20). Although variability between patients was observed, consistent effects of repeated antigen exposure on cd8+ Treg cell functional responses were observed in multiple data reads. These results indicate that cd8+ Treg cells may have an induced memory function.
Example 15: selection of the relevance of KIR proteins to cd8+ Treg cell HLA ligand expression in celiac disease
Celiac patient PBMCs were stained with antibodies to KIR2DL1/2/3 and KIR3DL1 and analyzed using flow cytometry. Narrow expression of specific inhibitory KIR proteins on cd8+ Treg cells was observed, and patients selectively expressed KIR2DL1/2/3, KIR3DL1 or KIR2DL1/2/3 and KIR3DL2 (fig. 21), this observation extended to patients with other AI indications, including systemic lupus erythematosus, ulcerative colitis, crohn's disease, multiple sclerosis and type 1 diabetes (fig. 13). This pattern of expression is associated with almost ubiquitous genomic expression of inhibitory KIR proteins, and over-expression of haplotypes of their respective HLA ligands in celiac disease patients compared to healthy control populations (table 2).
Table 2: receptor-ligand pairing overexpression in celiac disease patients
*http://www.allelefrequencies.net/hla.asp
Example 16: expression and interaction of T cells in PBMC and intestinal tissues of celiac patients before and after gluten challenge Mode of use
Formalin-fixed, paraffin-embedded, celiac patient's duodenum biopsies were sectioned and stained, and eight cell markers were detected simultaneously in fixed tissue using proprietary platform and custom antibody mixtures (developed in tandem with ultvue, inc.). The antibody mixture comprises antibodies to CD3, CD4, CD8, CD28, PD-1, ki-67, granzyme B and KIR2DL 1/2/3.
Cd8+kir+ Treg cells increased in celiac disease (fig. 22). Expression of inhibitory KIR in peripheral blood and tissues of celiac patients was demonstrated to be a marker for cd8+ Treg cells (fig. 23). Only a subset of cd8+ T cells are co-located with expression of KIR, so that co-expression of KIR and CD8 is indicative of cd8+ Treg cells in intestinal tissue.
A sample of duodenal tissue from a celiac patient on a gluten-free diet was analyzed for T cell markers and prior to challenge with gluten. Direct interaction of granzyme-positive cd8+ T cells with cd4+ T cells was observed (see fig. 24, where granzyme B appears white, cd8+ T cells appear green, cd4+ T cells appear ocher, and interactions between cd8+ T cells and cd4+ T cells appear yellow).
After challenge with gluten, cd8+ Treg cells increased in the peripheral blood (fig. 25) and tissues of celiac patients (fig. 26). After 14 days of gluten challenge, the number of cd4+ T cells, co-stimulatory molecule expression and proliferation were reduced in the tissue, while T cells expressing inhibitory KIR2DL were increased (relative to matched patient tissue prior to gluten challenge) (fig. 27).
Example 17: bispecific KIR binding agents
Bispecific KIR binders, such as bispecific antibodies or fragments thereof with binding domains that target KIR and another molecule, can be used to design signal intensities to reach functional thresholds of activation and achieve non-toxic functions (fig. 28).
Briefly, CD8 Tregs were enriched from celiac patient PBMC and incubated with autologous CD 4T cells pulsed with a prolamin peptide mixture and antigen presenting cells, and analyzed by flow cytometry (FIG. 29A, FIG. 29B and FIG. 29C), biocomposite assay for 34 analytes (ProCarta Plex; FIG. 30) and longitudinal imaging using incucyte, followed by flow cytometry (FIG. 31). Gluten restimulation of celiac patient-derived PBMCs in the presence of bispecific and monospecific KIR blockade showed that bispecific blockade (anti-KIR 2DL1/2/3 and anti-CD 8) had a greater effect on cd8+ Treg cell activity than blockade KIR alone (fig. 29A, 29B and 29C). These effects were also dose dependent (fig. 29B and 29C). Dose-dependent reduction of a number of pro-inflammatory cytokines (figure 30) and chemokines was also observed. Bispecific KIR-CD8 blockade was also associated with greater and dose-dependent effects on cd4+ T cell survival (fig. 31 and 32). Bispecific binding KIR3DL1 and CD8 was equally effective (data not shown). Although KIR expressing cd8+ T cells were reduced 10-fold compared to NK cells (fig. 33 and 34), preferential binding of bispecific blockade (anti KIR2DL1/2/3 and anti CD 8) to cd8+ T lymphocytes was demonstrated. Similar preferential binding was observed for dual specificity targeting KIR3DL1 instead of KIR2DL1/2/3 (data not shown).
While particular embodiments have been shown and described, it will be readily appreciated that the various embodiments described above may be combined to provide further embodiments and that various changes may be made therein without departing from the spirit and scope of the invention.
All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned in this specification or listed in the application data sheet, including U.S. provisional patent application number 63/145,394, U.S. provisional patent application number 63/148,016, U.S. provisional patent application number 63/161,325, U.S. provisional patent application number 63/209,949, and U.S. provisional patent application number 63/298,028, U.S. provisional patent application number 63/949, and U.S. 1/10, 2022, and U.S. 1. Aspects of the embodiments can be modified, if necessary, to employ concepts of the various patents, applications and publications to provide yet further embodiments.
These and other changes can be made to the embodiments in light of the above-detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and the claims, but should be construed to include all possible embodiments along with the full scope of equivalents to which such claims are entitled.

Claims (161)

1. A binding agent, comprising:
a first binding domain that specifically binds a first antigen selected from antigens expressed on cd8+kir+t regulatory cells (tregs) other than KIR proteins; and
a second binding domain that specifically binds an inhibitory KIR protein, wherein the binding agent binds cd8+kir+treg.
2. The binding agent of claim 1, wherein the first antigen is selected from the group consisting of: CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4 and 41BB.
3. The binding domain according to any one of the preceding claims, wherein the first antigen is selected from the group of antigens consisting of:
CD3, CD5, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD122, ICOS, OX-40, 2B4, 41BB and HLA-DR;
lag-3/CD223, TIM-3, PD-1, S1000A8/9 and TLT2;
CD3, CD5, CD8, CD27, CD38, CD39, CD40L, CD RA, CD45RB, CD45RO, CD73, CD103 (ITGAE), CD122, CD166, CD177, CCR7, CXCR3, CXCR5, HLA-DR, ICOS, LAG-3/CD223, OX-40, PD-1, S1000A8/9, TIM-3, TLT-2, 2B4 and 41BB;
CD103 (ITGAE), CD166, CD177, CXCR3 and S1000A8/9;
ccr7, CXCR3 and CXCR5;
pd-1, ICOS and CXCR3;
cd3, CD5 and CD8; and
cd3 and CD8.
4. The binding agent of any one of the preceding claims, wherein the binding agent is a bispecific antibody, diabody antibody, antibody Fc fusion protein, scFv1-scFv2, scFv1 2 -Fc-scFv2 2 IgG-scFv, DVD-Ig, trifunctional antibody/tetravalent body tumor, two-in-one IgG, scFv 2 -Fc, tandAb, scFv-HSA-scFv, scFv-VHH, fab-scFv-Fc, fab-VHH-Fc, dAb-IgG, igG-VHH, tandem scFv-Fc, (scFv 1) 2 -Fc-(VHH) 2 BiTe, DART, cross mab, scFv-Fc, single arm tandem scFv-Fc, DART-Fc, anticalin, affibody, avimer, DARPin or adnectin.
5. The binding agent according to any one of the preceding claims, wherein either of the first binding domain or the second binding domain is selected from an antibody or antigen binding portion thereof and the other binding domain is an antibody fragment.
6. The binding agent of claim 5, wherein the antigen binding portion is Fab, fab ', F (ab') 2 Fv, scFv or single domain antibodies.
7. The binding agent of any one of the preceding claims, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region.
8. The binding agent of any one of the preceding claims, wherein the second binding domain comprises a heavy chain variable region and a light chain variable region.
9. The binding agent of any one of the preceding claims, wherein the first binding domain specifically binds CD3 or a subunit of CD3, optionally CD3 epsilon.
10. The binding agent of claim 9, wherein the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL amino acid sequences being selected from the group consisting of amino acid sequence pairs set forth in seq id nos:
a. SEQ ID NO. 1 and SEQ ID NO. 2, respectively;
b. SEQ ID NO 9 and SEQ ID NO 10, respectively;
c. SEQ ID NO. 17 and SEQ ID NO. 18, respectively;
d. SEQ ID NO. 25 and SEQ ID NO. 26, respectively;
e. SEQ ID NO 33 and SEQ ID NO 34, respectively;
f. SEQ ID NO 41 and SEQ ID NO 34, respectively;
g. SEQ ID NO 45 and SEQ ID NO 34, respectively;
h. SEQ ID NO. 49 and SEQ ID NO. 50, respectively;
i. SEQ ID NO 57 and SEQ ID NO 58, respectively;
j. SEQ ID NO. 65 and SEQ ID NO. 66, respectively; and
k. SEQ ID NO. 65 and SEQ ID NO. 166, respectively.
11. A binding agent according to claim 9, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region, the heavy chain and light chain variable region comprising hCDR1, hCDR1 and hCDR3, respectively, and CDR1, CDR2 and CDR3, the CDRs having amino acid sequences selected from the group of amino acid sequences shown in seq id nos:
a. SEQ ID NO 3 to SEQ ID NO 8, respectively;
b. SEQ ID NO. 11 to SEQ ID NO. 16, respectively;
c. SEQ ID NO 19 through SEQ ID NO 24, respectively;
d. SEQ ID NO 27 through SEQ ID NO 32, respectively;
e. SEQ ID NO 35 to SEQ ID NO 40, respectively;
f. SEQ ID NO. 42 to SEQ ID NO. 44 and SEQ ID NO. 38 to SEQ ID NO. 40, respectively;
g. 46 to 48 and 38 to 40, respectively;
h. SEQ ID NO. 51 to SEQ ID NO. 56, respectively;
i. 59 to 64, respectively;
j. SEQ ID NO 67 to SEQ ID NO 72, respectively; and
k. SEQ ID NOS 67-69 and 167-169, respectively.
12. The binding agent of any one of claims 1 to 8, wherein the first binding domain specifically binds CD8 or a subunit of CD8, optionally CD8 a.
13. The binding agent of claim 12, wherein the first binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL regions having amino acid sequences selected from the group consisting of amino acid sequence pairs shown in seq id nos:
a. SEQ ID NO 73 and SEQ ID NO 74, respectively; and
b. SEQ ID NO. 81 and SEQ ID NO. 82, respectively;
or the first binding domain comprises a VHH chain having an amino acid sequence selected from the group of amino acid sequences shown in seq id no:
c.SEQ ID NO:89；
d, SEQ ID NO. 93; and
e.SEQ ID NO:97。
14. a binding agent according to claim 12, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region, said heavy chain and light chain variable region comprising hCDR1, hCDR1 and hCDR3, respectively, and CDR1, CDR2 and CDR3, respectively, said CDRs having amino acid sequences selected from the group of amino acid sequences shown in seq id nos:
a. 75 to 80 respectively; or (b)
b. 83 to 88 respectively;
or the first binding domain comprises a VHH chain having hCDR1, hCDR2 and hCDR3, the VHH CDR having an amino acid sequence selected from the group of amino acid sequences shown in the group consisting of seq id nos:
c. SEQ ID NO 90 to SEQ ID NO 92, respectively;
d. 94 to 96 respectively; and
e. 98 to 100, respectively.
15. The binding agent of any one of claims 1 to 8, wherein the first binding domain specifically binds ICOS.
16. The binding agent of claim 15, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID No. 170 and a light chain variable region (VL) having the amino acid sequence of SEQ ID No. 171.
17. The binding agent of claim 15, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region comprising hCDR1, hCDR1 and hCDR3 amino acid sequences according to SEQ ID NOs 172, 173 and 174, respectively, and cdr1, cdr2 and cdr3 amino acid sequences according to SEQ ID NOs 175, 176 and 177, respectively.
18. The binding agent of any one of claims 1 to 8, wherein the first binding domain specifically binds PD-1.
19. The binding agent of claim 18, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID No. 178 and a light chain variable region (VL) having the amino acid sequence of SEQ ID No. 179.
20. The binding agent of claim 18, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region comprising hCDR1, hCDR1 and hCDR3 amino acid sequences according to SEQ ID NOs 180, 181 and 182 respectively, and cdr1, cdr2 and cdr3 amino acid sequences according to SEQ ID NOs 183, 184 and 185 respectively.
21. The binding agent of any one of claims 1 to 8, wherein the first binding domain specifically binds CXCR3.
22. The binding agent of claim 21, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID No. 186 and a light chain variable region (VL) having the amino acid sequence of SEQ ID No. 187.
23. The binding agent of claim 21, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region comprising hCDR1, hCDR1 and hCDR3 amino acid sequences according to SEQ ID NOs 188, 189 and 190, respectively, and cdr1, cdr2 and cdr3 amino acid sequences according to SEQ ID NOs 191, 192 and 193, respectively.
24. The binding agent of any one of claims 1 to 8, wherein the first binding domain specifically binds CD5.
25. The binding agent of claim 24, wherein the first binding domain comprises a heavy chain variable region (VH) having the amino acid sequence of SEQ ID No. 194 and a light chain variable region (VL) having the amino acid sequence of SEQ ID No. 195.
26. The binding agent of claim 24, wherein the first binding domain comprises a heavy chain variable region and a light chain variable region comprising hCDR1, hCDR1 and hCDR3 amino acid sequences according to SEQ ID NOs 196, 197 and 198 respectively, and cdr1, cdr2 and cdr3 amino acid sequences according to SEQ ID NOs 199, 200 and 201 respectively.
27. The binding agent of any one of the preceding claims, wherein the inhibitory KIR protein is selected from KIR3DL1, KIR3DL2, KIR2DL1, KIR2DL2, and KIR2DL3, or a combination thereof.
28. The binding agent of claim 27, wherein the second binding agent specifically binds KIR2DL1/2/3 or KIR2DL1/2.
29. The binding agent of any one of the preceding claims, wherein the second binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL), the VH and VL having amino acid sequences selected from the group of amino acid sequence pairs shown:
a. SEQ ID NO. 101 and SEQ ID NO. 102, respectively;
b. SEQ ID NO 109 and SEQ ID NO 110, respectively;
c. SEQ ID NO 117 and SEQ ID NO 118, respectively;
d. SEQ ID NO. 125 and SEQ ID NO. 126, respectively;
e. SEQ ID NO 133 and SEQ ID NO 134, respectively;
f. 141 and 142, respectively;
g. 149 and 150, respectively; and
h. SEQ ID NO. 157 and SEQ ID NO. 158, respectively.
30. A binding agent according to any one of the preceding claims, wherein the second binding domain comprises a heavy chain variable region (VH) and a light chain variable region comprising hCDR1, hCDR1 and hCDR3, respectively, and CDR1, CDR2 and CDR3, the CDRs having amino acid sequences selected from the group of amino acid sequences shown in seq id nos:
a. 103 to 108 respectively;
b. SEQ ID NO 111 through SEQ ID NO 116, respectively;
c. 119 to 124 respectively;
d. 127 to 132 respectively;
e. SEQ ID NO. 135 to SEQ ID NO. 140, respectively;
f. SEQ ID NO 143 to SEQ ID NO 148, respectively;
g. 151 to 156 respectively; and
h. 159 to 164, respectively.
31. The binding agent of any one of the preceding claims, wherein the binding agent does not comprise an Fc domain.
32. The binding agent of any one of claims 1 to 30, further comprising an Fc domain.
33. The binding agent of claim 32, wherein the Fc domain is selected from an IgG1 and an IgG4 Fc domain.
34. The binding agent of claim 33, wherein the binding agent has substantially no effector function activity.
35. The binding agent of any one of claims 32 to 34, wherein the Fc domain is an IgG1 Fc domain.
36. The binding agent of any one of claims 32 to 35, wherein the Fc domain is an IgG1 Fc null.
37. The binding agent of any one of the preceding claims, wherein the binding agent is divalent or tetravalent.
38. The binding agent of any one of the preceding claims, wherein the binding agent is bispecific.
39. A pharmaceutical composition comprising the binding agent of any one of claims 1 to 38 and a pharmaceutically acceptable carrier.
40. A nucleic acid encoding the binding agent of any one of claims 1 to 38.
41. A vector comprising the nucleic acid of claim 40.
42. A cell line comprising the vector of claim 41.
43. A method of treating an autoimmune disease comprising administering to a subject in need thereof a binding agent according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 39 in an amount effective to reduce the number or activity of pathogenic immune cells and thereby ameliorate symptoms of the autoimmune disease.
44. A method of inhibiting an immune response mediated by pathogenic immune cells comprising contacting cd8+kir+t regulatory cells (tregs) with an amount of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 effective to activate or stimulate cd8+kir+tregs (activated tregs), thereby reducing the number or activity of pathogenic immune cells.
45. A method of suppressing an immune response to an antigen (such as an autoantigen) comprising administering to a subject in need thereof a binding agent according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 39 in an amount effective to activate or stimulate cd8+ kir+ tregs, thereby reducing the number or activity of pathogenic immune cells that are responsive to the antigen or autoantigen.
46. The method of claim 44, wherein the cd8+ kir+ Treg is contacted with the binding agent in vivo.
47. The method of claim 44, wherein the cd8+ kir+ Treg is contacted with the binding agent ex vivo.
48. The method of claim 47, wherein the activated cd8+ kir+ tregs are administered to a subject in need thereof in an effective amount.
49. The method of any one of claims 43 to 48, wherein the pathogenic immune cells are autoreactive cd4+ T cells, autoantibody producing B cells, or autoantigen presenting dendritic cells.
50. The method of any one of claims 43-48, wherein the pathogenic immune cells are autoantigen-presenting cells.
51. The method of claim 45, wherein the titer of autoantibodies in the subject is decreased.
52. The method of any one of claims 43-51, wherein the subject has an autoimmune disease selected from the group consisting of: celiac disease, crohn's disease, juvenile idiopathic arthritis, inflammatory Bowel Disease (IBD), insulin dependent diabetes mellitus (IDDM or type 1 diabetes), lupus nephritis, myasthenia gravis, myocarditis, multiple Sclerosis (MS), pemphigus/pemphigoid, rheumatoid Arthritis (RA), scleroderma/systemic sclerosis, sjogren's syndrome (SjS), systemic Lupus Erythematosus (SLE), and ulcerative colitis.
53. The method of claim 52, wherein the autoimmune disease is selected from the group consisting of: celiac disease, crohn's disease, inflammatory Bowel Disease (IBD), insulin dependent diabetes mellitus (IDDM or type 1 diabetes), lupus nephritis, multiple Sclerosis (MS), rheumatoid Arthritis (RA), scleroderma/systemic sclerosis, sjogren's syndrome (SjS), systemic Lupus Erythematosus (SLE) and ulcerative colitis.
54. The method of any one of claims 43-53, wherein the binding agent specifically binds to inhibitory KIR proteins on CD8 and cd8+ kir+ tregs.
55. The method of any one of claims 43-53, wherein the binding agent specifically binds to inhibitory KIR proteins on CD3 and cd8+ kir+ tregs.
56. The method of any one of claims 43-53, wherein the binding agent specifically binds to inhibitory KIR proteins on CD5 and cd8+ kir+ tregs.
57. The method of any one of claims 43-53, wherein the binding agent specifically binds to inhibitory KIR proteins on PD-1 and cd8+ kir+ tregs.
58. The method of any one of claims 43-53, wherein the binding agent specifically binds to inhibitory KIR proteins on ICOS and cd8+ kir+ tregs.
59. The method of any one of claims 43-53, wherein the binding agent specifically binds to inhibitory KIR proteins on CXCR3 and cd8+ kir+ tregs.
60. The method of any one of claims 43-59, wherein the cd8+kir+ Treg is MHC class I-restricted.
61. The method of any one of claims 43-60, wherein the cd8+kir+ Treg is not MHC HLA E (Qa-1 b) restricted.
62. The method of any one of claims 43-61, further comprising administering an immunosuppressant to the subject.
63. The method of any one of claims 43-62, wherein administering the binding agent to the subject results in improved therapeutic outcome in the subject.
64. The method of claim 63, wherein the improved therapeutic outcome is a reduced frequency and severity of disease onset, reduced systemic inflammatory cytokines, or reduced self-reporting of symptoms associated with the autoimmune disease.
65. The method of any one of claims 43 to 64, wherein the binding agent is administered intravenously.
66. The method of any one of claims 43-65, wherein the binding agent is administered subcutaneously.
67. The method of any one of claims 43-66, wherein the binding agent is administered at a dose of about 0.01mg/kg to about 20 mg/kg.
68. The method of any one of claims 43 to 67, wherein the binding agent has substantially no effector function activity.
69. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for treating an autoimmune disease in a subject by activating or stimulating cd8+kir+ tregs.
70. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for reducing the immune response of pathogenic immune cells by activating or stimulating cd8+kir+ tregs.
71. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for reducing autoantibody titers in a subject by activating or stimulating cd8+kir+ tregs.
72. A method of treating cancer comprising administering to a subject in need thereof a binding agent according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 39, wherein the binding agent has substantially no effector function activity, in an amount effective to activate or stimulate cd8+kir+ tregs and thereby ameliorate symptoms of the cancer.
73. A method of stimulating an immune response against an antigen associated with cancer (cancer antigen), comprising contacting cd8+kir+t regulatory cells (tregs) with the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 in an amount effective to activate or stimulate cd8+kir+tregs (activated tregs), thereby increasing the immune response to the cancer antigen, wherein the binding agent has substantially no effector function activity.
74. A method of treating cancer comprising administering to a subject in need thereof a binding agent according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 39, wherein the binding agent has effector function activity comprising at least ADCC, in an amount effective to deplete cd8+kir+ tregs and thereby ameliorate symptoms of the cancer.
75. A method of stimulating an immune response against an antigen associated with cancer (cancer antigen), comprising contacting cd8+kir+t regulatory cells (tregs) with the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 in an amount effective to deplete cd8+kir+tregs, thereby increasing the immune response against the cancer antigen, wherein the binding agent has effector function activity including at least ADCC.
76. The method of claim 73 or claim 75, wherein the cd8+ kir+ Treg is contacted with the binding agent in vivo.
77. The method of claim 73, wherein the cd8+ kir+ Treg is contacted with the binding agent ex vivo.
78. The method of claim 77, wherein the activated cd8+ kir+ Treg is administered to a subject in need thereof in an effective amount.
79. The method of any one of claims 73 and 75-78, wherein the increased immune response comprises cancer cytopenia or immunosuppressive immune cell depletion.
80. The method of any one of claims 72-79, wherein the cancer cells in the subject are reduced.
81. The method of any one of claims 72-80, wherein the cancer is selected from the group consisting of: carcinoma, lymphoma, blastoma, sarcoma, myeloma, and leukemia.
82. The method of any one of claims 72-81, wherein the cancer is selected from the group consisting of: a solid tumor of the type described above, such as breast, cervical, ovarian, lung, CRC (and other bowel cancers), skin, oesophageal, adenocarcinoma, bladder and prostate cancer; lymphoma.
83. The method of any one of claims 72-82, wherein the binding agent specifically binds to inhibitory KIR proteins on CD8 and cd8+ kir+ tregs.
84. The method of any one of claims 72-82, wherein the binding agent specifically binds to inhibitory KIR proteins on CD3 and cd8+ kir+ tregs.
85. The method of any one of claims 72-82, wherein the binding agent specifically binds to inhibitory KIR proteins on CD5 and cd8+ kir+ tregs.
86. The method of any one of claims 72-82, wherein the binding agent specifically binds to an inhibitory KIR protein on PD-1 and cd8+ kir+ tregs.
87. The method of any one of claims 72-82, wherein the binding agent specifically binds to inhibitory KIR proteins on ICOS and cd8+ kir+ tregs.
88. The method of any one of claims 72-82, wherein the binding agent specifically binds to inhibitory KIR proteins on CXCR3 and cd8+ kir+ tregs.
89. The method of any one of claims 72-88, wherein the cd8+kir+ Treg is MHC class I-restricted.
90. The method of any one of claims 72-89, wherein the cd8+kir+ Treg is not MHC HLA E (Qa-1 b) restricted.
91. The method of any one of claims 72-90, further comprising administering a chemotherapeutic agent to the subject.
92. The method of any one of claims 72-91, wherein administration of the binding agent to the subject results in improved therapeutic outcome in the subject.
93. The method of claim 92, wherein the improved therapeutic outcome is a partial response or a complete response.
94. The method of claim 92, wherein the improved therapeutic outcome is remission.
95. The method of any one of claims 72-94, wherein the binding agent is administered intravenously.
96. The method of any one of claims 72-94, wherein the binding agent is administered subcutaneously.
97. The method of any one of claims 72-96, wherein the binding agent is administered at a dose of about 0.01mg/kg to about 20 mg/kg.
98. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for treating cancer in a subject by activating or stimulating cd8+kir+ tregs, wherein the binding agent has substantially no effector function activity.
99. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for reducing immunosuppression of immunosuppressive immune cells by activating or stimulating cd8+kir+ tregs, wherein the binding agent has substantially no effector function activity.
100. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for reducing tumor burden in a subject by activating or stimulating cd8+kir+ tregs, wherein the binding agent has substantially no effector function activity.
101. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for treating cancer in a subject by depleting cd8+kir+ tregs, wherein the binding agent has effector function activity including at least ADCC.
102. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for depleting cd8+kir+ Treg, wherein said binding agent has effector function activity including at least ADCC.
103. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for reducing tumor burden in a subject, wherein the binding agent has effector function activity comprising at least ADCC.
104. A method of treating an infection comprising administering to a subject in need thereof the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 in an amount effective to activate or stimulate cd8+kir+ tregs and thereby ameliorate symptoms of the infection.
105. A method of stimulating an immune response against an infected cell resulting from an infection, comprising contacting a cd8+kir+t regulatory cell (Treg) with an amount of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 effective to activate or stimulate a cd8+kir+treg (activated Treg), thereby generating the immune response against the infected cell.
106. The method of claim 105, wherein the cd8+ kir+ Treg is contacted with the binding agent in vivo.
107. The method of claim 105, wherein the cd8+ kir+ Treg is contacted with the binding agent ex vivo.
108. The method of claim 107, wherein the activated cd8+ kir+ Treg is administered to a subject in need thereof in an effective amount.
109. The method of any one of claims 104 to 108, wherein the immune response comprises an infectious cytopenia or an immunosuppressive immunocytopenia selected from the group consisting of: cd4+ T regulatory cells and tolerogenic DCs.
110. The method of claim 109, wherein the number of infected cells in the subject is reduced.
111. The method of any one of claims 104 to 110, wherein the infection is selected from the group consisting of a bacterial disease, a systemic fungal disease, rickettsiosis, a parasitic disease, and a viral disease.
112. The method of claim 111, wherein the infection is selected from the group consisting of: HIV infection, hepatitis C Virus (HCV) infection, human Papilloma Virus (HPV) infection, epstein Barr Virus (EBV) infection, coronavirus infection such as SARS-COV2 infection (Covid-19), cytomegalovirus (CMV) infection, and influenza virus infection.
113. The method of any one of claims 104-112, wherein the binding agent specifically binds to inhibitory KIR proteins on CD8 and cd8+ kir+ tregs.
114. The method of any one of claims 104-112, wherein the binding agent specifically binds to an inhibitory KIR protein on CD3 and cd8+ kir+ tregs.
115. The method of any one of claims 104-112, wherein the binding agent specifically binds to an inhibitory KIR protein on CD5 and cd8+ kir+ tregs.
116. The method of any one of claims 104-112, wherein the binding agent specifically binds to an inhibitory KIR protein on PD-1 and cd8+ kir+ tregs.
117. The method of any one of claims 104-112, wherein the binding agent specifically binds to inhibitory KIR proteins on ICOS and cd8+ kir+ tregs.
118. The method of any one of claims 104-112, wherein the binding agent specifically binds to inhibitory KIR proteins on CXCR3 and cd8+ kir+ tregs.
119. The method of any one of claims 104-118, wherein the cd8+kir+treg is mhc class i restricted.
120. The method of any one of claims 104-119, wherein the cd8+kir+ Treg is not MHC HLA E (Qa-1 b) restricted.
121. The method of any one of claims 104-120, further comprising administering an antimicrobial or antiviral agent to the subject.
122. The method of any one of claims 104-121, wherein administering the binding agent to the subject results in improved therapeutic outcome in the subject.
123. The method of claim 122, wherein the improved therapeutic result is a reduction in infection or infected cells.
124. The method of any one of claims 104-123, wherein the binding agent is administered intravenously.
125. The method of any one of claims 104-123, wherein the binding agent is administered subcutaneously.
126. The method of any one of claims 104 to 125, wherein the binding agent is administered at a dose of about 0.01mg/kg to about 20 mg/kg.
127. The method of any one of claims 104 to 126, wherein the binding agent has substantially no effector function activity.
128. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for treating an infection in a subject by activating or stimulating cd8+kir+ tregs.
129. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for stimulating an immune response by activating or stimulating cd8+kir+ Treg and thereby inhibiting immunosuppressive immune cells.
130. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for reducing infection or infected cells in a subject by activating or stimulating cd8+kir+ tregs.
131. A method of reducing or preventing the onset of Graft Versus Host Disease (GVHD) after transplantation comprising administering to a subject in need thereof a binding agent according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 39, wherein the binding agent has substantially no effector function activity, in an amount effective to activate or stimulate cd8+kir+ tregs and thereby reduce or ameliorate at least one symptom of GVHD.
132. A method of treating a subject receiving a graft comprising contacting cd8+ kir+ T regulatory cells (tregs) with a binding agent according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 39 in an amount effective to activate or stimulate cd8+ kir+ tregs (activated tregs) so as to reduce or inhibit GVHD, wherein the binding agent has substantially no effector function activity.
133. A method of treating a subject receiving a graft, comprising administering to a subject in need thereof a binding agent according to any one of claims 1 to 38 or a pharmaceutical composition according to claim 39, wherein the binding agent has effector function activity comprising at least ADCC, in an amount effective to deplete cd8+kir+ tregs and thereby ameliorate symptoms of GVHD.
134. A method of inhibiting GVHD against a graft comprising contacting cd8+kir+t regulatory cells (tregs) with an amount of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 effective to deplete cd8+kir+tregs, thereby reducing GVHD or symptoms thereof, wherein the binding agent has effector function activity including at least ADCC.
135. The method of claim 132 or claim 134, wherein cd8+ kir+ tregs are contacted with the binding agent in vivo.
136. The method of claim 132, wherein the cd8+ kir+ Treg is contacted with the binding agent ex vivo.
137. The method of claim 136, wherein the activated cd8+ kir+ Treg is administered to a subject in need thereof in an effective amount.
138. The method of any one of claims 132 and 134-137, wherein reducing or reducing GVHD comprises reducing active cd4+ T cells in GVHD.
139. The method of any one of claims 131 to 138, wherein the graft is selected from the group consisting of: organ grafts, hematopoietic stem cell grafts, cord blood stem cell grafts, induced pluripotent stem cell-derived progenitor or differentiated cell grafts, and bone marrow grafts.
140. The method of claim 139, wherein the graft is a hematopoietic stem cell graft, a cord blood stem cell graft, a progenitor or differentiated cell graft or a bone marrow graft that induces multipotent stem cell derivation.
141. The method of any one of claims 131 to 140, wherein the graft is allogeneic.
142. The method of any one of claims 131-141, wherein the binding agent specifically binds to an inhibitory KIR protein on CD8 and cd8+ kir+ tregs.
143. The method of any one of claims 131-141, wherein the binding agent specifically binds to an inhibitory KIR protein on CD3 and cd8+ kir+ tregs.
144. The method of any one of claims 131-141, wherein the binding agent specifically binds to an inhibitory KIR protein on CD5 and cd8+ kir+ tregs.
145. The method of any one of claims 131-141, wherein the binding agent specifically binds to an inhibitory KIR protein on PD-1 and cd8+ kir+ tregs.
146. The method of any one of claims 131-141, wherein the binding agent specifically binds to inhibitory KIR proteins on ICOS and cd8+ kir+ tregs.
147. The method of any one of claims 131 to 141, wherein the binding agent specifically binds to inhibitory KIR proteins on CXCR3 and cd8+ kir+ tregs.
148. The method of any one of claims 131 to 147, wherein the cd8+kir+ Treg is mhc class i restricted.
149. The method of any one of claims 131 to 148, wherein the cd8+kir+ tregs are not MHC HLA E (Qa-1 b) restricted.
150. The method of any one of claims 131-149, further comprising administering to the subject an immunosuppressant.
151. The method of any one of claims 131-150, wherein administering the binding agent to the subject results in improved therapeutic outcome in the subject.
152. The method of claim 151, wherein the improved therapeutic outcome is a decrease in symptoms associated with GVHD, a decrease in systemic inflammatory cytokines, a decrease in pathology in tissue affected by GVHD, a decrease in self-reporting of symptoms associated with an immune response on host tissue, an improvement or prolongation of graft implantation, a decrease in one or more symptoms, and/or prevention, delay or slowing of the occurrence or progression of graft rejection, or prolonged implantation of a graft and a decrease in the use of a broad spectrum immunosuppressant (e.g., a corticosteroid).
153. The method of any one of claims 131 to 152, wherein the binding agent is administered intravenously.
154. The method of any one of claims 131 to 152, wherein the binding agent is administered subcutaneously.
155. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for treating graft-related GVHD in a subject, wherein the binding agent has substantially no effector function activity.
156. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for treating graft-related GVHD in a subject by activating or stimulating cd8+kir+ Treg, wherein the binding agent has substantially no effector function activity.
157. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for reducing graft-related GVHD by activating or stimulating cd8+kir+ Treg, wherein the binding agent has substantially no effector function activity.
158. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for reducing GVHD for a transplant, wherein the binding agent has substantially no effector function activity.
159. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for treating graft-related GVHD in a subject by depleting cd8+kir+ Treg, wherein the binding agent has effector function activity comprising at least ADCC.
160. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for depleting cd8+kir+ Treg, wherein said binding agent has effector function activity including at least ADCC.
161. Use of the binding agent of any one of claims 1 to 38 or the pharmaceutical composition of claim 39 for depleting cd8+kir+ tregs in a subject who has received an implant to reduce GVHD, wherein the binding agent has effector function activity including at least ADCC.
CN202280013079.4A 2021-02-03 2022-02-02 Binding agents and methods of use thereof Pending CN116964093A (en)

Applications Claiming Priority (7)

Application Number Priority Date Filing Date Title
US63/145,394 2021-02-03
US63/148,016 2021-02-10
US63/161,325 2021-03-15
US63/209,949 2021-06-11
US202263298028P 2022-01-10 2022-01-10
US63/298,028 2022-01-10
PCT/US2022/014881 WO2022169825A1 (en) 2021-02-03 2022-02-02 Binding agents and methods of using the same

Publications (1)

Publication Number Publication Date
CN116964093A true CN116964093A (en) 2023-10-27

Family

ID=88456864

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202280013079.4A Pending CN116964093A (en) 2021-02-03 2022-02-02 Binding agents and methods of use thereof

Country Status (1)

Country Link
CN (1) CN116964093A (en)

Similar Documents

Publication Publication Date Title
JP7134204B2 (en) Chimeric receptors and methods of use thereof
CN109072194B (en) Immune cell compositions and methods of use thereof
JP2022078179A (en) Chimeric antigen and t-cell receptor, and use method
JP2022088432A (en) Combination therapy of chimeric antigen receptor and pd-1 inhibitor
US11890301B2 (en) Methods and compositions for cells expressing a chimeric intracellular signaling molecule
JP2021527431A (en) Bispecific anti-PSMA x anti-CD28 antibody and its use
KR20170128234A (en) Antibodies specific for ROR1 and chimeric antigen receptors
TW202045547A (en) Dll3 targeting chimeric antigen receptors and binding agents
JP2022513778A (en) Chimeric antigen receptor and T cell receptor and how to use
JP2018505911A (en) Identification of VSIG8 as a putative VISTA receptor and its use to produce a VISTA / VSIG8 modulator
JP2011513479A (en) MONOCLONAL ANTIBODY AND METHOD THEREOF
KR20190004319A (en) CD40L-FC fusion polypeptides and methods for their use
JP2019521098A (en) Anti-GITR antibody and use thereof
JP7459046B2 (en) Chimeric receptors for STEAP1 and methods of use thereof
US20240026293A1 (en) Methods and Compositions for Cells Expressing a Chimeric Intracellular Signaling Molecule
US20240101673A1 (en) Binding agents and methods of using the same
JP2020530766A (en) Antigen binding region for fibronectin type III domain and its usage
CN116964093A (en) Binding agents and methods of use thereof
WO2023076876A1 (en) Modulation of immune responses to viral vectors
WO2024030956A2 (en) Cd39-specific binding agents and methods of using the same
WO2022235295A1 (en) Mesothelin isoform binding molecules and chimeric pd1 receptor molecules, cells containing the same and uses thereof
WO2023062604A1 (en) Anti-steap2 chimeric antigen receptors and uses thereof
TW202216751A (en) Receptors providing targeted costimulation for adoptive cell therapy

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination