Patent Application
20230051847
This document relates to methods and materials for assessing a mammal having or suspected of having cancer and/or for treating a mammal having cancer. For example, this document provides methods and materials for using a molecule including one or more antigen-binding domains (e.g., a single-chain variable fragment (scFv)) that can bind to a modified peptide (e.g., a tumor antigen) to treat a mammal having a cancer.
Somatic mutations in cancer are ideal targets for cancer therapy as they are expressed only in tumor cells and not in normal cells. Targeting driver gene proteins (broadly subdivided into oncogene proteins and tumor suppressor proteins) have added benefits. First, these mutations typically occur early during the development of the tumor, thus essentially all daughter cancer cells will contain the mutation. Second, the tumor's dependence on their oncogenic-endowing capacity makes resistance less likely. Finally, driver gene proteins tend to have hotspot mutations shared among many patients, thus a therapy targeting a single mutation could be applied to a broad patient population.
Most mutant proteins, including most mutant driver gene proteins, are intracellular. While small molecules can target intracellular proteins, developing small molecules that can specifically inhibit the activity of a mutant driver gene and not its wild-type (WT) counterpart has remained out of reach for the majority of such driver gene proteins. Antibodies, which can have the capacity to distinguish a single amino acid mutation, can typically only target extracellular epitopes.
The immune system samples the intracellular contents of cells through antigen processing and presentation. Following protein proteolysis, a fraction of the resulting peptides are loaded onto a human leukocyte antigen (HLA) and sent to the cell surface where they serve as a way for T cells, via their T cell receptor (TCR), to distinguish self from non-self peptides. For example, a virally-infected cell will present viral peptides in its HLA, triggering T cells to kill that cell. Similarly, in cancer, mutant peptides can be presented in an HLA on the cancer cell surface, referred to as MANAs, for Mutation-Associated NeoAntigens. In some cases, and to varying degrees, patients may mount an anti-cancer T cell response against these mutant-peptide-HLA neoantigens, and checkpoint blockade antibodies can further augment this response. However, many patients, particularly those with a low mutational burden, cannot mount a sufficient anti-cancer T cell response. A therapy or diagnostic specifically targeting MANAs could therefore provide a truly tumor-specific method to diagnose or treat cancer.
HLA class I proteins are present on all nucleated cells. There are three classical HLA class I genes, A, B, and C, each of which are highly polymorphic. Each HLA allele has a particular peptide-binding motif, and as a result, only certain peptides will bind to certain HLA alleles.
There is a continuing need in the art to develop new methods to diagnose, monitor, and effectively treat cancers.
Identification of therapeutic targets highly specific to cancer cells is one of the greatest challenges for developing an effective cancer therapy.
This document provides methods and materials for treating a mammal having cancer. For example, this document provides methods and materials for using one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide (e.g., a modified peptide present in a peptide-HLA-beta-2 microglobulin (b2M or β2M) complex) to treat a mammal having a cancer (e.g., a cancer expressing the modified peptide). In some cases, one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide (e.g., a modified peptide present in a peptide-HLA-β2M complex) can be administered to a mammal having a cancer (e.g., a cancer expressing the modified peptide) to treat the mammal.
As demonstrated herein, scFvs were identified that target (e.g., bind to) numerous MANAs present in HLA-restricted MANAs derived from common cancer driver mutations, including RAS Q61H/L/R and p53 R175H. Also as demonstrated herein, the scFvs were used to design bispecific antibodies capable of inducing MANA-dependent T cell activation that can lead to recognition and killing of cells (e.g., cancer cells) expressing MANAs.
MANAs can be used as highly specific cancer targets because they are not present in normal tissue(s). The ability to specifically target MANAs provides a tumor-specific method to diagnose and/or treat cancer. For example, scFvs specifically targeting MANAs can be used in full-length antibodies or fragments thereof, antibody drug conjugates (ADCs), antibody radionuclide conjugates, T cells expressing a chimeric antigen receptor (CARTs), or bispecific antibodies to diagnose and/or treat a mammal having cancer. Further, an antibody that can bind to a MANA (a MANAbody), or a fragment thereof capable of binding to a MANA, have the potential of becoming widely applicable and genetically predictable off-the-shelf targeted cancer immunotherapy.
In general, one aspect of this document features molecules having an antigen-binding domain that can bind to a peptide-HLA-β2M complex, where the peptide can be derived from a modified p53 polypeptide. The modified p53 polypeptide can include from 7 amino acids to 25 amino acids (e.g., the modified p53 polypeptide can include 9 amino acids). The modified p53 polypeptide can include the amino acid sequence set forth in SEQ ID NO:1. The antigen binding domain can include an amino acid sequence set forth in any one of SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, and SEQ ID NO:141. The molecule can be any one of an antibody, an antibody fragment, a single chain variable fragment (scFv), a chimeric antigen receptor (CAR), a T cell receptor (TCR), a TCR mimic, a tandem scFv, a bispecific T cell engager, a diabody, a single-chain diabody (scDb), an scFv-Fc, a bispecific antibody, and a dual-affinity re-targeting antibody (DART). The molecule also can include an antigen-binding domain that can bind to an effector cell receptor selected from any one of CD3, CD28, CD4, CD8, CD16a, NKG2D, PD-1, CTLA-4, 4-1BB, OX40, ICOS, CD27, and an Fc receptor. In some cases, the antigen-binding domain that can bind to an effector cell can bind to CD3, and the antigen-binding domain can include an amino acid sequence set forth in any one of SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO:176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, and SEQ ID NO:183.
In another aspect, this document features molecules that have an antigen-binding domain that can bind to a peptide-HLA-β2M complex, where the peptide can be derived from a modified RAS polypeptide. The modified RAS peptide can include from 7 amino acids to 25 amino acids (e.g., the modified RAS peptide can include 10 amino acids). The modified RAS peptide can include an amino acid sequence set forth in any one of SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4. In some cases, the modified RAS peptide can include the amino acid set forth in SEQ ID NO:2, and the antigen binding domain can include an amino acid sequence set forth in any one of SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, and SEQ ID NO:149. In some cases, the modified RAS peptide can include the amino acid sequence set forth in SEQ ID NO:3, and the antigen binding domain can include an amino acid sequence set forth in any one of SEQ ID NO:150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, and SEQ ID NO:160. In some cases, the modified RAS peptide can include the amino acid sequence set forth in SEQ ID NO:4, and the antigen binding domain can include an amino acid sequence set forth in any one of SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, and SEQ ID NO:169. The molecule can be any one of an antibody, an antibody fragment, a scFv, a CAR, a TCR, a TCR mimic, a tandem scFv, a bispecific T cell engager, a diabody, a scDb, an scFv-Fc, a bispecific antibody, and a DART. The molecule also can include an antigen-binding domain that can bind to an effector cell receptor selected from any one of CD3, CD28, CD4, CD8, CD16a, NKG2D, PD-1, CTLA-4, 4-1BB, OX40, ICOS, CD27, and an Fc receptor. In some cases, the antigen-binding domain that can bind to an effector cell can bind to CD3, and the antigen-binding domain can include an amino acid sequence set forth in any one of SEQ ID NO:170, SEQ ID NO:171, SEQ ID NO:172, SEQ ID NO:173, SEQ ID NO:174, SEQ ID NO:175, SEQ ID NO: 176, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:179, SEQ ID NO:180, SEQ ID NO:181, SEQ ID NO:182, and SEQ ID NO:183.
In another aspect, this document features methods for treating a mammal having a cancer. The methods can include, or consist essentially of, administering to a mammal having cancer one or more molecules described herein (e.g., a molecule having an antigen-binding domain that can bind to a peptide-HLA-β2M complex, where the peptide can be derived from a modified p53 polypeptide or a modified RAS polypeptide), where the cancer includes cancer cells expressing the modified peptide. The mammal can be a human. The cancer can be any one of Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia, acute lymphoblastic leukemia, multiple myeloma, a myelodysplastic syndrome (MDS), a myeloproliferative disease, lung cancer, pancreatic cancer, gastric cancer, colorectal cancer, ovarian cancer, endometrial cancer, biliary tract cancer, liver cancer, breast cancer, prostate cancer, esophageal cancer, stomach cancer, kidney cancer, bone cancer, soft tissue cancer, head and neck cancer, glioblastoma multiforme, astrocytoma, thyroid cancer, germ cell tumor, and melanoma.
Unless otherwise defined, 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 pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document provides methods and materials for assessing a mammal having cancer or suspected of having cancer and/or treating a mammal having cancer. For example, one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can target (e.g., bind to) one or more modified peptides (e.g., peptides present in a peptide-HLA complex such as a peptide-HLA-β2M complex) can be used to assess a mammal having cancer or suspected of having cancer and/or to treat a mammal having a cancer (e.g., a cancer expressing one or more modified peptides). In some cases, one or more molecules includes one or more antigen-binding domains that can bind to a modified peptide can be used to detect the presence or absence of one or more modified peptides in a sample obtained from a mammal having cancer or suspected of having cancer. In some cases, one or more molecules including one or more antigen-binding domains that can bind to a modified peptide can be administered to a mammal having a cancer (e.g., a cancer expressing the modified peptide) to treat the mammal.
As used herein, a modified peptide is a peptide derived from a modified polypeptide. A modified polypeptide can be any appropriate modified polypeptide (e.g., a polypeptide having a disease-causing mutation such as a mutation in an oncogenic or a mutation in a tumor suppressor gene). A modified peptide can have one or more amino acid modifications (e.g., substitutions) relative to a WT peptide (e.g., a peptide derived from a WT polypeptide from which the modified polypeptide is derived). A modified peptide also can be referred to as a mutant peptide. In some cases, a modified peptide can be a tumor antigen. Examples of tumor antigens include, without limitation, MANAs, tumor-associated antigens, and tumor-specific antigens. A modified peptide can be any appropriate length. In some cases, a modified peptide can be from about 7 amino acids to about 25 amino acids (e.g., from about 8 amino acids to about 25 amino acids, from about 9 amino acids to about 25 amino acids, from about 10 amino acids to about 25 amino acids, from about 11 amino acids to about 25 amino acids, from about 12 amino acids to about 25 amino acids, from about 13 amino acids to about 25 amino acids, from about 15 amino acids to about 25 amino acids, from about 18 amino acids to about 25 amino acids, from about 20 amino acids to about 25 amino acids, from about 7 amino acids to about 22 amino acids, from about 7 amino acids to about 20 amino acids, from about 7 amino acids to about 18 amino acids, from about 7 amino acids to about 15 amino acids, from about 7 amino acids to about 12 amino acids, from about 7 amino acids to about 10 amino acids, from about 7 amino acids to about 9 amino acids, from about 8 amino acids to about 22 amino acids, from about 10 amino acids to about 18 amino acids, from about 12 amino acids to about 15 amino acids, from about 8 amino acids to about 12 amino acids, from about 12 amino acids to about 18 amino acids, from about 18 amino acids to about 22 amino acids, or from about 9 amino acids to about 10 amino acids) in length. For example, a modified peptide can be about 9 amino acids in length. For example, a modified peptide can be about 10 amino acids in length. A modified peptide can be derived from any modified polypeptide. Examples of modified polypeptides from which modified peptides described herein can be derived include, without limitation, p53 and RAS (e.g., KRAS, HRAS, and NRAS). A modified peptide can include any appropriate modification. In some cases, modified peptides described herein can include one or more modifications (e.g., mutations) shown in Table 1.
A modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4) can be in a complex with any appropriate HLA. An HLA can be any appropriate HLA allele. In some cases, an HLA can be a class I HLA (e.g., HLA-A, HLA-B, and HLA-C) allele. In some cases, an HLA can be a class II HLA (e.g., HLA-DP, HLA-DM, HLA-DOA, HLA-DOB, HLA-DQ, and HLA-DR) allele. Examples of HLA alleles that a modified peptide described herein can complex with include, without limitation, HLA-A1 and HLA-A2. Exemplary HLA alleles for particular modified peptides are shown in Table 1. For example, a modified peptide derived from a modified p53 polypeptide (e.g., HMTEVVRHC (SEQ ID NO:1)) can be in a complex with HLA-A2 and β2M. For example a modified peptide derived from a modified H/K/N RAS polypeptide (e.g., ILDTAGHEEY (SEQ ID NO:2), ILDTAGLEEY (SEQ ID NO:3), and ILDTAGREEY (SEQ ID NO:4)) can be in a complex with HLA-A1 (e.g., can be in a complex with HLA-A1 and β2M).
This document provides molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4). In some cases, a molecule including one or more antigen-binding domains that can bind to a modified peptide described herein does not target (e.g., does not bind to) an uncomplexed modified peptide described herein (e.g., a modified peptide described herein that is not present in a complex (e.g., a peptide-HLA-β2M complex)). In some cases, a molecule including one or more antigen-binding domains that can bind to a modified peptide described herein does not target (e.g., does not bind to) a WT peptide (e.g., a peptide derived from a WT polypeptide from which the modified polypeptide is derived).
A molecule including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein can be any appropriate type of molecule. In some cases, a molecule can be a monovalent molecule (e.g., containing a single antigen-binding domain). In some cases, a molecule can be a multivalent molecule (e.g., containing two or more antigen-binding domains and simultaneously targeting two or more antigens). For example, a bispecific molecule can include two antigen-binding domains, a trispecific molecule can include three antigen-binding domains, a quadruspecific molecule can include four antigen-binding domains, etc. Examples of molecules that contain antigen-binding domains include, without limitation, antibodies, antibody fragments, scFvs, chimeric antigen receptors (CARs), T cell receptors (TCRs), TCR mimics, tandem scFvs, bispecific T cell engagers, diabodies, scDbs, scFv-Fcs, bispecific antibodies, bispecific single-chain Fcs, dual-affinity re-targeting antibodies (DARTs), and any other molecule that includes at least one variable heavy chain (VH) and at least one variable light chain (VL). Any of these molecules can be used in accordance with materials and methods described herein. In some cases, an antigen-binding domain can be a scFv. For example, a molecule including one or more antigen-binding domains (e.g., one or more scFvs) that can bind to a modified peptide described herein can be a CAR. For example, a molecule including two scFvs that can bind to a modified peptide described herein can be a single-chain diabody (scDb).
In some cases, when a molecule including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4) is a multivalent molecule (e.g., a bispecific molecule), a first antigen-binding domain can bind to a modified peptide described herein and a second antigen-binding domain can bind to an effector cell (e.g., an antigen present on an effector cell). Examples of effector cells include, without limitation, T cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, plasma cells, macrophages, monocytes, microglia, dendritic cells, neutrophils, fibroblasts, and mast cells. Examples of antigens present on effector cells include, without limitation, CD3, CD4, CD8, CD28, NKG2D, PD-1, CTLA-4, 4-1BB, OX40, ICOS, CD27, Fc receptors (e.g., CD16a), and any other effector cell surface receptors. In some cases, a molecule described herein can include a first antigen-binding domain that can bind to a modified peptide described herein and a second antigen-binding domain that can bind to an antigen present on a T cell (e.g., CD3). In some cases, sequences (e.g., scFv sequences) that can bind to CD3 can be as shown in Table 4. In some cases, sequences (e.g., scFv sequences) that can bind to CD3 can be as described elsewhere (see, e.g., Rodrigues et al., 1992 Int J Cancer Suppl. 7:45-50; Shalaby et al., 1992 J Exp Med. 175:217-25; Brischwein et al., 2006 Mol Immunol. 43:1129-43; Li et al., 2005 Immunology. 116:487-98; WO2012162067; US20070065437; US20070065437; US20070065437; US20070065437; US20070065437; and US20070065437). In some cases, a molecule described herein can include a first antigen-binding domain that can bind to a modified peptide described herein and a second antigen-binding domain that can bind to an antigen present on a NK cell (e.g., CD16a or NKG2D). In some cases, sequences (e.g., scFv sequences) that can bind to CD16a can be as shown in Table 5. By binding both the modified peptide and the effector cell, the multivalent molecule can bring the cell expressing the modified peptide (e.g., as part of the HLA complex) into proximity with the effector cell, permitting the effector cell to act on the cell expressing the modified peptide.
In some cases, when a molecule including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4) is a multivalent molecule (e.g., a bispecific molecule), a molecule can be in any appropriate format which includes at least one VH and at least one VL. For example, a VH and a VL can be in any appropriate orientation. In some cases, a VH can be N-terminal to the VL. In some cases, a VH can be C-terminal to the VL. In some cases, a linker amino acid sequence can be positioned between the VH and VL.
In some cases, when a bispecific molecule includes tandem scFvs, the tandem scFvs can be in any appropriate orientation. Examples of tandem scFv orientations including scFv-A and scFv-B include, without limitation, VLA-LL-VHA-SL-VLB-LL-VHB, VLA-LL-VHA-SL-VHB-LL-VLB, VHA-LL-VLA-SL-VLB-LL-VHB, VHA-LL-VLA-SL-VHB-LL-VLB, VLB-LL-VHB-SL-VLA-LL-VHA, VLB-LL-VHB-SL-VHA-LL-VLA, VHB-LL-VLB-SL-VLA-LL-VHA, and VHB-LL-VLB-SL-VHA-LL-VLA, where SL is a short linker and LL is a long linker. A short linker can be from about 3 amino acids to about 10 amino acids in length. A short linker can include any appropriate amino acids (e.g., glycines and serines) in any appropriate combination. A long linker can be from about 10 amino acids to about 25 amino acids in length. A long linker can include any appropriate amino acids (e.g., glycines and serines) in any appropriate combination.
In some cases, when a bispecific molecule is a diabody, the diabody can be in any appropriate orientation. Examples of diabody orientations including scFv-A and scFv-B include, without limitation, VLA-SL-VHB and VLB-SL-VHA, VLA-SL-VLB and VHB-SL-VHA, VHA-SL-VLB and VHB-SL-VLA, VLB-SL-VHA and VLA-SL-VHB, VLB-SL-VLA and VHA-SL-VHB, and VHB-SL-VLA and VHA-SL-VLB, where SL is a short linker. A short linker can be from about 3 amino acids to about 10 amino acids in length. A short linker can include any appropriate amino acids (e.g., glycines and serines) in any appropriate combination.
In some cases, when a bispecific molecule is a scDb, the scDb can be in any appropriate orientation. Examples of scDb orientations including scFv-A and scFv-B include, without limitation, VLA-SL-VHB-LL-VLB-SL-VHA, VHA-SL-VLB-LL-VHB-SL-VLA, VLA-SL-VLB-LL-VHB-SL-VHA, VHA-SL-VHB-LL-VLB-SL-VLA, VLB-SL-VHA-LL-VLA-SL-VHB, VHB-SL-VLA-LL-VHA-SL-VLB, VLB-SL-VLA-LL-VHA-SL-VHB, and VHB-SL-VHA-LL-VLA-SL-VLB, where SL is a short linker and LL is a long linker. A short linker can be from about 3 amino acids to about 10 amino acids in length. A short linker can include any appropriate amino acids (e.g., glycines and serines) in any appropriate combination. A long linker can be from about 10 amino acids to about 25 amino acids in length. A long linker can include any appropriate amino acids (e.g., glycines and serines) in any appropriate combination.
In some cases, when a bispecific molecule is a scFv-Fc, the scFv-Fc can be in any appropriate orientation. Examples of scFv-Fc orientations including scFv-Fc-A, scFv-Fc-B, and an Fc domain include, without limitation, VLA-LL-VHA-hinge-Fc and VLB-LL-VHB-hinge-Fc, VHA-LL-VLA-hinge-Fc and VHB-LL-VLB-hinge-Fc, VLA-LL-VHA-hinge-Fc and VHB-LL-VLB-hinge-Fc, VHA-LL-VLA-hinge-Fc and VLB-LL-VHB-hinge-Fc, where LL is a long linker. A long linker can be from about 10 amino acids to about 25 amino acids in length. A long linker can include any appropriate amino acids (e.g., glycines and serines) in any appropriate combination. In some cases, an Fc domain in a scFv-Fc can include one or more modifications to increase heterodimerization and/or to decrease homodimerization of the scFv-Fc. In some cases, an Fc domain in a scFv-Fc can exclude a hinge domain. In some cases, an Fc domain in a scFv-Fc can be at the N-terminus of the scFv.
In some cases, when a bispecific molecule is a bispecific single-chain Fc, the bispecific single-chain Fc can be in any appropriate orientation. Examples of bispecific single-chain Fe orientations include, without limitation, VLA-LL-VHA-SL-VHB-LL-VLB-SL-hinge-CH2-CH3-LL-hinge-CH2-CH3, VLA-LL-VHA-SL-VLB-LL-VHB-SL-hinge-CH2-CH3-LL-hinge-CH2-CH3, VHA-LL-VLA-SL-VLB-LL-VHB-SL-hinge-CH2-CH3-LL-hinge-CH2-CH3, VHA-LL-VLA-SL-VHB-LL-VLB-SL-hinge-CH2-CH3-LL-hinge-CH2-CH3, and VLA-SL-VHB-LL-VLB-VHA-SL-hinge-CH2-CH3-LL-hinge-CH2-CH3, where SL is a short linker and LL is a long linker. A short linker can be from about 3 amino acids to about 8 amino acids in length. A short linker can include any appropriate amino acids (e.g., glycines and serines) in any appropriate combination. A long linker can be from about 10 amino acids to about 25 amino acids in length. A long linker can include any appropriate amino acids (e.g., glycines and serines) in any appropriate combination. Any appropriate Fc domain can be used in a bispecific single-chain Fc. In some cases, an Fc domain can include an amino acid sequence derived from an IgG (e.g., a natural IgG). In some cases, an Fc domain can include an amino acid sequence that includes one or more modifications (e.g., one or more modifications to increase stability of the molecule and/or to increase or decrease binding to one or more Fc receptors). In some cases, an Fc domain that can be used in a bispecific single-chain Fc can exclude a hinge domain. In some cases, an Fc domain that can be used in a bispecific single-chain Fc can be at the N-terminus of the scFvs. In some cases, an Fc domain that can be used in a bispecific single-chain Fc can be as described elsewhere (see, e.g., International Patent Application Publication No. WO 2017/134134 A1 at, for example, SEQ ID NOs:25-32; and International Patent Application Publication No. WO 2017/134158 A1 at, for example, Table 38; and SEQ ID NOs:25-32).
A molecule including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4) can include any appropriate complementarity determining regions (CDRs). For example, a molecule including one or more antigen-binding domains that can bind to a modified peptide described herein can include a variable heavy chain (VH) having three VH complementarity determining regions (CDR-VHs) and a variable light chain (VL) having three VL CDRs (CDR-VLs). For example, a molecule that can bind to a modified peptide derived from a modified p53 polypeptide (e.g., HMTEVVRHC (SEQ ID NO:1)) can include one of each of the CDRs set forth below:
For example, a molecule that can bind to a modified peptide derived from a modified H/K/N RAS polypeptide Q61H (e.g., ILDTAGHEEY (SEQ ID NO:2)) can include one of each of the CDRs set forth below:
For example, a molecule that can bind to a modified peptide derived from a modified H/K/N RAS polypeptide Q61L (e.g., ILDTAGLEEY (SEQ ID NO:3) can include one of each of the CDRs set forth below:
For example, a molecule that can bind to a modified peptide derived from a modified H/K/N RAS polypeptide Q61R (e.g., ILDTAGREEY (SEQ ID NO:4) can include one of each of the CDRs set forth below:
In some cases, a molecule including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4) can include any appropriate set of CDR sequences (e.g., any of the CDR sequence sets described herein).
A molecule including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4) can include any appropriate sequence. For example, a molecule that can bind to a modified peptide derived from a modified p53 polypeptide (e.g., HMTEVVRHC (SEQ ID NO:1)) can include, without limitation, the scFv sequence set forth in any one of SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, or SEQ ID NO:141. For example, a molecule that can bind to a modified peptide derived from a modified H/K/N RAS polypeptide (e.g., ILDTAGHEEY (SEQ ID NO:2), ILDTAGLEEY (SEQ ID NO:3), or ILDTAGREEY (SEQ ID NO:4)) can include, without limitation, the scFv sequence set forth in any one of SEQ ID NO:142, SEQ ID NO:143, SEQ ID NO:144, SEQ ID NO:145, SEQ ID NO:146, SEQ ID NO:147, SEQ ID NO:148, SEQ ID NO:149, SEQ ID NO: 150, SEQ ID NO:151, SEQ ID NO:152, SEQ ID NO:153, SEQ ID NO:154, SEQ ID NO:155, SEQ ID NO:156, SEQ ID NO:157, SEQ ID NO:158, SEQ ID NO:159, SEQ ID NO:160, SEQ ID NO:161, SEQ ID NO:162, SEQ ID NO:163, SEQ ID NO:164, SEQ ID NO:165, SEQ ID NO:166, SEQ ID NO:167, SEQ ID NO:168, or SEQ ID NO:169. Examples of sequences (e.g., scFv sequences) that can bind to particular modified peptides are shown in Table 3 and Table 12. In some cases, a molecule including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4) can have a sequence that deviates from a sequence shown in Table 3 and Table 12, sometimes referred to as a variant sequence. For example, a molecule including one or more antigen-binding domains that can bind to a modified peptide described herein can have at least 75% sequence identity (e.g., at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, at least 96% sequence identity, at least 97% sequence identity, at least 98% sequence identity, at least 99% sequence identity, or more) to any of the sequences shown in Table 3 and Table 12, provided the variant sequence maintains the ability to bind to a modified peptide described herein. For example, a molecule including one or more antigen-binding domains that can bind to a modified peptide described herein can have one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more) modifications (e.g., one or more amino acid substitutions) as compared to the sequences shown in Table 3 and Table 12, provided the variant sequence maintains the ability to bind to a modified peptide described herein. In some cases, a molecule including one or more antigen-binding domains that can bind to a modified peptide described herein can include any appropriate set of CDR sequences described herein, and any sequence deviations from a sequence shown in Table 3 and Table 12 can be in the scaffold sequence(s).
A molecule including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4) can be attached (e.g., covalently or non-covalently attached) to a label (e.g., a detectable label). A detectable label can be any appropriate label. In some cases, a label can be used to assist in detecting the presence or absence of one or more modified peptides described herein. For example, a molecule described herein that is labelled can be used in vitro to detect cancer cells (e.g., cancer cells expressing a modified peptide described herein) in a sample obtained from a mammal. In some cases, a label (e.g., a detectable label) can be used to assist in determining the location of one or more modified peptides described herein. For example, molecule described herein that is labelled can be used in vivo to monitor antitumor therapy and/or to detect cancer cells (e.g., cancer cells expressing a modified peptide described herein) in a mammal. Examples of labels that can be attached to a molecule described herein include, without limitation, radionuclides, contrast agents used in magnetic resonance imaging (MRI), computed tomography (CT), ultrasound (US), and other imaging modalities, chromophores, enzymes, and fluorescent molecules (e.g., green fluorescent protein and near-IR fluorescence).
A molecule including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4) can be attached (e.g., covalently or non-covalently attached) to a therapeutic agent. A therapeutic agent can be any therapeutic agent. In some cases, a therapeutic agent can be an anti-cancer agent. Examples of therapeutic agents that can be attached to a molecule described herein include, without limitation, anti-cancer agents such as monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), maytansine, mertansine/emtansine (DM1), ravtansine/soravtansine (DM4), SN-38, calicheamicin, D6.5, dimeric pyrrolobenzodiazepines (PBDs), α-amantin (AAMT), PNU-159682, ricin, pseudomonas exotoxin A, diphtheria toxin, and gelonin.
This document also provides methods for using one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4). For example, one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can target (e.g., bind to) one or more modified peptides can be used to assess a mammal having cancer or suspected of having cancer and/or to treat a mammal having a cancer (e.g., a cancer expressing one or more modified peptides such as a p53 R175H MANA, a RAS Q61H/L/R MANA, and/or a RAS G12V MANA). In some cases, one or more molecules includes one or more antigen-binding domains that can bind to a modified peptide can be used to detect the presence or absence of one or more modified peptides in a sample obtained from a mammal having cancer or suspected of having cancer. In some cases, one or more molecules including one or more antigen-binding domains that can bind to a modified peptide can be administered to a mammal having a cancer (e.g., a cancer expressing the modified peptide) to treat the mammal. Administration of one or more molecules including one or more antigen-binding domains that can bind to a modified peptide described herein to a mammal (e.g., human) having a cancer can be effective to treat the mammal.
Any type of mammal can be assessed and/or treated as described herein. Examples of mammals that can be assessed and/or treated as described herein include, without limitation, primates (e.g., humans and non-human primates such as chimpanzees, baboons, or monkeys), dogs, cats, pigs, sheep, rabbits, mice, and rats. In some cases, a mammal can be a human.
A mammal can be assessed and/or treated for any appropriate cancer. In some cases, a cancer can express one or more modified peptides (e.g., one or more MANAs) described herein. A cancer can be a primary cancer. A cancer can be a metastatic cancer. A cancer can include one or more solid tumors. A cancer can include one or more non-solid tumors. Examples of cancers that can be assessed as described herein (e.g., based at least in part on the presence of one or more modified peptides described herein) and/or treated as described herein (e.g., by administering one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein) include, without limitation, blood cancers (e.g., Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), multiple myeloma, MDS, and myeloproliferative diseases), lung cancers, pancreatic cancers, gastric cancers, colon cancers (e.g., colorectal cancers), ovarian cancers, endometrial cancers, biliary tract cancers, liver cancers, bone and soft tissue cancers (e.g., sarcomas), breast cancers, prostate cancers, esophageal cancers, stomach cancers, kidney cancers, head and neck cancers, brain cancers (e.g., glioblastoma multiforme and astrocytomas), thyroid cancers, germ cell tumors, and melanomas.
When assessing a mammal having cancer or suspected of having cancer, one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) can be used to assess for the presence or absence of one or more modified peptides described herein. For example, the presence, absence, or level of one or more modified peptides described herein in a sample obtained from a human can be used to determine whether or not the human has a cancer. In some cases, the presence of one or more modified peptides described herein in a sample obtained from a mammal can be used to identify the mammal as having a cancer. For example, a mammal can be identified as having a cancer when a sample obtained from the mammal has one or more modified peptides described herein.
Any appropriate sample obtained from a mammal can be assessed for the presence, absence, or level of one or more modified peptides described herein. For example, biological samples such as tissue samples (e.g., breast tissue, and cervical tissue such as from a Papanicolaou (Pap) test), fluid samples (e.g., blood, serum, plasma, urine, saliva, sputum, and cerebrospinal fluid), and solid samples (e.g. stool) can be obtained from a mammal and assessed for the presence, absence, or level of one or more modified peptides described herein. Any appropriate method can be used to detect the presence, absence, or level of one or more modified peptides described herein. For example, sequencing techniques including, but not limited to, Sanger sequencing, chemical sequencing, nanopore sequencing, sequencing by ligation (SOLiD sequencing), sequencing with mass spectrometry, whole exome sequencing, whole genome sequencing, and/or next-generation sequencing can be used to determine the presence, absence, or level of one or more modified peptides described herein in a sample obtained from a mammal.
When treating a mammal having cancer, one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) can be administered to a mammal having cancer to treat the mammal. In some cases, a mammal can have a cancer expressing one or more modified peptides described herein. For example, one or more molecules including one or more antigen-binding domains that can bind to a modified peptide described herein can be administered to a mammal having a cancer expressing that modified peptide to treat the mammal. For example, one or more molecules including one or more scFvs that can bind to a modified peptide described herein (e.g., one or more scDbs) can be administered to a mammal having a cancer expressing that modified peptide to treat the mammal.
In some cases, one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) can be administered to a mammal (e.g., a mammal having a cancer) once or multiple times over a period of time ranging from days to weeks.
In some cases, one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) can be formulated into a composition (e.g., a pharmaceutically acceptable composition) for administration to a mammal (e.g., a mammal having a cancer). For example, one or more antigen-binding domains that can bind to a modified peptide described herein can be formulated together with one or more pharmaceutically acceptable carriers (additives), excipients, and/or diluents. In some cases, a pharmaceutically acceptable carrier, excipient, or diluent can be a naturally occurring pharmaceutically acceptable carrier, excipient, or diluent. In some cases, a pharmaceutically acceptable carrier, excipient, or diluent can be a non-naturally occurring (e.g., an artificial or synthetic) pharmaceutically acceptable carrier, excipient, or diluent. Examples of pharmaceutically acceptable carriers, excipients, and diluents that can be used in a composition described herein include, without limitation, sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses such as microcrystalline cellulose and cellulose ethers like hydroxypropyl cellulose (HIPC) and cellulose ether hydroxypropyl methylcellulose (IPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), crosslinked polyvinylpyrrolidone (crospovidone), carboxymethyl cellulose, polyethylene-polyoxypropylene-block polymers, and crosslinked sodium carboxymethyl cellulose (croscarmellose sodium)), titanium oxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), citric acid, sodium citrate, benzyl alcohol, lysine hydrochloride, trehalose dihydrate, sodium hydroxide, parabens (e.g., methyl paraben and propyl paraben), petrolatum, dimethyl sulfoxide, mineral oil, serum proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g., saline, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylates, waxes, wool fat, lecithin, and corn oil. In some cases, a pharmaceutically acceptable carrier, excipient, or diluent can be an antiadherent, a binder, a colorant, a disintegrant, a flavor (e.g., a natural flavor such as a fruit extract or an artificial flavor), a glidant, a lubricant, a preservative, a sorbent, and/or a sweetener.
A composition (e.g., a pharmaceutical composition) containing one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) can be formulated into any appropriate dosage form. Examples of dosage forms include solid or liquid forms including, without limitation, gums, capsules, tablets (e.g., chewable tablets, and enteric coated tablets), suppositories, liquids, enemas, suspensions, solutions (e.g., sterile solutions), sustained-release formulations, delayed-release formulations, pills, powders, and granules.
A composition containing one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) can be designed for oral, parenteral (including subcutaneous, intramuscular, intravenous, and intradermal), or intratumoral administration. Compositions suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. The formulations can be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.
A composition containing one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) can be administered using any appropriate technique and to any appropriate location. A composition including one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein can be administered locally (e.g., intratumorally) or systemically. For example, a composition provided herein can be administered locally by intratumoral administration (e.g., injection into tumors) or by administration into biological spaces infiltrated by tumors (e.g. intraspinal administration, intracerebellar administration, intraperitoneal administration and/or pleural administration). For example, a composition provided herein can be administered systemically by oral administration or by intravenous administration (e.g., injection or infusion) to a mammal (e.g., a human).
Effective doses can vary depending on the risk and/or the severity of the cancer, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and the judgment of the treating physician. An effective amount of a composition containing one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) can be any amount that treats a cancer present within the subject without producing significant toxicity to the subject. If a particular subject fails to respond to a particular amount, then the amount of one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein can be increased (e.g., by two-fold, three-fold, four-fold, or more). After receiving this higher amount, the mammal can be monitored for both responsiveness to the treatment and toxicity symptoms, and adjustments made accordingly. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the subject's response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., cancer) may require an increase or decrease in the actual effective amount administered.
The frequency of administration of one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) can be any frequency that effectively treats a mammal having a cancer without producing significant toxicity to the mammal. For example, the frequency of administration of one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein can be from about two to about three times a week to about two to about three times a year. In some cases, a subject having cancer can receive a single administration of one or more antibodies described herein. The frequency of administration of one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein can remain constant or can be variable during the duration of treatment. A course of treatment with a composition containing one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein can include rest periods. For example, a composition containing one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein can be administered every other month over a two-year period followed by a six-month rest period, and such a regimen can be repeated multiple times. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the condition (e.g., cancer) may require an increase or decrease in administration frequency.
An effective duration for administering a composition containing one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) can be any duration that effectively treats a cancer present within the mammal without producing significant toxicity to the mammal. In some cases, the effective duration can vary from several months to several years. In general, the effective duration for treating a mammal having a cancer can range in duration from about one or two months to five or more years. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the condition being treated.
In certain instances, a cancer within a mammal can be monitored to evaluate the effectiveness of the cancer treatment. Any appropriate method can be used to determine whether or not a mammal having cancer is treated. For example, imaging techniques or laboratory assays can be used to assess the number of cancer cells and/or the size of a tumor present within a mammal. For example, imaging techniques or laboratory assays can be used to assess the location of cancer cells and/or a tumor present within a mammal.
In some cases, one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4) can be administered to a mammal having a cancer as a combination therapy with one or more additional cancer treatments (e.g., anti-cancer agents). A cancer treatment can include any appropriate cancer treatments. In some cases, a cancer treatment can include surgery. In some cases, a cancer treatment can include radiation therapy. In some cases, a cancer treatment can include administration of one or more therapeutic agents (e.g., one or more anti-cancer agents). Examples of anti-cancer agents include, without limitation, platinum compounds (e.g., a cisplatin or carboplatin), taxanes (e.g., paclitaxel, docetaxel, or an albumin bound paclitaxel such as nab-paclitaxel), altretamine, capecitabine, cyclophosphamide, etoposide (vp-16), gemcitabine, ifosfamide, irinotecan (cpt-11), liposomal doxorubicin, melphalan, pemetrexed, topotecan, vinorelbine, luteinizing-hormone-releasing hormone (LHRH) agonists (e.g., goserelin and leuprolide), anti-estrogens (e.g., tamoxifen), aromatase inhibitors (e.g., letrozole, anastrozole, and exemestane), angiogenesis inhibitors (e.g., bevacizumab), poly(ADP)-ribose polymerase (PARP) inhibitors (e.g., olaparib, rucaparib, and niraparib), radioactive phosphorus, anti-CTLA-4 antibodies, anti-PD-1 antibodies, anti-PD-L1 antibodies, IL-2 and other cytokines, other bispecific antibodies, and any combinations thereof. In cases where one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein are used in combination with one or more additional cancer treatments, the one or more additional cancer treatments can be administered at the same time or independently. For example, a composition including one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein can be administered first, and the one or more additional cancer treatments administered second, or vice versa.
Also provided herein are kits that include one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein (e.g., a modified peptide including an amino acid sequence set forth in any one of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4). For example, a kit can include a composition (e.g., a pharmaceutically acceptable composition) containing one or more molecules including one or more antigen-binding domains (e.g., scFvs) that can bind to a modified peptide described herein. In some cases, a kit can include instructions for performing any of the methods described herein. In some cases, a kit can include at least one dose of any of the compositions (e.g., pharmaceutical compositions) described herein. In some cases, a kit can provide a means (e.g., a syringe) for administering any of the compositions (e.g., pharmaceutical compositions) described herein.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
In this study, a phage display library was designed and built, which displayed a single chain variable fragment (scFv) on the phage surface. The scFvs present in the library were based on the humanized 4D5 (trastuzumab) framework with amino acid variability introduced at key positions of the scFv's complementarity determining regions (CDRs).
Phage display library was used to identify scFvs that specifically recognized mutation-containing peptides folded into a complex with a recombinant HLA allele alpha chain and beta-2 microglobulin (β2M). These complexes, also referred to herein as monomers, mimic the natural peptide/HLA complexes on a cancer cell surface.
Peptide-HLA targets can include mutant peptides (e.g., MANAs) shown in Table 1. scFvs that can specifically bind to peptide-HLA targets in Table 1 are shown in Table 3. These scFvs can also be referred to as MANAbodies for their ability to bind to MANAs.
Flow cytometry data for scFvs that specifically recognized a p53 peptide containing the R175H mutation in complex with HLA-A2 (HMTEVVRHC; SEQ ID NO: 1) are shown in
To demonstrate that MANAbody clones can be utilized as a therapeutic modality, selected MANAbody clones were engineered into bispecific antibodies having one antibody-fragment binding to a mutant peptide presented in the context of HLA and having one antibody-fragment binding to a CD3 protein on the T cell surface (Table 4). The bispecific antibodies are referred to as single-chain diabody (scDb) hereafter. Specifically, bispecific antibodies targeting a mutant p53 R175H peptide presented in the context of HLA-A2 and CD3 were engineered, and bispecific antibodies targeting mutant H/K/N RAS Q61H, Q61L, and Q61R peptides presented in the context of HLA-A1 and CD3 were engineered.
Representative scDb co-culture results are shown in
To evaluate whether the H/K/N RAS Q61H, Q61L, and Q61R scDbs can react against all 3 RAS isoforms with the cognate Q61 mutations, COS-7 cells were transfected with HLA-A1 and plasmids encoding for full-length HRAS, KRAS, or NRAS that are WT, or harbored Q61H, Q61K, Q61L, or Q61R mutation. The transfected COS-7 cells were then co-cultured with T cells and representative H/K/N RAS Q61H, Q61L, and Q61R scDbs.
To evaluate the ability of MANAbody clones to recognize tumor cells, tumor cell lines with endogenous cognate HLA and mutations were co-cultured with T cells and scDbs. An endogenous p53 R175H HLA-A2 positive cell line TYKnu, along with its isogenic p53 knockout control, was cultured with T cells and p53 R175H HLA-A2 scDb. IFNγ release was only induced against the parental TYKnu cell line but not the p53 knockout TYKnu (
To evaluate the efficacy of using MANAbody clones as a therapeutic modality, target cell viability of p53 R175H HLA-A2 and H/K/N RAS Q61L HLA-A1 scDb co-cultures was assayed using Promega's CellTiter-Glo reagent. CellTiter-Glo measures ATP concentration in a well, which is proportional to the number of viable cells. Percent target cell viability was measured by subtracting the CellTiter-Glo value from T cell only wells and normalizing to target cell only wells. Only when parental TYKnu cells were incubated with T cells in the presence of the p53 R175H HLA-A2 scDb, was there significant target cell death (
Together, these findings demonstrate that MANAbodies can be used to redirect and activate T cells to kill tumor cells expressing particular mutant protein and HLA allele pairs (e.g., p53 R175H with HLA-A2 and H/K/N RAS Q61L with HLA-A1).
RPMI-6666 cells (ATCC, Manassas, Va.) were cultured in RPMI-1640 (ATCC) with 20% FBS (GE Hyclone, Logan, Utah, USA), and 1% penicillin streptomycin (Life Technologies). T2 cells (ATCC) and TYKnu (JCRB, Japan) were cultured in RPMI-1640 (ATCC) with 10% FBS (GE Hyclone), and 1% penicillin streptomycin (Thermo Fisher). SigM5 cells (DSMZ, Brunswick, Germany) and HL-60 cells (ATCC) were cultured in Iscove's MDM (ATCC) with 20% FBS (GE Hyclone), and 1% penicillin streptomycin (Thermo Fisher). COS-7 cells (ATCC) was cultured in McCoy's 5A (Modified) Medium (Thermo Fisher) with 10% FBS (GE Hyclone), and 1% penicillin streptomycin (Thermo Fisher). COS-7 cells (ATCC, CRL-1651™) were cultured in DMEM (high glucose, pyruvate; Thermo Fisher) with 10% FBS (GE Hyclone), and 1% Penicillin-Streptomycin (Thermo Fisher). 293FT cells (Thermo Fisher) were cultured in high-glucose D-MEM (Thermo Fisher), with 10% FBS (GE Hyclone), 0.1 mM MEM Non-Essential Amino Acids (NEAA, Thermo Fisher), 6 mM L-glutamine (Thermo Fisher), 1 mM MEM Sodium Pyruvate (Thermo Fisher), 500 μg/ml geneticin (Thermo Fisher), and 1% Penicillin-Streptomycin (Thermo Fisher). All cell lines were maintained at 37° C. under 5% CO2.
PBMCs were obtained by Ficoll-Paque PLUS (GE Healthcare) gradient centrifugation of whole blood from healthy volunteer donors. To activate and expand T cells, PBMCs were cultured with 15 ng/ml OKT3 (BioLegend, San Diego, Calif.), 100 IU/mL recombinant human interleukin-2 (Aldesleukin, Prometheus Therapeutics and Diagnostics, San Diego, Calif.), and 5 ng/ml recombinant human interleukin-7 (BioLegend, San Diego, Calif.) in RPMI-1640 (ATCC) with 10% FBS (GE Hyclone), 1% Penicillin-Streptomycin (Life Technologies) at 37° C. under 5% CO2 for 3 days. After 3 days, the expanded T cells were kept in the same cytokine-containing media without OKT3.
Oligonucleotides were synthesized by GeneArt (Thermo Fisher Scientific) using trinucleotide mutagenesis (TRIM) technology. The oligonucleotides were incorporated into the pADL-10b phagemid (Antibody Design Labs, San Diego, Calif.). This phagemid contains an F1 origin, a transcriptional repressor to limit uninduced expression, a lac operator, and a lac repressor. The scFv was synthesized with a pelB periplasmic secretion signal and was subcloned downstream of the lac operator. A FLAG (DYKDDDDK; SEQ ID NO:190) epitope tag was placed immediately downstream of the variable heavy chain, which was followed in frame by the full-length M13 pIII coat protein sequence.
Ten ng of the ligation product was mixed on ice with 10 μL of electrocompetent SS320 cells (Lucigen, Middleton, Wis.) and 14 μL of double-distilled water. This mixture was electroporated (200 ohms, 25 microFarads, 1.8 kV) using a Gene Pulser electroporation system (Bio-Rad, Hercules, Calif.) and allowed to recover in Recovery Media (Lucigen) for 45 minutes at 37° C. Cells transformed with 60 ng of ligation product were pooled and plated on a 24-cm×24-cm plate containing 2×YT medium supplemented with carbenicillin (100 μg/mL) and 2% glucose. Cells were grown at 37° C. for 6 hours and placed at 4° C. overnight. To determine the transformation efficiency for each series of electroporations, aliquots were taken and titered by serial dilution. Cells grown on plates were scraped into 850 mL of 2×YT medium with carbenicillin (100 μg/mL) plus 2% glucose for a final OD600 of 5-15. Two mL of the 850 mL culture were taken and diluted ˜1:200 to reach a final OD600 of 0.05-0.07. To the remaining culture, 150 mL of sterile glycerol were added before snap freezing to produce glycerol stocks. The diluted bacteria were grown to an OD600 of 0.3-0.5, infected with M13K07 Helper phage at an MOI of 4 (Antibody Design Labs) and shaken at 37° C. for 1 hour. The culture was centrifuged and the cells were resuspended in 2×YT medium with carbenicllin (100 μg/mL), kanamycin (50 μg/mL), and IPTG (50 mM, Thermo Fisher) and grown overnight at 30° C. for phage production. The following morning, the bacterial culture was aliquoted into 50 mL Falcon tubes and pelleted twice at high speed to obtain clarified supernatant. The phage-laden supernatant was precipitated on ice for 40 minutes with a 20% PEG-8000/2.5M NaCl solution at a 4:1 ratio of PEG/NaCl:supernatent. After precipitation, phage was centrifuged at 12,000 g for 40 minutes and resuspended in 1×TBS, 2 mM EDTA, and 1× Complete Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, Mo.). Phages from multiple tubes were pooled and re-precipitated. The total number of transformants obtained was determined to be 3.6×1010. The library was aliquoted and stored in 15% glycerol at −80° C.
DNA from the libraries was amplified using primers that flank the CDR-H3 region. The sequences at the 5′-ends of these primers incorporated molecular barcodes to facilitate unambiguous enumeration of distinct phage sequences. The protocols for PCR-amplification and sequencing are described by Kinde et al. (2011 PNAS. 108:9530-35). Sequences processed and translated using a custom SQL database and both the nucleotide sequences and amino acid translations were analyzed using Microsoft Excel.
Mutant and WT peptides (listed in Table 1) were predicted to bind to HLA alleles using NetMHC version 4.0. All peptides were synthesized at a purity of >90% by Peptide 2.0 (Chantilly, Va.). Peptides were resuspended in DMSO or DMF at 10 mg/mL and stored at −20° C. HLA monomers were synthesized by refolding recombinant HLA with peptide and beta-2 microglobulin, purified by gel-filtration, and biotinylated (Fred Hutchinson Immune Monitoring Lab, Seattle, Wash.). Monomers were confirmed to be folded prior to selection by performing an ELISA using W6/32 antibody (BioLegend, San Diego, Calif.).
scFv-bearing phage clones specific to the mutant pHLA complexes were identified similar to methods described elsewhere (see, e.g., Skora et al, 2015 PNAS.112:9967-72). The panning schema involved an enrichment phase, a competition phase, and a final selection phase.
The phage display library stored at −20° C. in 15% glycerol, were regrown within a week of starting the panning process. A colony of phage-competent SS320 cells (Lucigen, Middleton, Wis.) was inoculated in a 37° C. overnight culture of 2×YT medium (Sigma-Aldrich, St. Louis, Mo.) supplemented with tetracycline (20 μg/mL), and the next day grown to 2 L of mid-log phase (OD600 of 0.3-0.5) bacteria. Bacteria were infected with the phage library at an MOI of 0.5 and M13K07 Helper phage (Antibody Design Labs, San Diego, Calif.) at an MOI of 4 along with the addition of 2% (W/V) glucose (Sigma-Aldrich, St. Louis, Mo.) and allowed to shake for 1 hour at 37° C. The culture was centrifuged and the cells were resuspended in 2×YT medium with carbenicillin (100 μg/mL), kanamycin (50 μg/mL), and 50 μM IPTG and subsequently shaken and grown overnight at 30° C. for phage production. The following morning, the bacterial culture was aliquoted into 50 mL Falcon tubes and pelleted twice at high speed to obtain clarified supernatant. The phage-laden supernatant was precipitated on ice for 40 minutes with a 20% PEG-8000/2.5M NaCl solution at a 1:4 ratio of PEG/NaCl:supernatant. After precipitation, phage was centrifuged at 12,000×g for 40 minutes and resuspended in 1 mL of 1×TBS with 2 mM EDTA, 0.1% sodium azide, and 1×Complete Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, Mo.).
Biotinylated pHLA monomer complexes were conjugated to M-280 streptavidin magnetic Dynabeads (Life Technologies, Carlsbad, Calif.). The biotinylated pHLA were incubated with either 25 μL of Dynabeads beads per 1 μg of pHLA in blocking buffer (PBS, 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide) for 1 hour at room temperature (RT). After the initial incubation, the complexes were washed and resuspended in 100 μL of blocking buffer.
During the enrichment phase (Round 1), approximately 2×1013 phage, representing ˜500-fold coverage of the library, were negatively selected for 1 hour at RT with a mixture of 1 mL unconjugated washed Dynabeads, 1 mg free streptavidin protein (RayBiotech, Norcross, Ga.) to remove any phage recognizing either the unconjugated Dynabead and streptavidin. After negative selection, beads were isolated with a DynaMag-2 magnet (Life Technologies, Carlsbad, Calif.) and the supernatant containing unbound phage was transferred for positive selection for 1 hour at RT against the 1 μg of mutant pHLA conjugated to Dynabeads. Prior to elution, beads were washed 10 times with 1 mL of TBST (1×TBS with 0.5% Tween-20). Phage were eluted by resuspending the beads in 1 mL of 0.2 M glycine, pH 2.2. After a 10 minutes incubation, the solution was neutralized by the addition of 150 μL of 1 M Tris, pH 9.0. Neutralized phages were used to infect 10 mL cultures of mid-log-phage SS320s, with the addition of M13K07 helper phage (MOI of 4) and 2% glucose. Bacteria were then incubated as previously described and the phages were precipitated the next morning with PEG/NaCl.
During the selection phase (Rounds 2-5), phage from the previous round was subjected to negative selection against HLA-matched cell lines without the mutations of interest, corresponding WT pHLA monomer conjugated to Dynabeads, unrelated pHLA monomerconjugated to Danybeads, and free streptavidin. After negative selection, beads were isolated with a DynaMag-2 magnet and unbound phage was transferred for positive selection. This was performed by incubating phage with 1 μg (Round 2), 0.5 μg (Rounds 3, 4), or 0.25 μg (Round 5) mutant pHLA conjugated to the magnetic Dynabeads. Prior to elution, beads were washed 10 times in 1 mL TBST. Phage were eluted from magnetic Dynabeads and used to infect mid-log phase SS320 cells as described above.
Monoclonal phage flow cytometry staining was performed by selecting individual colonies of SS320 cells transformed with a limiting dilution of phage obtained from the final selection round. Individual colonies were inoculated into 200 μl of 2×YT medium containing 100 μg/mL carbenicillin and 2% glucose and grown for three hours at 37° C. The cells were then infected with 1.6×107 M13K07 helper phage and incubated for 1 hour at 37° C. with shaking. The cells were pelleted, resuspended in 300 μL of 2×YT medium containing carbenicillin (100 μg/mL), kanamycin (50 μg/mL), and 50 μM IPTG, and grown overnight at 30° C. for phage production. Cells were pelleted and the phage-laden supernatant was used for staining.
For peptide pulsing, HLA-matched cells were washed once with PBS and once with serum-free RPMI-1640 before incubation at 106 cells per mL in serum-free RPMI-1640 containing 50 μg/mL peptide and 10 μg/mL human beta-2 microglobulin (ProSpec, East Brunswick, N.J.) overnight at 37° C. The pulsed cells were pelleted, washed once in cold staining buffer (PBS containing 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide), and resuspended in 50 μL of stain buffer. Phage staining was performed on ice with 50 μL monoclonal phage supernatant for 1 hour, followed by one 800 μL wash in cold staining buffer. Cells were then stained with 1 μg of rabbit anti-M13 antibody (Novus Biologics, Centennial, Colo.) in 100 μL total volume on ice for 1 hour and washed once with 800 μL of cold staining buffer. Cells were then stained with 5 μL anti-rabbit-PE (Biolegend) on ice for 1 hour in 100 μL total volume, followed by incubation with 200 μL LIVE/DEAD Fixable Near-IR Dead Cell Stain (Thermo Fisher) for 10 minutes at room temperature per manufacturer's instructions. Cells were washed once in 800 μL of staining buffer. Stained cells were analyzed using an iQue Screener (IntelliCyt, Albuquerque, N. Mex.).
gBLOCKs encoding bispecific antibodies were ordered from IDT (Skokie, Ill.). gBLOCKs were cloned into the pcDNA3.4 plasmid (Thermo Fisher) by NEBuilder HiFi DNA Assembly (New England Biolabs, Ipswich, Mass.) following the manufacturer's protocol. 293FT cells (Thermo Fisher) were transfected with the bispecific antibody pcDNA3.4 plasmids using Lipofectamine 3000 (Life Technologies) per the manufacturer's instructions in a T75 flask. Following a 5-8 day incubation, media was harvested and centrifuged at 500 g for 10 minutes at 4° C. Bispecific antibody protein was purified using a Clontech Capturem™ His-Tagged Purification Mixiprep Kit (Takara Bio, Mountain View, Calif.) per manufacturer's instructions. Bispecific antibody protein was desalted into PBS using Zeba spin 7 k MWCO desalting columns per the manufacturer's instructions. Bispecific antibody concentration was quantified using Mini-PROTEAN® TGX Stain-Free™ Precast Gels (Biorad, Hercules, Calif.) using a standard curve of protein of known concentration. Stain-free gels were imaged using the ChemiDoc XRS+ Imager (Biorad).
COS-7 cells were transfected with various combinations of pcDNA3.1 or pcDNA3.4 (Life Technologies) plasmids encoding HLA-A2, HLA-A1, p53(WT, R175H), HRAS(WT, Q61H, Q61K, Q61L, Q61R), KRAS(WT, Q61H, Q61K, Q61L, Q61R), NRAS(WT, Q61H, Q61K, Q61L, Q61R) with Lipofectamine 3000 (Life Technologies) per manufacturer's instructions in a T75 flask. A total of 50,000 T cells were combined with transfected 50,000 COS-7 cells, 25,000 TYKnu cells, or 25,000 HL-60 cells and the specified concentration of bispecific antibody in a 96-well plate, and the co-culture was allowed to incubate for 20 hours at 37° C. under 5% CO2. Following co-culture, conditioned media was collected and assayed for secreted IFNγ by Quantikine® ELISA (R&D Systems, Minneapolis, Minn.). Alternatively, following coculture, target cell viability was measured using CellTiter-Glo (Promega, Madison, Wis.) per the manufacturer's instructions.
The Alt-R CRISPR system (Integrated DNA Technologies, IDT) was used to modify the p53 of the TYKnu cell line and the HLA allele of HL-60 cell line. Alt-R® CRISPR Cas9 crRNAs (IDT) targeting TP53 exon 3 (CCCCGGACGATATTGAACAA; SEQ ID NO:191), and HLA-A exon 2 (CAGACTGACCGAGCGAACCT; SEQ ID NO:192) as well as Alt-R® CRISPR-Cas9 tracrRNA (IDT) were resuspended at 100 μM with Nuclease-Free Duplex Buffer (IDT). The crRNAs and tracrRNA were duplexed at a 1:1 molar ratio for 5 minutes at 95° C. according to the manufacturer's instructions. The duplexed RNA was allowed to cool to room temperature prior to mixing with Cas9 Nuclease (IDT) at a 1.2:1 molar ratio for 15 minutes. To knock out p53 of TYKnu cells, 40 μmols of the Cas9 RNP complexed with p53 gRNA were mixed with 200,000 TYKnu cells in 20 μL of OptiMEM. This mixture was loaded into a 0.1 cm cuvette (Biorad) and electroporated at 120V and 16 ms using an ECM 2001 (BTX). To knock out the HLA-A alleles in HL-60 cells, 40 pmols of the Cas9 RNP complexed with HLA-1 gRNA were mixed with 200,000 HL-60 cells in 20 μL of OptiMEM. This mixture was loaded into a 0.1 cm cuvette (Biorad) and electroporated at 150V and 16 ms using an ECM 2001 (BTX). After electroporation, cells were immediately transferred to complete growth medium and cultured for 10 days, changing media and passaging as needed.
p53 and HLA-A modified polyclonal pools were plated at a density of 0.5 cells per well in 96 well plates and cultured for 2 weeks. Single colonies were harvested and plated into 2 replicate 96-well plates. Genomic DNA was harvested from one of the plates using the Quick-DNA™ 96 Kit (Zymo Research, Irvine, Calif.), PCR amplified using Q5@ Hot Start High-Fidleity 2× Master Mix (New England Biolabs), and Sanger sequenced (Genewiz, South Plainfield, N.J.) to select for clones with the desired modifications.
TP53 is the most commonly mutated cancer driver gene, but despite extensive efforts, no drug targeting mutant TP53 has been approved for treatment of the large number of patients whose tumor contain p53 mutations. This Example describes the identification of an antibody highly specific to the most common TP53 mutation (R175H) in complex with a common HLA-A allele on the cell surface. For example, this Example describes the identification of a TCRm antibody specific to the HLA-A*02:01-restricted p53R175H neoantigen, the structural basis of its specificity, and its conversion to a bispecific antibody that can lyse cancer cells in a fashion dependent on the presence of the neoantigen. Such an immunotherapeutic agent that targets a common TP53 mutation can be used to target cancers containing other tumor suppressor gene mutations.
The p53R175H Neoantigen is Presented on the Surface of Cancer Cells
The p53R175H (aa 168-176, HMTEVVRHC; SEQ ID NO:1) and p53WT(HMTEVVRRC; SEQ ID NO:135) peptides were predicted on the NetMHCpan 4.0 server to bind HLA-A*02:01 at 5177.6 nM (rank 9.6%) and 7121.5 nM (11.6%), respectively. To provide experimental evidence of and to quantify such presentation, peptides eluted from HLA molecules were analyzed in four different cell culture systems using a mass spectrometry (MS)-based method. First, the human HLA-A*02:01 and either p53R175H or p53WT were co-expressed in monkey COS-7 cells. MS analysis of the peptides immunopurified with an anti-HLA antibody detected the p53R175H peptide at approximately 1500 copies per cell (
Identification of scFv-Expressing Phage Clones Specific for the HLA-A*02:01-Restricted p53R175H Peptide and Conversion to scDb Format
To identify TCR-mimic single-chain variable fragments (scFvs) selectively targeting mutant pHLA complexes, an scFv-displaying phage library was screened with an estimated complexity >1×1010. Positive selection against HLA-A*02:01 pHLA monomers containing the p53R175H peptide were combined with negative selection against pHLA monomers containing the p53WT and irrelevant peptides. Selected phage clones were amplified and assessed for binding to T2 cells presenting the mutant or wild-type (WT) peptide via flow cytometry (
Twenty-three phage clones with median fluorescence intensity (MFI) ratios of p53R175H to p53WT>4 were then converted to T cell-retargeting bispecific antibodies (
Next, it was examined whether anti-CD3 arms of the scDb other than the original UCHT1, could influence the ability of H2 to induce T-cell activation. The H2 scFv was linked to a panel of commonly used anti-CD3E scFvs, including UCHT1, hUCHT1v9, OKT3, TR66, and hXR32. It was found that, among the anti-CD3 scFvs tested, UCHT1 activated T cells at the lowest p53R175H peptide concentration when linked to the H2 scFv (
H2-scDb Specifically Recognizes Cancer Cells Expressing the p53R175H Neoantigen
It was next evaluated the ability of H2-scDb to recognize cancer cell lines harboring the p53R175H mutations and expressing various levels of HLA-A*02:01. H2-scDb elicited T-cell responses in a dose-dependent manner when T cells were co-cultured with three lines that expressed moderate to high levels of HLA-A*02:01 (
It was further validated the specificity of H2-scDb using nine pairs of isogenic cell lines that differed with respect to HLA-A*02:01 expression or p53R175H mutation. First, human HEK293FT (TP53WT/HLA-A*02:01) or Saos-2 (TP53null/HLA-A*02:01) cells were transfected with plasmids expressing either full-length p53R175H or p53WT. H2-scDb induced robust T-cell activation when co-cultured with both cell lines overexpressing p53R175H but not with p53WT-overexpressing or parental cells (
To understand the structural basis for the high specificity of the H2 clone for p53R175H/HLA-A*02:01, the H2 fragment antigen-binding (H2-Fab)-p53R175H/HLA-A*02:01 complex was purified (
Binding of the p53R175H Peptide to HLA-A*02:01
The p53R175H neoantigen occupies the binding cleft α1-α2 of HLA-A*02:01, burying a solvent accessible surface area of 870 Å2, slightly larger than other peptide/HLA-A*02:01 complexes (
Structural Basis for the Recognition of p53R175H HLA-A*02:01 by the H2-Fab
The recognition of the HLA-A*02:01 by the H2-Fab was mediated by all six CDRs. There were a total of 79 contacts between the H2-Fab CDRs and the α1 and α2 of HLA-A*02:01, with the light chain contributing to 61% of those contacts. The H2-Fab buried a solvent accessible surface area of 818 Å2 within the HLA, of which 427 Å2 were contributed by the light chain and 391 Å2 by the heavy chain (Table 8). In contrast, only four of the six H2-Fab CDRs (H1, H2, H3 and L3) interacted with the p53R175H peptide. Overall, the H2-Fab made 36 contacts with the p53R175H neoantigen, including five hydrogen bonds and numerous van der Waals interactions. Importantly, His175 at P8 made 47% of all direct contacts with the H2-Fab. The CDR-H1, H2, and H3 of the heavy chain and CDR-L3 of the light chain formed a cage-like configuration around the C-terminus of the p53R175H peptide, trapping Arg174 at P7 and His175 at P8 into position by providing a stable interaction (
1, 4
(bold ≥ 10)
61, 65,
65, 66, 72, 73
58, 62, 63, 65,
(bold ≥ 10)
66, 155, 161,
158, 163
169, 170
Viewed from the axis of the C-terminus to the N-terminus of the p53R175H peptide, the CDRs were arranged in the order H2, H1, L3, H3, L1, L2 (
One of the major challenges confronting new immunotherapeutic antibodies is off-target binding, which can result in toxicity to normal cells. Several powerful approaches to profile TCR and TCRm specificity have been developed to address this important issue. Scanning mutagenesis was employed to identify peptides in the human proteome to which H2-scDb might cross-react. A peptide library was generated by systemically substituting amino acids at each position of the target p53R175H peptide (HMTEVVRHC; SEQ ID NO:1) with each of the remaining 19 common amino acids. T2 cells loaded with each of the 171 variant peptides were then used to assess T-cell activation by measuring IFN-γ release following incubation with H2-scDb (
Next, a nonamer binding motif, x-[AILMVNQTC]-[ST]-[DE]-[IV]-[IMVST]-R-H-[AILVGHSTYC] (SEQ ID NO:197), was generated using 20% target peptide reactivity as a cutoff for permissive amino acids at each position (
To determine whether H2-scDb could control tumor growth in vivo, KMS26 cells were engrafted into NOD-SCID-Il2rg−/− (NSG) mice through intravenous injection, establishing widespread, actively growing cancer cells throughout the body. Two models were used to assess the effects of the H2-scDb in combination with human T cells. In an early treatment model, mice were randomized based on luminescence quantification of tumor burden and H2-scDb was subsequently administered through intraperitoneal infusion pumps at 0.3 mg/kg/day, starting one day after tumor inoculation. An irrelevant isotype scDb was administered in parallel as control. H2-scDb markedly suppressed the growth of parental KMS26 cells (
Taken together, these results demonstrate that --- can be used to target p53, the most common mutation of the most commonly mutated tumor suppressor gene in human cancers, and, as such, can specifically target cancer cells harboring the mutation.
COS-7, T2 (174×CEM.T2), Raji, HH, AU565, SK-BR-3, KLE, HCT116, SW480, NCI-H441, Saos-2, and CCRF-CEM cells were purchased from American Type Culture Collection (ATCC, Manassas, Va.). KMS26, TYK-nu, and HuCCT1 were purchased from Japanese Collection of Research Bioresources Cell Bank (JCRB, Osaka, Japan). SigM5 was obtained from DSMZ (Braunschweig, Germany). HEK293FT was obtained from Invitrogen (Thermo Fisher Scientific, Waltham, Mass.). T2, Raji, Jurkat, HH, AU565, NCI-H441, TOV-112D, CCRF-CEM, KMS26, TYK-nu, and HuCCT1 were cultured in RPMI-1640 (ATCC, 30-2001) with 10% FBS (GE Healthcare, SH30070.03) and 1% Penicillin-Streptomycin (Thermo Fisher Scientific, 15140163). COS-7, SK-BR-3, HCT116, SW480, and Saos-2 were culture in McCoy's 5A modified media (Thermo Fisher Scientific, 16600108) with 10% FBS and 1% Penicillin-Streptomycin. SigM5 was cultured in IMDM (Thermo Fisher Scientific, 12440061) with 20% FBS and 1% Penicillin-Streptomycin. HEK293FT was cultured in DMEM (high glucose, pyruvate, Thermo Fisher Scientific, 11995065) with 10% FBS, additional 2 mM GlutaMAX (Thermo Fisher Scientific, 35050061), 0.1 mM MEM non-essential amino acids (Thermo Fisher Scientific, 11140050), 1% Penicillin-Streptomycin, and 500 μg/mL Geneticin (Thermo Fisher Scientific, 10131027). PBMCs were isolated from leukapheresis samples (Stem Cell Technologies, Vancouver, BC) by standard density gradient centrifugation with Ficoll Paque Plus (GE Healthcare, 17-1440-03). T cells were expanded from PBMCs with addition of the anti-human CD3 antibody (OKT3, BioLegend, San Diego, 317347) at 15 ng/mL for three days. T cells were cultured in RPMI-1640 with 10% FBS, 1% Penicillin-Streptomycin, 100 IU/mL recombinant human IL-2 (aldesleukin, Prometheus Therapeutics and Diagnostics, San Diego, Calif.), and 5 ng/mL recombinant human IL-7 (BioLegend, 581908). All cells were grown at 37° C. in 5% CO2 with humidification.
HLA-A*02:01-restricted p53R175H peptide was directly detected and quantified in COS-7 cells transfected with HLA-A*02:01 and p53R175H and in human cancer cell lines expressing HLA-A*02:01 and p53R175H through MANA-SRM. In particular, the dual-reduction approach described in MANA-SRM was critical for this detection because a cystine and a methionine coexist in the p53R175H peptide. 100 femtomole heavy-isotope labeled peptide HMTEVVRHC (SEQ ID NO:1; New England Peptide Inc, Gardner, Mass.) were spiked into each sample before the assay. The MANA-SRM assays were performed at Complete Omics (Baltimore, Md.).
The scFv-bearing phage library was described elsewhere (see, e.g., Miller et al., J. Biol. Chem. 294:19322-19334 (2019); and Skora et al., Proc. Natl. Acad. Sci. U.S.A 112:9967-9972 (2015)). Oligonucleotides were synthesized by GeneArt (Thermo Fisher Scientific) using trinucleotide mutagenesis (TRIM) technology to diversify complementarity-determining region (CDR)-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3. A FLAG (DYKDDDDK; SEQ ID NO:190) epitope tag was placed immediately downstream of the scFv, which was followed in frame by the full-length M13 pIII coat protein sequence. The total number of transformants obtained was determined to be 3.6×1010.
All peptides were synthesized at a purity of >90% by Peptide 2.0 (Chantilly, Va.) or ELIM Biopharm (Hayward, Calif.), except for the positional scanning library, where crude peptides were used. Peptides were resuspended in dimethylformamide at 10 mg/mL and stored at −20° C. Peptide-HLA monomers were synthesized by refolding recombinant HLA with peptide and β2 microglobulin (β2M), purified by gel-filtration, and biotinylated (Fred Hutchinson Immune Monitoring Lab, Seattle, Wash.). Monomers were confirmed to be folded prior to selection by performing an ELISA using W6/32 antibody (BioLegend, 311402), which recognizes only folded HLA.
Selection of Mutant pHLA Specific Phage Clone
Phage clones bearing scFvs specific to p53R175H/HLA-A*02:01 pHLA were identified using an approach described elsewhere (Skora et al., Proc. Natl. Acad. Sci. U.S.A 112:9967-9972 (2015)). Biotinylated HLA-A*02:01 pHLA monomer complexes were conjugated to 25 μL of M-280 streptavidin magnetic Dynabeads (Thermo Fisher Scientific, 11206D) per 1 μg of pHLA. During the enrichment phase (Round 1), approximately 4×1013 phage, representing ˜1000-fold coverage of the library, were negatively selected with a mixture of unconjugated Dynabeads and free streptavidin protein (RayBiotech, Norcross, Ga., 228-11469). After negative selection, supernatant containing unbound phage was transferred for positive selection using 1 μg of p53R175H/HLA-A*02:01 pHLA. Beads were then washed and phage was eluted to infect mid-log-phage SS320 bacteria, with the addition of M13K07 helper phage (MOI of 4). Bacteria were then grown overnight at 30° C. for phage production and the phage was precipitated the next morning with PEG/NaCl.
During the selection phase (Rounds 2-5), phage from the previous round was subjected to two stages of negative selection: 1) against cell lines without p53R175H/HLA-A*02:01 (RPMI-6666, Jurkat, Raji, SigM5, HH, T2, and NCI-H441) and 2) against p53WT/HLA-A*02:01 pHLA, unrelated HLA-A*02:01 pHLA, and free streptavidin. For negative selection using cell lines, phage was incubated with a total number of 5×106-1×107 of cells at 4° C. overnight. After negative selection, beads were isolated and unbound phage was transferred for positive selection by incubating with 1 μg (Round 2), 0.5 μg (Round 3), or 0.25 μg (Round 4, 5) of p53R175H/HLA-A*02:01 pHLA. Phage was then eluted and amplified by infecting SS320 as described above.
After five rounds of selection, SS320 cells were infected with a limiting dilution of the enriched phage. A total of 190 individual colonies of SS320 were picked and phage DNA was PCR amplified by primers flanking the CDRs (Forward: GGCCATGGCAGATATTCAGA (SEQ ID NO:198), Reverse: CCGGGCCTTTATCATCATC (SEQ ID NO:199)) using Q5 Hot Start High-Fidelity 2× Master Mix (New England BioLabs, M0494L) and Sanger sequenced by GENEWIZ (South Plainfield, N.J.). Sequences flanking the CDRs were trimmed using DNA Baser Sequence Assembler v4 (Arges, Romania) and the sequences spanning the CDRs were clustered using the CD-HIT Suite. Colonies containing unique phage clones were selected and grown overnight in 400 μL of media in deep 96-well plates (Thermo Fisher Scientific, 278743) with the addition of M13K07 helper phage. Bacteria were pelleted the next day and the phage-laden supernatants were used for downstream analysis.
For peptide pulsing, T2 cells were washed with serum-free RPMI-1640 media before incubation at 5×105-1×106 cells per mL in serum-free RPMI-1640 containing specified concentration of peptide for 2 hours at 37° C. For experiments assessed by flow cytometry, 10 μg/mL human β2M (ProSpec, East Brunswick, N.J., PRO-337) was added with the peptides and is specified in the figure legend of such experiments.
Phage staining of peptide-pulsed T2 cells was performed with 50 μL phage supernatant on ice for 1 hour, followed by staining with 1 μg of rabbit anti-M13 antibody (Novus Biologicals, NB100-1633), and anti-rabbit-PE (BioLegend, 406421). HLA-A*02 staining was performed by staining cells with fluorescently labeled anti-human HLA-A*02 (BB7.2, BioLegend, 343308) or mouse isotype IgG2b, K (BioLegend), 402206). Stained cells were analyzed using an LSRII flow cytometer (Becton Dickinson, Mansfield, Mass.) or an iQue Screener (IntelliCyt, Albuquerque, N. Mex.).
Streptavidin-coated, 96-well plates (R&D Systems, Minneapolis, Minn., CP004) were coated with 50 ng of biotinylated HLA-A*02:01 pHLA monomers in 50 μL of blocking buffer (PBS with 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide) at 4° C. overnight. Plates were washed with 1×TBST (TBS+0.05% Tween-20) using a BioTek 405 TS plate washer (BioTek, Winooski, Vt.). Serial dilutions of single chain diabodies (scDbs) were incubated on the plate for 1 hour at RT, washed then incubated with 1 μg/mL recombinant protein L (Thermo Fisher Scientific, 77679) for 1 hour at RT, washed, then incubated with anti-protein L HRP (1:10000, Abcam, ab63506) for 1 hour at RT. Plates were washed, 50 μL of 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (BioLegend, San Diego, Calif., 4211101) was added to each well, and the reaction was quenched with 50 μl 2N sulfuric acid (Thermo Fisher Scientific). Absorbance at 450 nm was measured with a Synergy H1 Multi-Mode Reader (BioTek).
Single chain diabodies (scDbs) were produced by cloning gBlocks (IDT, Coralville, Iowa) encoding each of the variants in the format of, from N- to C-terminus: IL-2 signal sequence, anti-pHLA variable light chain (VL), GGGGS linker (SEQ ID NO:200), anti-CD3 variable heavy chain (VH), (GGGGS)3 linker (SEQ ID NO:201), anti-CD3 VL, GGGGS linker (SEQ ID NO:200), anti-pHLA VH, and 6×HIS tag into linearized pcDNA3.4 vector (Thermo Fisher Scientific, A14697). The proteins were expressed by the Eukaryotic Tissue Culture Core Facility of Johns Hopkins University. Briefly, 1 mg of plasmid DNA was transfected with PEI at a ratio of 1:3 into 1 L of FreeStyle 293-F cells at a density of 2-2.5×106 cells per mL and incubated at 37° C. Five days after transfection, culture media was collected and filtered through a 0.22-μm unit. The scDbs were purified using HisPur Ni-NTA Resin (Thermo Fisher Scientific, 88222) and desalted into PBS pH 7.4 or 20 mM Tris pH 9.0, 150 mM NaCl using 7 k MWCO Zeba Spin desalting columns (Thermo Fisher Scientific, 89890). Proteins were quantified using a 4-15% Mini-PROTEAN TGX gel (Bio-Rad, Hercules, Calif., 4568085) and/or nanodrop (Thermo Fisher Scientific). Proteins were stored at 4° C. for short term storage or snap frozen with the addition of 7% glycerol and stored at −80° C. for long term storage. Alternatively, the scDb protein was produced by GeneArt (Thermo Fisher Scientific) in Expi293s, purified with a HisTrap column (GE Healthcare, 17-5255-01) followed by size exclusion chromatography with a HiLoad Superdex 200 26/600 column (GE Healthcare, 28989335).
Surface Plasmon Resonance Affinity Measurements of p53R175H HLA-A *02:01 to H2-scDb
Biotinylated p53R175H/HLA-A*02:01, p53WT/HLA-A*02:01, and H2-scDb binding experiments were performed at 25° C. using a Biacore T200 SPR instrument (GE Healthcare). Approximately 100-110 response units (RU) of biotinylated p53R175H/HLA-A*02:01 and p53WT/HLA-A*02:01 were captured in flow cells (Fc) 2 and 4, respectively, using a streptavidin chip. Single-cycle kinetics were performed by injecting increasing concentrations (3, 12, 50, 200 to 800 nM) of purified clone H2-scDb flowed over Fe 1-4. Binding responses for kinetic analysis were both blank- and reference-subtracted. Both binding curves were fit with a 1:1 binding model using Biacore Insight evaluation software.
The Alt-R CRISPR system (IDT) was used to knock out the TP53 gene from KMS26, TYK-nu, and KLE cell lines. CRISPR Cas9 crRNAs targeting TP53 exon 3 (p53-5: CCCCGGACGATATTGAACAA (SEQ ID NO:191) or p53-6: CCCCTTGCCGTCCCAAGCAA (SEQ ID NO:202)) as well as CRISPR-Cas9 tracrRNA were resuspended at 100 μM with Nuclease-Free Duplex Buffer. The crRNAs and tracrRNA were duplexed at a 1:1 molar ratio for 5 minutes at 95° C. The duplexed RNA was allowed to cool to room temperature prior to mixing with Cas9 Nuclease at a 1.2:1 molar ratio for 15 minutes. A total of 40 μmols of the Cas9 RNP complexed with TP53 gRNA were mixed with 200,000 cells in 20 μL of OptiMEM. This mixture was loaded into a 0.1 cm cuvette (Bio-Rad, 1652089) and electroporated at 120 V and 16 ms using an ECM 2001 (BTX, Holliston, Mass.). Cells were immediately transferred to complete growth medium and cultured for 7 days. Single cell clones were established by limiting dilution and genomic DNA was harvested using a Quick-DNA 96 Kit (Zymo Research, Irvine, Calif., D3012). A region flanking the CRISPR cut site was PCR amplified (forward primer: GCTGCCCTGGTAGGTTTTCT (SEQ ID NO:203), reverse primer: GAGACCTGTGGGAAGCGAAA (SEQ ID NO:204)) and Sanger Sequenced to select for clones with the desired TP53 status.
Cells were lysed in cold RIPA buffer (Thermo Fisher Scientific, 89901) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, 87785). Protein concentration was determined using a BCA assay (Thermo Fisher Scientific, 23227). Equal amounts of total protein (20-50 μg) were loaded in each lane of a 4-15% Mini-PROTEAN TGX gel (Bio-Rad, 4568085) and transferred to polyvinylidene difluoride membranes after electrophoresis. The membranes were incubated with appropriate primary antibodies (p53 [DO-1], 1:1000, Santa Cruz, sc-126; STAT2, 1:1000, Thermo Fisher Scientific, 44-362G; ZFP3, 1:1000, Thermo Fisher Scientific, PA5-62726; 3-actin [13E5], 1:1000, Cell Signaling Technology, 5125S; β-actin [8H10D10], 1:1000, Cell Signaling Technology, 3700S) and species-specific HRP-conjugated secondary antibodies (1:5000-10000). Signal was detected by a ChemiDoc MP chemiluminescence system (Bio-Rad).
gBlocks (IDT) encoding HLA and target proteins were cloned into pcDNA3.1 or pcDNA3.4 vectors (Thermo Fisher Scientific, V79020, A14697). COS-7, Saos-2, and HEK293FT cells were transfected at 70-80% confluency using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015) and incubated at 37° C. overnight. A total of 15 μg and 30 μg plasmid (1:1 ratio of HLA plasmid/target protein plasmid in co-transfections) was used for T25 and T75 flasks, respectively.
HLA-A*02:01-encoding retrovirus was produced using the MSCV retroviral expression system (Clontech, Mountain View, Calif., 634401). In brief, a gBlock encoding HLA-A*02:01-T2A-GFP (IDT) was cloned into the pMSCVpuro retroviral vector by HiFi DNA assembly (New England Biolabs, Ipswich, Mass., E2621L). The pMSCVpuro-HLA-A*02:01-T2A-GFP plasmid was then co-transfected with a pVSV-G envelope vector into the GP2-293 packaging cell line. Viral supernatant was harvested 48 hours after transfection and concentrated 20-fold using Retro-X Concentrator (Clontech, 631456). RediFect Red-Fluc-GFP lentivirus particles (Perkin Elmer, Waltham, Mass., CLS960003) was used for generating luciferase-expressing cell lines.
For transduction, non-tissue culture-treated 48-well plates were coated with 200 μL of 10 μg/mL RetroNectin (Clontech, T100B) per well overnight at 4° C. and blocked with 10% FBS for 1 hour at RT. Viral particles and 2×105 target cells were added to each well in a total volume of 500 μL cell culture media and spun at 2000×g for 1 hour then incubated at 37° C. Selection with 1 μg/mL puromycin (Thermo Fisher Scientific, A1113803) began three days later. Transduced cells were sorted based on presence of GFP using FACSAria Fusion (BD Biosciences, San Jose, Calif.) 10-14 days after transduction.
In Vitro scDb Co-Incubation Assays
To each well of a 96-well flat-bottom plate, the following components were combined in a final volume of 100 μL RPMI-1640 with 10% FBS, 1% Penicillin-Streptomycin, and 100 IU/mL IL-2: bispecific antibody diluted to the specified concentration, 5×104 human T cells, and 1-5×104 target cells (COS-7, T2, or other tumor cell lines). The effector to target cell ratio is specified in the figure legend for each experiment. The co-culture plate was incubated for 20 hours at 37° C. and conditioned media was assayed for cytokine and cytotoxic granule protein secretion using the Human IFN-γ Quantikine Kit (R&D Systems, Minneapolis, Minn., SIF50), Human IFN-γ Flex Set Cytometric Bead Array (BD, 558269), or the MILLIPLEX Luminex assays (Millipore Sigma, HSTCMAG28SPMX13, HCD8MAG-15K) read on the Bioplex 200 platform (Bio-Rad). Cytotoxicity was assayed by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, Wis., G7571) or Bio-Glo Luciferase Assay (Promega, G7941) per manufacturer's instructions. For CellTiter-Glo assays, percent cytotoxicity was calculated by subtracting the luminescence signal from the average of the T cell only wells and normalizing to the no scDb condition: 1−(scDb well−T cell only)/(no scDb well−T cell only)×100. For Bio-Glo assays, percent cytotoxicity was calculated by normalizing luminescence signal to the no scDb condition: 1−(scDb well)/(no scDb well)×100.
A total of 1×104 CellTracker Green CMFDA (Thermo Fisher Scientific, C7025)-labeled target cells were plated in each well of a 96-well flat bottom plate and allowed to attach for 8 hours before adding T cells and scDb at the indicated E:T ratio and concentrations, respectively. Each condition was plated in triplicate. Plates were imaged every 3 hours using the IncuCyte ZOOM Live-Cell analysis system (Essen Bioscience, Ann Arbor, Mich.) for a total of 60 hours. Four images per well at 10× zoom were collected at each time point. Cell confluence in each well was quantified using the phase contrast channel.
Expression, purification and refolding of p53R175H HLA-A*02:01
Plasmids for HLA-A*02:01 and β2M were received from the NIH Tetramer Facility (Atlanta, Ga.) and separately transformed into BL21(DE3) cells. Each was expressed in inclusion bodies using auto-induction media as described elsewhere (Skora et al., Proc. Natl. Acad. Sci. U.S.A 112:9967-9972 (2015); Martayan et al., The Journal of Immunology 182:3609-3617 (2009); and Huang et al., Bioinformatics 26:680-682 (2010)). Purification of the HLA-A*02:01 and β2M inclusion bodies was achieved with a series of detergent washes followed by solubilization with 8 M urea. Refolding of the HLA-A*02:01, β2M, and mutant p53R175H peptide was performed as described elsewhere (Myszka, J. Mol. Recognit. 12:279-284 (1999)). Briefly, HLA-A*02:01 and β2M inclusion bodies were combined in a refolding buffer containing 100 mM Tris pH 8.3, 400 mM L-arginine, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 2 mM PMSF, and 30 mg of the mutant p53R175H peptide (amino acid 168-176, HMTEVVRHC (SEQ ID NO:1)) dissolved in 1 mL of DMSO. The resultant solution was stirred at 4° C. for 2 days, with two further additions of HLA-A*02:01 inclusion bodies on day 2, concentrated to 10 mL and purified by size exclusion chromatography on a HiLoad 26/60 Superdex 75 Prep grade column (GE Healthcare, 28989334). For incubation with the H2-Fab, purified pHLA-A*02:01 was concentrated to ˜1-3 mg/mL and stored at −80° C. until use.
The light chain (LC) and heavy chain (HC) variable region sequences of H2 scFv were grafted, linked with the respective constant region sequences of human IgG1 and separately cloned into a pcDNA3.4 vector (Thermo Fisher Scientific, A14697). Both chains were preceded by a mouse IgKVIII signal peptide. Before large-scale expression of full-length antibody, optimization of the LC:HC DNA ratio for transfection was performed to determine optimal recombinant protein yields. For a 1 L expression, a total of 50 μg of purified plasmids (1:1 LC:HC ratio) were transfected with PEI at a ratio of 1:3 into Freestyle 293-F cells at a density of 2-2.5×106 cells per mL and incubated at 37° C. for 7 days. The media was harvested via centrifugation, filtered through a 0.22-μm unit and the full-length antibody was purified via protein A affinity chromatography on a HiTrap MabSelect™ SuRe™ column (GE Healthcare, 29-0491-04). Full-length antibody was eluted using a linear gradient of 0-100 mM sodium citrate, pH 3.5. The protein A fractions containing pure H2 antibody were pooled, quantified by SDS-PAGE gel electrophoresis and dialyzed into 20 mM sodium phosphate buffer, pH 7.0, 10 mM EDTA.
For generation of H2-Fab fragments, ˜1-3 mg of full-length antibody was mixed with 0.5 mL of a 50% Immobilized Papain slurry (Thermo Fisher Scientific, 20341) pre-activated with digestion buffer (20 mM sodium phosphate buffer, pH 7.0, 10 mM EDTA) containing 20 mM cysteine-HCl. The mixture was incubated at 37° C. overnight with constant shaking at 200 rpm. The H2 antibody digest was separated from the immobilized resin by a gravity resin separator and washed with 10 mM Tris-HCl, pH 7.5. Newly generated H2-Fab fragments were further purified by cation-exchange chromatography using a Mono-S column (GE Healthcare, 17516801) and eluted using a linear gradient of 0-500 mM NaCl.
The H2-Fab fragments were concentrated, mixed with equimolar p53R175H/HLA-A*02:01 and incubated at 4° C. overnight. The H2-Fab-p53R175H/HLA-A*02:01 mixture was evaluated by size exclusion chromatography on a Superdex™ 200 Increase 10/300 column (GE Healthcare, 28990944). The fractions of ˜98% pure pHLA-A*02:01-H2-Fab complex were pooled, concentrated to 12.6 mg/mL and exchanged into a buffer containing 25 mM HEPES, pH 7.0, 200 mM NaCl.
Crystals of the ternary complex H2-Fab-p53R175H/HLA-A*02:01 were grown in hanging drop by vapor diffusion in drops set up with a TTP mosquito robot with a reservoir solution of 0.2 M ammonium chloride and 20% (w/v) PEG 3350 MME. Crystals were flash-frozen in mother liquor. Data were collected at National Synchrotron Light Source-II at beamlines 17-ID-1(AMX) on a DECTRIS Eiger X 9M detector and 17-ID-2 (FMX) on a DECTRIS Eiger X 16M detector. Datasets were indexed, integrated and scaled using fastdp, XDS, and aimless. Monoclinic crystals of H2-Fab-p53R175H/HLA-A*02:01 diffracted to 3.5 Å. The structure for the H2-Fab-p53R175H/HLA-A*02:01 complex was determined by molecular replacement with PHASER using PDB ID 604Y (11) and 6UJ9 as the search models. The data was refined to a final resolution of 3.5 Å using iterative rounds of refinement with REFMAC5 and manual rebuilding in Coot. Structures were validated using Coot and PDB Deposition tools. The model has 94.11% of the residues in preferred and allowed regions according to Ramachandran statistics (Table 7). Figures were rendered in PyMOL (v2.2.3, Schrödinger, LLC, New York, N.Y.). Buried areas were calculated with PDBePISA. The angle that determines the relative orientation between the pHLA and the Fab/TCR was calculated by the dot product of the vector defined by the position of the alpha carbon of the P1-P9 of the peptide and the vector defined by the disulfide bonds in the VH and VL domains.
Female NOD.Cg-PrkdcscidIl2rgtmlWjl/SzJ (NSG) mice at 6-10 weeks were acquired from the Jackson Laboratory (Bar Harbor, Me., 005557) and treated in compliance with the institutional Animal Care and Use Committee approved protocol. In the early treatment model, mice were inoculated intravenously with 1×106 luciferase-expressing KMS26 or KMS26-TP53 KO cells and 1×107 in vitro expanded human T cells via lateral tail vein injection on day 0. On day 1, mice were randomized based on luminescence quantification using the IVIS imaging system and Living Image software (Perkin Elmer) to ensure similar pretreatment tumor burden. After randomization, two-week micro-osmotic pumps (ALZET, Cupertino, Calif., 1002) filled with H2-scDb or isotype control scDb (scFv against an irrelevant pHLA linked with UCHT1scFv) that had been primed in 1 mL PBS overnight at 37° C. were placed intraperitoneally using sterile surgical technique. Tumor growth was serially monitored by bioluminescent imaging. In the established tumor model, mice were inoculated with 3.5×105 luciferase-expressing KMS26 cells and 1×107 human T cells via lateral tail vein injection on day 0. On day 6, H2-scDb or isotype control scDb was administered similarly as in the early treatment model.
Data are presented as means±SD. Statistical analyses were carried out using specific tests indicated in the figure legend. A P value of <0.05 was used to denote statistical significance. All analyses were performed using Prism version 8.0 (GraphPad, San Diego, Calif.).
Mutations in the RAS oncogenes occur in multiple cancers and new approaches to target these mutations have been the subject of intense research for decades. Most of these efforts have been focused on conventional small molecule drugs rather than antibody-based therapies because the RAS proteins are intracellular.
This Example identifies scFvs specific for peptides derived from two recurrent RAS mutations, G12V and Q61H/L/R, which are presented on cancer cells in the context of two common human leukocyte antigen alleles, HLA-A3 and HLA-A1, respectively. The scFvs did not recognize the peptides derived from the wildtype (WT) form of RAS proteins or other related peptides. Given their extremely low antigen density on cancer cells, a very sensitive immunotherapeutic agent was developed to kill cells harboring RAS gene mutations. Single chain diabodies (scDbs) specific for peptides derived from G12V or Q61H/L/R were capable of inducing T cell activation and killing of target cancer cells expressing endogenous levels of the mutant RAS proteins and cognate HLA alleles.
MANAs Derived from Clinically Relevant RAS Gene Mutations
In silico predictions suggested that the 10-mer peptide from codons 5 to 14 (KLVVVGAVGV, “G12V[5-14]”; SEQ ID NO:209), in which the underlined valine residue (V) represents the G12V mutation, would bind to HLA-A*02:01 (henceforth referred to as HLA-A2) (see, e.g., Skora et al., Proc Natl Acad Sci USA 112:9967-9972 (2015); and Andreatta, Bioinformatics 32:511-517 (2016)). A MANAbody (called D10) that bound to this pHLA-A2 complex and did not bind to the wildtype (WT) counterpart, with selective binding demonstrated using pHLA complexes attached to artificial surfaces as well as using cells pulsed with mutant peptides, was previously reported (Skora et al., Proc Natl Acad Sci USA 112:9967-9972 (2015)). However, it could not be demonstrated that the D10 MANAbody could bind to cells that expressed endogenous levels of the mutant KRAS gene or even to cells that overexpressed an exogenous mutant KRAS gene. It was hypothesized that despite the predictions of the in silico algorithms, the KRAS protein was not processed to the G12V[5-14] peptide and transported to the cell surface. There is indeed precedent for such in silico predictions to be inaccurate (Schmidt et al., J Biol Chem 292:11840-11849 (2017)). To evaluate this possibility, a highly-sensitive mass spectrometry (MS)-based approach (MANA-SRM) was developed to analyze HLA-bound peptides. Human HLA-A2 and full-length human KRAS containing G12V were expressed in SV40 virus-immortalized monkey kidney COS-7 cells and HLA-bound peptides were immunopurified. MS analysis of eluted peptides showed that the G12V[5-14] peptide could not be detected, even when the mutant KRAS gene was overexpressed (Table 10). It was concluded that this peptide was not processed and presented on PLA-A2+ cells harboring a KRAS G12V mutant gene.
Thus, attention was to two peptides, a 9-mer (VVGAVGVGK; SEQ ID NO:206) from codons 8 to 16 (“G12V[8-16]”) and a 10-mer (VVVGAVGVGK; SEQ ID NO:205) from codons 7 to 16 (“G12V[7-16]”), containing the RAS G12V mutation that were predicted by NetMHCv4.0 to bind HLA-A*03:01 (henceforth referred to as HLA-A3) with high affinity (Andreatta, Bioinformatics 32:511-517 (2016)). HLA-A3 is one of the most common HLA-A alleles. MANA-SRM was used to confirm the in silico predictions that these peptide complexes would be displayed on the cell surface before screening for MANAbodies. Human HLA-A3 and full-length human KRAS G12V were expressed in COS-7 cells, then immunopurified with an anti-human HLA antibody. MS analysis of peptides eluted from the captured peptide-HLA complexes detected the G12V[7-16] peptide at 102 copies per cell and the G12V[8-16] peptide at considerably lower levels (24 copies per cell) (
Another commonly mutated residue in RAS genes is the glutamine at codon 61. RAS codon 61 mutations are found in a wide variety of cancers, including melanomas, multiple myelomas, thyroid, and bladder cancers. Using NetMHCv4.0, it was predicted that a 10-mer RAS peptide (codons 55 to 64) could bind with high affinity to HLA-A*01:01 (henceforth referred to as HLA-A1), another common HLA-A allele (Maiers et al., Hum Immunol 68:779-788 (2007)). This peptide (ILDTAGQEEY; SEQ ID NO:136) as well as the flanking amino acids are conserved across all three RAS proteins. Using MANA-SRM, cell surface expression of 10-mer peptides from codons 55 to 64 containing the Q61H, Q61L, and Q61R mutations (ILDTAGHEEY (SEQ ID NO:2), ILDTAGLEEY (SEQ ID NO:3), and ILDTAGREEY (SEQ ID NO:4), respectively) were previously evaluated, and in the transfected COS-7 overexpression system, an average of 583, 512, and 127 copies of the Q61H, Q61L, and Q61R peptides per cell were found (Wang et al., Cancer Immunol Res 7:1748-1754 (2019)). These peptides were also identified to be presented on cell lines expressing endogenous levels of Q61 mutant RAS proteins, notably with four copies of the Q61L peptide per cell found in the acute promyelocytic leukemia cell line HL-60 (Table 10).
Together, these data show that G12V and Q61 mutant RAS proteins can be processed into peptides that are presented on the surface of cancer cell lines, albeit at antigen densities below what is considered the minimum required for recognition by conventional antibody-based immunotherapeutic agents.
Identification of scFv-Expressing Phage Clones Targeting HLA-Restricted RAS Mutant Peptides
To develop MANAbodies targeting the above-named RAS peptides, a second phage library displaying scFvs was built. This library was designed based on principles described elsewhere (Skora et al., Proc Natl Acad Sci USA 112:9967-9972 (2015)), but with important modifications. In particular, precursor library DNA was synthesized using trinucleotide mutagenesis (TRIM) technology, permitting fine tuning of the amino acid diversity at particular codons considered most critical for antigen binding. Diversity was introduced in five of the six complementarity-determining regions (CDRs), with the most amino acid diversity as well as length diversity incorporated into the third CDR of the heavy chain (CDR-H3) (
For the RAS G12V HLA-A3 MANA target, HLA-A3 and beta-2-microglobulin were folded together with the chemically synthesized G12V peptide or its WT [7-16] (G12WT) counterpart to form pHLA complexes (Table 11). These pHLA-A3 were then used to screen the phage display library described above. The screening process consisted of four to six rounds of phage selection, similar to the scheme described previously, with key differences outlined in the Materials and Methods. Over the course of this selection, phage were negatively selected against soluble streptavidin, streptavidin magnetic beads, denatured HLA-A3, unrelated pHLA-A3, and G12WT pHLA-A3 and positively selected against the G12V pHLA-A3. Following the screening process, individual phage clones were amplified and subjected to enzyme-linked immunosorbent assay (ELISA) to assess their binding specificity (
The scFv component of the phage was then expressed and purified in bacteria for further characterization. The recombinant V2 scFv retained the same binding profile as its phage counterpart (
A similar screening procedure was used for RAS Q61H, Q61L, and Q61R pHLA-A1 MANA targets (see Materials and Methods). Using phage staining and T cell-based assays, one phage clone was identified displaying high specificity for each of the three targets: clone H1 for Q61H, clone L2 for Q61L, and clone R6 for Q61R (
T Cell-Engaging Bispecific Antibodies can Recognize Mutant RAS-Derived pHLA Complexes
A variety of T cell-engaging bispecific antibody formats have been developed for targeting T cells to specific ligands. However, there are little data available on whether any of these formats can recognize targets when they are presented on the cell surface at low densities. To inform this point, six bispecific formats were evaluated: diabodies, single chain diabodies (scDbs), bispecific T cell engagers (BiTEs), dual affinity retargeting molecules (DARTs), bivalent scFv-Fcs, and trivalent scFv-Fcs (
In sum, 42 recombinant proteins were expressed in HEK293 cells to identify the most effective format and configuration (
In the initial tests of formats and configurations, five different anti-CD3 scFvs were used. Based on the results indicating that the scDb performed best, seven additional anti-CD3 clones were tested (Tables 12 and 13). A total of twelve V2 scDbs were expressed, purified (
Based off of this work with the G12V clone V2, the Q61 scFv clones H1, L2, and R6 were grafted into the optimized scDb format to generate H1-U, L2-U, and R6-U scDbs. All three scDbs retained binding to their cognate mutant-derived Q61 pHLA-A1 and recombinant CD3ε/δ on ELISA and none exhibited appreciable binding to the Q61WT pHLA or to other control pHLA including an unrelated CMV pHLA (
scDbs Recognize Cells Pulsed with Low Nanomolar Concentrations of Exogenous Peptides
To assess the minimal concentration of target antigen required for activating T cells, T2A3 cells were pulsed with G12V or G12WT peptides and then co-cultured with healthy donor T cells in the presence of the V2-U scDb. T cells were activated at G12V peptide concentrations as low as 1 nM, as evidenced by IFNγ and TNFα secretion (
To determine the approximate antigen density on peptide-pulsed T2A3 cells, the cells were stained with the V2 scFv and assessed by flow cytometry. Antigen density was determined using QIFIKIT and Quantibrite Beads, which allowed quantitative determination of the number of cell surface antigenic molecules (
Similar experiments were performed on the RAS Q61 targeting scDbs using peptide-pulsed SigM5 cells and iDCs. The L2-U scDb elicited T cell activation as shown by IFNγ secretion and SigM5 cytotoxicity when the target cells were pulsed with the Q61L peptide at concentrations as low as 1 nM, without cross-reactivity to the Q61WT peptide (
scDbs Recognize COS-7 Cells Overexpressing HLA and Mutant RAS Genes
COS-7 cells were co-transfected with plasmids encoding HLA-A3 and either mutant or WT full-length KRAS (
Analogous experiments were performed with the mutant RAS Q61-targeting H1-U, L2-U, and R6-U scDbs. COS-7 cells were co-transfected with plasmids encoding HLA-A1 and full-length WT or mutant HRAS, KRAS, or NRAS to assess whether each scDb was capable of recognizing the cognate mutant peptides derived from each of the RAS proteins. Each scDb elicited T cell responses highly specific for the COS-7 cells expressing the RAS gene with the Q61 mutation of interest, regardless of the RAS gene assessed (
scDbs Activate T Cells when Exposed to Cancer Cells Harboring Endogenous Mutant RAS Genes
As noted above, the NCI-H441 cancer cell line presents only ˜9 copies of mutant-derived KRAS G12V pHLA complexes per cell (Table 10). Despite this extremely low level of the target peptide, T cells could be activated by NCI-H441 cells in the presence of the V2-U scDb, as evidenced by the secretion of IFNγ and cytotoxicity (
To further assess the specificity of the V2-U scDb, a second cell line, NCI-H358, was used. This lung cancer cell line contains the HLA-A3 allele and a KRAS G12C mutation. Using CRISPR, the G12V mutation was introduced in the KRAS locus in three independent clones (
IFNγ secretion in co-cultures of T cells with several other HLA-A3+ cancer cell lines without RAS G12V mutations was assessed. These lines included A-427 (lung adenocarcinoma), COLO 741 (melanoma), Hs 578T (breast invasive ductal carcinoma), Jurkat (acute T cell leukemia), SK-MES-1 (lung squamous cell carcinoma), and SW780 (bladder transitional cell carcinoma). CFPAC-1, the KRAS G12V and HLA-A3+ pancreatic adenocarcinoma cell line that presents an average of only ˜3 copies of the G12V peptide per cell were also assessed (Table 10). Expression of HLA-A3 in all these cell lines was confirmed via flow cytometry (
To study the ability of the L2-U scDb to induce T cell activation, co-culture with a panel of cell lines that differed in RAS mutation status and HLA-A1 expression were employed (
To investigate whether the V2 scFv could bind to similar peptides derived from other proteins, a protein BLAST (BLASTp) search of the human RefSeq proteome was performed with the amino acid sequences of the G12V peptide or its G12WT, G12C, or G12D counterparts. Thirty-two proteins containing similar peptides were identified through this search. Of these, NetMHC v4.0 predicted that 17 peptides had strong or weak binding of HLA-A3 (Table 14). Each of these 17 peptides were synthesized and used to pulse T2A3 cells. While the majority of these peptides bind to HLA-A3, as assessed by GAP.A3 antibody staining, V2 phage only recognized the peptide IIVGAIGVGK (“Blast2”; SEQ ID NO:260), a peptide derived from the protein Rab-7b (
sapiens]
sapiens]
sapiens]
sapiens]
Rab-7b is a RAS-related protein expressed in monocytic cells and keratinocytes. To investigate whether this peptide represents an authentic alternative target and thus could cause off-target toxicity of the V2-U scDb, T cells first co-cultured with peripheral blood mononuclear cells (PBMC), monocytes, iDCs, and mature dendritic cells (mDC) prepared from an HLA-A3+ donor (
As a final assessment of the potential cross-reactivity with the Rab-7b peptide, plasmids encoding full length Rab-7b, KRAS WT, or KRAS G12V, in combination with HLA-A3, were transfected into COS-7. Cells overexpressing the mutant KRAS induced robust T cell activation in the presence of V2-U scDb (
To further assay for potential cross-reactivity of the V2-U and L2-U scDbs, their binding to libraries of positional scanning variant peptides was evaluated. The library of peptides was generated by systematically substituting each amino acid of the original peptides with the other 19 amino acids. This resulted in 190 variants for each of the G12V and Q61L peptides for the V2 and L2 scDbs. The variant peptides were pulsed on to T2A3 cells (for V2-U scDb) or SigM5 cells (for L2-U scDb) and co-cultured with T cells. Recognition of the variant peptides was evaluated through IFNγ release (
Evaluation of scDbs in Mouse Models
To determine whether the L2-U scDb could control tumor growth in vivo, HL-60 leukemia cells (NRAS Q61L/WT) and human T cells were intravenously injected into NSG mice to establish widespread leukemic infiltrates. As controls, HL-60 cells engineered to harbor Q61H instead of Q61L/WT alleles were used in a separate group of mice. Bioluminescence established tumor uptake, and mice were randomized to receive the L2-U scDb or a control scDb through intraperitoneal 14-day infusion pumps. The L2-U scDb slowed the growth of the Q61L leukemic cells (
Together these results demonstrate that highly specific bispecific antibodies can be generated against pHLA complexes resulting from common mutations occurring in cancer cells. The format and configuration of the bispecific antibodies developed here are highly specific and sensitive scDbs against protein products containing mutations occurring in cancer cells.
The objective of this study was to generate therapeutic agents that target common mutations in RAS genes. This was accomplished by using phage display to identify scFvs specific to MANAs that had been confirmed to be presented via mass spectrometry. These scFvs were grafted into an optimized bispecific antibody format, the scDb. The scDbs were then shown to mediate MANA-specific T cell activation and target cell cytotoxicity in overexpression and endogenous-level expression model systems. All data presented were representative of data collected during this study. All experiments were performed in triplicate with three technical replicates unless otherwise noted. All experiments were performed in a way to minimize confounding variables, such as plate layout effects.
Plasmids encoding KRAS (isoform B), HRAS (isoform 1), and NRAS variants (WT and mutant) and HLA class I alleles A*01:01 and A*03:01 were synthesized and cloned into pcDNA3.1 by GeneArt (Thermo Fisher Scientific, Waltham, Mass.) or synthesized into gBlocks (IDT, Coralville, Iowa) and assembled into pcDNA3.4 using NEBuilder® HiFi DNA Assembly Cloning Kit (NEB, Ipswich, Mass.).
All cells were grown at 37° C. under 5% C02. HEK293F cells (Thermo Fisher Scientific) were cultured in FreeStyle Expression media. T2A3 cells (a kind gift from Eric Lutz and Elizabeth Jaffee, JHU) were cultured in RPMI-1640 (ATCC, Manassas, Va.) with 10% HyClone FBS (GE Healthcare, Chicago, Ill.), 1% Penicillin-Streptomycin (Thermo Fisher Scientific), 500 μg/mL Geneticin (Thermo Fisher Scientific), and 1× Non-Essential Amino Acids (Thermo Fisher Scientific). COS-7, NCI-H441, CFPAC-1, NCI-H358, and HCT 116 cells (all from ATCC, Manassas, Va.) were cultured in McCoy's 5A (Modified) (Thermo Fisher Scientific) with 10% HyClone FBS and 1% Penicillin-Streptomycin. Jurkat (Clone E6-1, ATCC), COLO 741 (Sigma-Aldrich, St. Louis, Mo.), and SW780 (ATCC) cells were cultured in RPMI-1640 with 10% HyClone FBS and 1% Penicillin-Streptomycin. KMS-21-BM (JCRB Cell Bank, Osaka, Japan) cells were cultured in RPMI-1640 with 20% HyClone FBS and 1% Penicillin-Streptomycin. A-427, Hep G2, Hs 695T, SK-MES-1 (all from ATCC) cells were cultured in Eagle's Minimum Essential Medium (ATCC) with 10% HyClone FBS and 1% Penicillin-Streptomycin. SigM5 (DSMZ) and HL-60 (ATCC) cells were cultured in Iscove's Modified Dulbecco's Medium (ATCC) with 20% HyClone FBS and 1% Penicillin-Streptomycin. RD (ATCC) cells were cultured in Dulbecco's Modified Eagle's Medium (ATCC) with 10% HyClone FBS and 1% Penicillin-Streptomycin. Hs 578T (ATCC) cells were cultured in Dulbecco's Modified Eagle's Medium (ATCC) with 10% HyClone FBS, 1% Penicillin-Streptomycin, and 0.01 mg/ml bovine insulin (Sigma-Aldrich).
Peripheral blood cells were obtained from healthy volunteer donors or purchased as leukapheresis samples (Stem Cell Technologies, Vancouver, BC). PMBCs were purified by density gradient centrifugation with Ficoll Paque Plus (GE Healthcare). T cells were expanded from PBMCs with addition of the anti-human CD3 antibody (clone OKT3, BioLegend, San Diego, Calif.) at 15 ng/mL, or with Human T-Activator CD3/CD28 Dynabeads (Thermo Fisher Scientific) at a 1:5 bead:cell ratio for three days, after which beads were removed with a magnet and the medium was changed. T cells were cultured in RPMI-1640 with 10% HyClone FBS, 1% Penicillin-Streptomycin, 100 IU/mL recombinant human IL-2 (aldesleukin, Prometheus Therapeutics and Diagnostics, San Diego, Calif.), and 5 ng/mL recombinant human IL-7 (BioLegend). The culture medium was changed every 3-4 days and cells were maintained at ˜1 million cells/mL.
To generate dendritic cells, monocytes were negatively isolated from PBMCs using microbeads (Miltenyi) and cultured in Mo-DC differentiation media (Miltenyi) for 5 days to induce differentiation into immature dendritic cells. To generate mature dendritic cells, immature dendritic cells were cultured with 0.5 mg/mL CD40 ligand oligomer (Enzo) in Mo-DC differentiation media for 2 more days.
All cloning was modeled in SnapGene (GSL Biotech LLC, San Diego, Calif.). Oligonucleotides were synthesized by GeneArt (Thermo Fisher Scientific) using trinucleotide mutagenesis (TRIM) technology. The oligonucleotides were incorporated into the pADL-10b phagemid (Antibody Design Labs, San Diego, Calif.) (
Ten ng of the ligation product was mixed on ice with 10 μL of electrocompetent SS320 cells (Lucigen, Middleton, Wis.) and 14 μL of double-distilled water. This mixture was electroporated (200 ohms, 25 microFarads, 1.8 kV) using a Gene Pulser electroporation system (Bio-Rad, Hercules, Calif.) and allowed to recover in Recovery Media (Lucigen) for 45 minutes at 37° C. Cells transformed with 60 ng of ligation product were pooled and plated on a 24-cm×24-cm plate containing 2×YT medium supplemented with carbenicillin (100 μg/mL) and 2% glucose. Cells were grown at 37° C. for 6 hours and placed at 4° C. overnight. To determine the transformation efficiency for each series of electroporations, aliquots were titered by serial dilution. Cells grown on plates were scraped into 850 mL of 2×YT medium with carbenicillin (100 μg/mL) plus 2% glucose for a final OD600 of 5-15. Two mL of the 850 mL culture were taken and diluted ˜1:200 to reach a final OD600 of 0.05-0.07. To the remaining culture, 150 mL of sterile glycerol was added before snap freezing to produce glycerol stocks. The diluted bacteria were grown to an OD600 of 0.3-0.5, transduced with M13K07 helper phage at an MOI of 4 (Antibody Design Labs) and shaken at 37° C. for 1 hour. The culture was centrifuged and the cells were re-suspended in 2×YT medium with carbenicllin (100 μg/mL), kanamycin (50 μg/mL), and IPTG (50 mM, Thermo Fisher Scientific) and grown overnight at 30° C. for phage production. The following morning, the bacterial culture was aliquoted into 50 mL Falcon tubes and pelleted twice at high speed to obtain clarified supernatant. The phage-laden supernatant was precipitated on ice for 40 minutes with a 20% PEG-8000/2.5 M NaCl solution at a 1:4 ratio of PEG/NaCl:supernatant. After precipitation, phage were centrifuged at 12,000 g for 40 minutes and re-suspended in 1×TBS (25 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 2 mM EDTA. Phage from multiple tubes were pooled and re-precipitated to achieve a higher concentration. Final phage were re-suspended in 1×TBS, 2 mM EDTA, and 1× Complete Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, Mo.). The total number of transformants obtained was determined to be 3.6×1010. The library was aliquoted and stored in 15% glycerol at −80° C. and in 50% glycerol at −20° C.
DNA from the library was amplified using the following primers (Forward: CGACGTAAAACGACGGCCAGTNNNNNNNNNNNNNNCGTGCAGAGGATACAGC AGTG (SEQ ID NO:681), Reverse: CACACAGGAAACAGCTATGACCATGCTAACGGTAACCAGGGTGCCCTG (SEQ ID NO:682)) which flank the CDR-H3 region. (All oligonucleotide sequences listed in this manuscript begin with the most 5′ nucleotide.) The sequences at the 5′-ends of these primers incorporated molecular barcodes to facilitate unambiguous enumeration of distinct phage sequences as well as universal primer sites. The protocols for PCR-amplification and sequencing were described elsewhere (Kinde et al., Proc Natl Acad Sci USA 108:9530-9535 (2011)). Sequences were processed and translated using a custom SQL database and both the nucleotide sequences and amino acid translations were assessed.
Peptides and pHLAs
Peptides (Table 11) were synthesized at a purity of >90% by Peptide 2.0 (Chantilly, Va.) or ELIM Biopharm (Hayward, Calif.), with the exception of the crude peptides that were used for the positional scanning library. Peptides were re-suspended in DMF at 10 mg/mL and stored at −80° C. pHLAs were synthesized by refolding recombinant HLA-A*01:01 (HLA-A1) or HLA-A*03:01 (HLA-A3) with peptide and beta-2 microglobulin, purified by gel-filtration, and biotinylated (Fred Hutchinson Immune Monitoring Lab, Seattle, Wash.; or Baylor MHC Tetramer Production Lab, Houston, Tex.). These pHLA were confirmed to be folded prior to selection via ELISA using the W6/32 antibody (BioLegend, San Diego, Calif.), which recognizes only folded HLA. Blast peptides (Table 14) were synthesized as described above, re-suspended in DMF at 10 mg/mL and stored at −80° C. Cognate peptide reactivity search of the UniProtKB human protein database using ScanProsite was performed using binding motifs with a 20% parental peptide IFNγ value as a cutoff. The V2 motif was {FWDY}-[ILMVTC]-{RE}-{ILV}-x-[ILV]-[GNST]-[VP]-[AG]-[HKY] (SEQ ID NO:683). The L2 motif was x-{PWRHDEY}-[APRDEQSC]-{DE}-[AMFPGHDNQSTYC]-[AG]-[ILM]-[AIMGRDENQ]-[DE]-[AY] (SEQ ID NO:684).
Selection of Mutant pHLA-Specific Phage Clones
scFv-bearing phage specific to the RAS G12V[7-16] “G12V” pHLA-A3 and RAS Q61H, Q61L, Q61R, referred to collectively as “Q61X”, in pHLA-A1 were identified using methods similar to those described elsewhere (Skora et al., Proc Natl Acad Sci USA 112:9967-9972 (2015); and Miller et al., J Biol Chem 294:19322-19334 (2019)). The phage display library was regrown within a week of starting the selection process. A colony of phage-competent SS320 cells (Lucigen, Middleton, Wis.) was inoculated in 2×YT medium (Sigma-Aldrich, St. Louis, Mo.) supplemented with tetracycline (20 μg/mL) and cultured at 37° C. overnight, then grown to 2 L of mid-log phase (OD600 of 0.3-0.5) bacteria. Bacteria were transduced with the phage library at an MOI of 0.5 and M13K07 helper phage (Antibody Design Labs, San Diego, Calif.) at an MOI of 4 along with the addition of 2% (W/V) glucose (Sigma-Aldrich, St. Louis, Mo.) and shaken, not stirred, for 1 hour at 37° C. The cells were pelleted and re-suspended in 2×YT medium with carbenicillin (100 μg/mL), kanamycin (50 μg/mL), and 50 μM IPTG and subsequently shaken and grown overnight at 30° C. for phage production. The following morning, the bacterial culture was aliquoted into 50 mL Falcon tubes and pelleted twice by centrifugation at 12,000×g to obtain clarified supernatant. The phage-laden supernatant was precipitated on ice for 40 minutes with a 20% PEG-8000/2.5 M NaCl solution at a 1:4 ratio of PEG/NaCl:supernatant. After precipitation, phage were pelleted by centrifugation at 12,000×g for 40 minutes and re-suspended in 1 mL of 1×TBS with 2 mM EDTA, 0.1% sodium azide, and 1× Complete Protease Inhibitor Cocktail (Sigma-Aldrich, St. Louis, Mo.).
For the G12V pHLA-A3 target MANA, the selection scheme involved an enrichment phase (one round), a competition phase (up to three rounds), and a final selection phase (two rounds). The biotinylated pHLA were incubated with 25 μL of M-280 streptavidin Dynabeads (Invitrogen, Thermo Fisher Scientific) or 100 μL of streptavidin-coated agarose beads (Novagen EMD Millipore, Burlington, Mass.) per 1 μg of pHLA in blocking buffer (phosphate-buffered saline or PBS, 0.5% BSA, 0.1% sodium azide) for 1 hour at room temperature (RT). After the initial incubation, the complexes were washed and re-suspended in 100 μL of blocking buffer. During the enrichment phase (Round 1), approximately 4×1012 phage, representing ˜100-fold coverage of the library, were negatively selected overnight at 4° C. against 500 μL unconjugated washed Dynabeads, 500 μg free streptavidin protein (RayBiotech, Norcross, Ga.), and 3 μg heat-denatured, allele-matched HLA conjugated to Dynabeads. This step was designed to remove phage recognizing Dynabeads, streptavidin or denatured HLA-A3. After negative selection, beads were isolated with a DynaMag-2 magnet (Life Technologies, Carlsbad, Calif.) and the supernatant containing unbound phage was used for positive selection by incubation with 0.5 μg G12V pHLA-A3 conjugated to Dynabeads for 1 hour at RT. The beads were washed 10 times with 1×TBS with 0.5% Tween-20 using the DynaMag-2 magnet, and the phage were eluted from the beads by re-suspension in 1 mL of 0.2 M glycine, pH 2.2. After a 10-minute incubation, the solution was pH-neutralized by the addition of 150 μL of 1 M Tris, pH 9.0. This neutralized solution was used to transduce 10 mL cultures of mid-log-phase SS320s to which M13K07 helper phage (MOI of 4) and 2% glucose was added. Bacteria were then incubated as described above and the phage precipitated the next morning with PEG/NaCl.
During the competitive phase (Rounds 2-4), the amount of input phage used in each round was decreased to 5%, 1%, and 0.1% of the total precipitated phage from the previous round, respectively. These phage were subjected to negative selection against 1 μg heat-denatured HLA-A3, 1 μg total of unrelated pHLA-A3, and 20 μg free streptavidin for 1 hour at RT. After negative selection, beads were isolated with a DynaMag-2 magnet and the unbound phage were used for positive selection. This was accomplished by simultaneously co-incubating phage with 0.5 μg G12V pHLA-A3 conjugated to the magnetic Dynabeads and corresponding 1 μg G12WT pHLA-A3 conjugated to streptavidin-coated agarose beads as competitor. Prior to elution, beads were washed 10 times in 1 mL 1×TBS with 0.5% Tween-20. Phage were eluted from magnetic Dynabeads and used to transduce mid-log phase SS320 cells as described above.
During the final selection phase, phage resulting from rounds 2, 3, and 4 were separately subjected to additional, more stringent rounds of selection. 0.1% of the precipitated phage from these rounds underwent two negative selections against 0.5 μg G12WT pHLA-A3, followed by a positive selection against 0.5 μg of G12V pHLA-A3. The beads were washed 10 times in 1 mL 1×TBS with 0.5% Tween-20, and phage were eluted and used to transduce mid-log phase SS320 cells as described above. The final selection steps described above was repeated a second time, thus the phage underwent a total of four to six total rounds of negative/positive selection.
scFv-bearing phage specific to RAS Q61X-HLA-A1 MANA targets were selected as described above with the following differences. The Q61X pHLA-A1 panning scheme involved one round of enrichment and four rounds of more stringent selection. In the enrichment round, ˜2.6×1013 phage, representing ˜720-fold coverage of the library, were negatively selected against 1 mL unconjugated washed Dynabeads and 1 mg free streptavidin protein. This was followed by positive selection of the unbound phage using 2 μg of the mutant Q61X pHLA-A1. For the four subsequent selection rounds, 10%, 1%, 0.1%, and 0.02% of the phage from the previous round were used as input for panning, respectively. In each of these rounds, phage were negatively selected using 2 μg Q61WT pHLA-A1, 2 μg unrelated pHLA-A1, and 5×108 cells from HLA-A1+ cell lines lacking the RAS mutation of interest. Unbound phage were used for positive selection with 1 μg, 0.5 μg, 0.5 μg, and 0.25 μg Q61X pHLA-A1 in the four sequential rounds, respectively.
To obtain monoclonal phage, individual colonies of SS320 cells transduced with a limiting dilution of phage were inoculated into 200 μL of 2×YT medium containing 100 μg/mL carbenicillin and 2% glucose and grown for 3 hours at 37° C. The cells were then transduced with 1.6×107 M13K07 helper phage and incubated for 1 hour at 37° C. with shaking. The cells were pelleted, re-suspended in 300 μL of 2×YT medium containing carbenicillin (100 μg/mL), kanamycin (50 μg/mL), and 50 μM IPTG, and grown overnight at 30° C. for phage production. Cells were pelleted and the phage-laden supernatant was used for downstream analysis.
Monoclonal phage DNA was PCR amplified using 1 μL of monoclonal phage supernatant in a reaction with primers flanking the CDRs (Forward: GGCCATGGCAGATATTCAGA (SEQ ID NO:198), Reverse: CCGGGCCTTTATCATCATC (SEQ ID NO:199)) and Q5 Hot Start High-Fidelity 2× Master Mix (New England BioLabs). The PCR product was Sanger-sequenced by Genewiz (South Plainfield, N.J.). Sequences flanking the CDRs were trimmed using DNA Baser Sequence Assembler v4 (Arges, Romania) and the sequences spanning the CDRs were clustered using the CD-HIT Suite. Colonies containing unique phage clones were selected for further assays.
Streptavidin-coated, 96-well plates (R&D Systems, Minneapolis, Minn.) were coated with 50 ng of biotinylated pHLA-A3 or pHLA-A1 (unless otherwise specified) or 25 ng of biotinylated recombinant heterodimeric CD3/6 (Abcam, Cambridge, Mass.) in 50 μL of blocking buffer (PBS with 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide) at 4° C. overnight. Plates were washed with 1×TBST (1×TBS+0.05% Tween-20) using a BioTek 405 TS plate washer (BioTek, Winooski, Vt.).
The phage clones resulting from the RAS G12V pHLA-A3 panning were characterized via monoclonal ELISA, where individual monoclonal phage clones were separately interrogated for their binding to G12V pHLA-A3 and G12WT pHLA-A3 via ELISA. 50 μL of phage supernatant was added to washed streptavidin ELISA plates coated with either G12V or G12WT pHLA-A3. Plates were incubated for 2 hours at RT and then washed 6 times. The bound phage were then incubated with 50 μL of rabbit anti-fd/M13 bacteriophage antibody (Novus Biologicals, Abingdon, UK) diluted 1:3000 in 1×TBST for 1 hour at RT, followed by washing and incubation with 50 μL of goat anti-rabbit HRP (Thermo Fisher Scientific) diluted 1:10,000 in 1×TBST for 1 hour at RT. After washing, 50 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate (BioLegend) was added to each well and the reaction was quenched with 1 N sulfuric acid (Fisher Scientific, Thermo Fisher Scientific). Absorbance at 450 nm was measured with a Synergy H1 Multi-Mode Reader (BioTek).
ELISA with purified scFvs, scDbs, and other bispecific antibody formats was performed essentially as above, with serial dilutions of the recombinant protein of interest incubated for 1 hour at RT, followed by incubation with 1 μg/mL recombinant protein L (Pierce, Thermo Fisher) for 1 hour at RT, followed by incubation with anti-protein L HRP (Abcam). Plates were washed, exposed, and read as described above. pHLA titration ELISAs assessing the binding of the scFvs (at a fixed concentration) to the mutant and WT pHLA (at varying concentrations) were performed by AxioMx Inc (Abcam).
Sandwich ELISAs were performed by coating biotinylated pHLA-A3 on a streptavidin plate and incubating with scDbs as described above, followed by incubation with recombinant heterodimeric CD3ε/δ protein containing a human Fc domain at 1 μg/mL for 1 hour at RT, followed by detection with anti-human Fc HRP (Abcam) at 1:10,000 for 1 hour at RT. Plates were washed, exposed, and read as described above.
For peptide pulsing of cells, cells were washed once with PBS and once with RPMI-1640 containing 1% Penicillin-Streptomycin without serum. The cells were then incubated at 5×105-1×106 cells per mL in serum-free RPMI-1640 containing 50 μg/mL or specified concentration of peptide and 10 μg/mL human beta-2 microglobulin (ProSpec, East Brunswick, N.J.) for 4 hours at 37° C. Prior to staining, cells were spun and re-suspended in cold stain buffer (PBS containing 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide).
The phage clones resulting from the RAS Q61X pHLA-A1 selection were characterized via flow cytometry, where individual monoclonal phage clones were separately interrogated for their binding to mutant Q61X and Q61WT peptide-pulsed SigM5 cells. Monoclonal phage were grown and sequenced as described above. Phage supernatant from representative wells of each unique clone was selected for flow cytometry analysis. In each well of a deep 96-well 2 mL plate, 50 μL of monoclonal phage was incubated with 2.5×105 peptide-pulsed SigM5 cells in 50 μL of stain buffer. Plates were incubated on ice for 1 hour, followed by washing with 1 mL of stain buffer. Cells were then stained with 1 μg of rabbit anti-M13 antibody (Novus Biologicals), washed, stained with anti-rabbit-PE (BioLegend), incubated with an additional 100 μL of LIVE/DEAD Fixable Near-IR dye diluted 1:1000 in PBS for 10 minutes at RT in the dark, followed by washing in stain buffer before analysis. Stained cells were analyzed using an Intellicyt iQue3 flow cytometer (Sartorius, Gottingen, Germany).
Peptide-pulsed T2A3 phage staining assays were performed by incubating 5×105-1×106 cells with 1×1010 phage in 100 μL stain buffer on ice for 1 hour, followed by one wash in stain buffer. Cells were then stained with 1 μg of rabbit anti-M13 antibody (Novus Biologicals), washed, stained with anti-rabbit-PE (BioLegend), incubated with an additional 500 μL of LIVE/DEAD Fixable Near-IR dye diluted 1:1000 in PBS for 10 minutes at RT in the dark, and washed in stain buffer before analysis. V2 scFv staining was performed using 0.33 μg of V2 scFv premixed with 1 μg anti-FLAG-PE antibody (BioLegend) overnight at 4° C. followed by incubation with peptide-pulsed T2A3 cells as described above. Anti-HLA-A3 staining was performed by staining 5×105 cells with 0.125 μg clone GAP.A3-PE (eBioscience, Thermo Fisher Scientific) or mouse isotype IgG2a-PE (BioLegend). Anti-HLA-A1 staining was performed by staining 5×105 cells with 0.5 μg anti-HLA-A1/A11/A26 antibody clone 8.L.101 (Abcam) or mouse isotype IgM (Thermo Fisher) followed by 0.25 μg anti-mouse-PE (BioLegend). Stained cells were analyzed using an LSRII flow cytometer (Becton Dickinson, Mansfield, Mass.).
Quantification of cell surface-bound G12V peptide was performed using two commercial kits: PE Quantibrite Beads (BD, Franklin Lakes, N.J.) and QIFIKIT (Agilient, Santa Clara, Calif.). For Quantibrite-based quantification, peptide-pulsed T2A3s were stained with 0.5 μg V2 scFv preconjugated to 1.5 μg clone L5 anti-FLAG-PE (BioLegend), followed by flow cytometry and quantification according to the manufacturer's instructions. For QIFIKIT-based quantification, peptide-pulsed T2A3s were stained with 0.5 μg V2 scFv preconjugated to 1.5 μg clone M2 anti-FLAG (Sigma Aldrich), followed by staining with anti-mouse-PE (BioLegend), flow cytometry and quantification according to the manufacturer's instructions.
Recombinant scFv and Bispecific Antibody Production
Recombinant scFv proteins were produced and purified by AxioMx Inc (Abcam). In brief, the V2, H1, L2, and R6 scFv sequences were subcloned into a vector containing a periplasmic localization sequence, and C-terminal Flag and His tags. ScFvs were expressed in E. coli and purified via nickel chromatography.
Bispecific antibodies were produced after subcloning gBlocks (IDT, Coralville, Iowa) encoding each of the variants with an IL-2 signal sequence and C-terminal 6×HIS tag into the pcDNA3.4 vector (Thermo Fisher Scientific). Bispecific antibodies were produced by the Eukaryotic Tissue Culture Core Facility of Johns Hopkins University. In brief, 1 mg of plasmid was transfected using polyethylenimine (PEI) into HEK293F cells which were then grown as suspension culture at a density of 2×106 cells/mL in FreeStyle 293 expression media (Thermo Fisher Scientific) at 37° C., 170 rpm, and 5% C02. Protein was expressed for 5 days, after which cells were harvested by centrifugation, and the resulting supernatant filtered using a 0.22 μm filter. To each 1 L of supernatant, 2 mL of Ni-NTA His-Bind (Millipore Sigma) resin slurry was added and allowed to incubate at 4° C. overnight on an orbital shaker. The supernatant containing the slurry was passed through a centrifuge column (Pierce, Thermo Fisher Scientific), whereby the slurry was washed with 20 mM imidazole in PBS, and eluted in 50 mM, 100 mM, and 250 mM imidazole fractions. Protein fractions were run on mini-PROTEAN TGX stain-free gels (Bio-Rad, Hercules, Calif.) and appropriate fractions were combined for desalting into PBS, pH 7.4 or 20 mM Tris, 150 mM NaCl, pH 9 using 7 k MWCO Zeba Spin desalting columns (Thermo Fisher Scientific). Proteins were quantified via stain free gel and BCA protein assay (Pierce, Thermo Fisher Scientific). For scDb western blots, protein was transferred from the stain-free gel to a Trans-Blot Turbo Mini 0.2 μm PVDF membrane (Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad). Membrane was blocked with blocking buffer (5% Bio-Rad Blotting-Grade Blocker in 1×TBST) on an orbital shaker (VWR) for 1 hour at RT, followed by incubation with anti-6×His tag antibody clone ab9108 at 1:1000 in blocking buffer at 4° C. overnight, washing in 1×TBST, and incubation with anti-rabbit-HRP (Thermo Fisher Scientific) at 1:10,000 in blocking buffer for 1 hour at RT. Membrane was washed in 1×TBST followed by ddH2O, then imaged using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) on a ChemiDoc XRS+ imager (Bio-Rad). Proteins were stored at 4° C. for short term storage or snap frozen with the addition of 7% glycerol and stored at −80° C. for long term storage. Alternatively, the V2-U scDb and L2-U scDb proteins were produced by GeneArt in Expi293s, purified with a HisTrap column followed by size exclusion chromatography using a HiLoad Superdex 200 26/600 column.
RAS G12V pHLA-A3, G12WT pHLA-A3, and V2 scFv binding experiments were performed at 25° C. using a Biacore T200 SPR instrument (GE Healthcare, Chicago, Ill.). Approximately 130-140 response units (RU) of biotinylated pHLA-A3 were captured in flow cells (Fc) 2 and 4, respectively, using a streptavidin chip. Single-cycle kinetics were performed by injecting increasing concentrations (1.56, 6.25, 25, 100, 400 nM) of purified V2 scFv flowed over Fc 1-4. Binding responses for kinetic analysis were both blank and reference subtracted. Both binding curves were fit with a 1:1 binding model using Biacore Insight evaluation software. Measurements for L2-U scDb were done similarly, using biotinylated Q61L pHLA-A1 and Q61WT pHLA-A1.
pHLA Immunoprecipitation and Mass Spectrometry
pHLA immunoprecipitation and mass spectrometry were performed as described elsewhere (Wang et al., Cancer Immunol Res 7:1748-1754 (2019)). Briefly, COS-7 cells seeded into 24.5×24.5 cm2 plates were transfected at 95% confluency using Lipofectamine 3000 Reagent (Thermo Fisher Scientific). For each plate, 125 μg of plasmids (50 μg of HLA plasmid and 75 μg of mutant or WT protein plasmid) were mixed with 200 μL of Lipofectamine P3000 in 6 mL of Opti-MEM (Thermo Fisher Scientific). In a separate tube, 200 μL of Lipofectamine 3000 Reagent was mixed with 6 mL of Opti-MEM. The contents of the two tubes were mixed and allowed to complex for 10 minutes. Medium bathing cells were removed and 50 mL of fresh complete medium was added followed by the Lipofectamine-DNA mixture. Cells were harvested 48 hours post-transfection. The transfection efficiency of COS-7 was >90% as assessed by GFP+ cell fraction on flow cytometry (BD LSRII).
Cells (transfected or untransfected) were grown to near confluency in 24.5×24.5 cm2 plates. Cultured cells were washed with PBS two times, followed by another wash with PBS pre-chilled at 4° C. containing 1× protease inhibitor. Cells were scraped and collected in a 500-mL centrifuge bottle. The bottle was centrifuged at 1,000 g for 5 minutes and the supernatant discarded. Cell pellets were snap frozen in liquid nitrogen and stored at −80° C. for future experiments.
Neoantigen-expressing cells were processed as described elsewhere (Wang et al., Cancer Immunol Res 7:1748-1754 (2019)). In brief, a total of 500 million cells were lysed and pHLA complexes were immunoprecipitated using Protein G Dynal Magnetic Beads (Thermo Fisher Scientific) pre-conjugated with anti-human HLA-A, B, C antibody clone W6/32 (Bio-X-Cell). After elution, dissociation, and filtration, peptides were lyophilized before further analysis. HPLC fractionation and a Dual-Reduction procedure were then performed (Wang et al., Cancer Immunol Res 7:1748-1754 (2019)). Controls for detection of the RAS G12V [7-16] and [8-16] peptides were established using AQUA™ heavy isotope labeled peptides of the same sequence (Sigma-Aldrich). These AQUA peptides were added to the cell lysates in every experiment. Transition parameters were manually examined and curated to exclude ions with excessive noise due to co-elution with impurities. Absolute copy numbers of peptides presented on the cell surface were calculated based on the MANA-SRM quantification using the AQUA™ heavy isotope labeled peptides and the recovery ratios of the pipeline, as described elsewhere (Wang et al., Cancer Immunol Res 7:1748-1754 (2019)).
The Alt-R CRISPR system (Integrated DNA Technologies, IDT) was used to modify the HLA alleles, the KRAS mutation status of the NCI-H358 and NCI-H441 cell lines, and the NRAS mutation status of the HL-60 cell line. Alt-R® CRISPR Cas9 crRNAs (IDT) and Alt-R® CRISPR-Cas9 tracrRNA (IDT) were re-suspended at 100 μM with Nuclease-Free Duplex Buffer (IDT). The crRNAs and tracrRNA were mixed at a 1:1 molar ratio and denatured for 5 minutes at 95° C., followed by slow cooling to room temperature to duplex prior to mixing with Cas9 Nuclease (IDT) at a 1.2:1 molar ratio for 15 minutes. To knock out the HLA-A3 allele in NCI-H441 cells, 40 pmoles of the Cas9 ribonucleoprotein (RNP) containing tracrRNA/HLA-A crRNA (GCTGCGACGTGGGGTCGGAC; SEQ ID NO:685) duplex were mixed with 2×105 NCI-H441 cells in 20 μL of OptiMEM. This mixture was loaded into a 0.1 cm cuvette (Bio Rad) and electroporated at 150 V for 10 ms using an ECM 2001 (BTX). HLA-A1 was similarly knocked out in HL-60 cells by mixing Cas9 RNP containing tracrRNA/HLA-A1 crRNA (CAGACTGACCGAGCGAACCTG; SEQ ID NO:686) duplex with 5×105 HL-60 cells, and electroporated at 120V for 16 ms. Cells were immediately transferred to complete growth medium and cultured for 10 days.
To change (“KI”) the KRAS mutation status of NCI-H358 from G12C/WT to G12V and NCI-H441 from G12V/WT to G13D/WT, Cas9 RNPs were co-electroporated with single-strand DNA homology directed repair templates. The G12V repair template was ATTAGCTGTATCGTCAAGGCACTCTTGCCTACGCCAACGGCGCCGACAACGACG AGTTTATATTCAGTCATTTTCAGCAGGCCTTATAA (SEQ ID NO:687) for KRAS-G12V crRNA (AATGACTGAATATAAACTTG; SEQ ID NO:688). The G13D repair template was AACAAGATTTACCTCTATTGTTGGATCATATTCGTCCACAAAATGATTCTGAATT AGCTGTATCGTCAAGGCACTCTTGCCTACGTCACCAGCTCCAACTACCACAAGTT TATATTCAGTCATTTTC (SEQ ID NO:689) for KRAS-G13D-1 crRNA (CTTGTGGTAGTTGGAGCTGT; SEQ ID NO:690). Both repair templates were obtained as Ultramer® Oligos from IDT. To improve rates of homology directed repair, NCI-H441 cells were pre-treated with 200 ng/mL of nocodazole (Sigma Aldrich) and 1 μM of SCR7 pyrazine (Sigma Aldrich) for 17 hours prior to electroporation. The electroporation mixture contained 40 pmoles of Cas9 RNP, 20 pmoles of repair template, and 2×105 cells in 20 μL of OptiMEM. NCI-H441 cells were electroporated at 150 V for 10 ms, while NCI-H358 cells were electroporated at 120 V for 16 ms. Both cell types were transferred to complete growth media containing 1 μM SCR7 for 72 hours following electroporation. Cells were grown in culture for 5 to 10 more days before use.
The NRAS mutation status in HL-60 was modified similarly. 5×105 HL-60 cells were co-electroporated at 120 V for 16 ms with Cas9 RNPs containing tracrRNA/NRAS crRNA (CCTCATGTATTGGTCTCTCATGG; SEQ ID NO:691) duplex and either the repair template for Q61H (AAACCTGTTTGTTGGACATACTGGATACAGCTGGACATGAGGAATATTCTGCAA TGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCT; SEQ ID NO:692) or Q61R (AAACCTGTTTGTTGGACATACTGGATACAGCTGGAAGAGAGGAATATTCTGCAA TGAGAGACCAATACATGAGGACAGGCGAAGGCTTCCT; SEQ ID NO:693).
HLA-A and RAS modified polyclonal pools were plated at a density of 0.5 to 2 cells per well in 96 well plates and cultured for 3 weeks. Single colonies were transferred into 2 or 3 replica 96-well plates. For HLA-A modified NCI-H441 cells, two replica plates were used. One plate was used to stain cells with the HLA-A3 specific antibody GAP.A3-PE and the other with anti-HLA-A2 specific antibody BB7.2-PE (BioLegend). Comparison of the staining allowed identification of clones with only the HLA-A3 allele knocked out, as NCI-H441 normally expresses both HLA-A2 and HLA-A3. HL-60 cells were stained with anti-HLA-A1 (clone 8.L.101) to select clones with HLA-A1 knocked out. For the RAS modified cells, genomic DNA was harvested from one of the plates using the Quick-DNA™ 96 Kit (Zymo Research), PCR amplified using Q5@ Hot Start High-Fidelity 2× Master Mix (New England BioLabs), and Sanger sequenced to identify clones with the desired RAS mutation status. Targeted next generation sequencing was performed to confirm the mutation status of selected clones.
COS-7 cells were plated in a T25 flask and transfected at 70% confluency. For each flask, 10 μg of plasmid DNA (1:1 ratio of HLA plasmid:RAS plasmid, or control plasmid DNA, was mixed with 20 μL of P3000 Reagent in 250 μL of Opti-MEM (Thermo Fisher Scientific). In a separate tube, 20 μL of Lipofectamine 3000 Reagent was mixed with 250 μL of Opti-MEM. The contents of the two tubes were mixed and allowed to complex for 10 minutes at room temperature. Existing medium on pre-plated COS-7 cells was removed and fresh medium was added followed by the Lipofectamine-DNA mixture. Cells were harvested 24 hour post-transfection for co-culture after washing once with PBS, adding 1 mL 0.05% trypsin (Thermo Fisher Scientific) and incubation at 37° C. for 5-10 minutes. Trypsin was quenched with serum-containing media and cells were counted. The co-culture was set up in 96-well flat-bottom tissue culture treated plates. To each well, the following components were combined: 50 μL of antibody diluted to the desired concentration in complete RPMI-1640 with a final IL-2 concentration of 100 IU/mL, 1×104 COS-7s in 100 μL complete RPMI-1640, 5×104 human T cells in 50 μL complete RPMI-1640. The co-cultures were incubated for 24 hours at 37° C. The resultant supernatant was assayed for cytokines using a Human IFNγ Quantikine and Human TNFα Quantikine ELISA Kits (both R&D Systems Bio-techne, Minneapolis, Minn.) according to the manufacturer's instructions.
For co-cultures with pulsed cells, cells were peptide-pulsed in a 96-well plate by combining the specified number of target cells in 50 μL of serum-free RPMI-1640 media (for T2A3, SigM5, Hs 695T) or serum-containing RPMI-1640 media (for PBMCs, monocytes, iDCs, mDCs) with 50 μL of serially diluted peptide in serum-free RPMI-1640 media. Cells were incubated for 4 hours at 37° C., after which 5×104 human T cells and antibodies were added in an additional 100 μL of serum-containing RPMI-1640 medium, with a final IL-2 concentration of 100 IU/mL. The co-cultures were incubated for 24 hours at 37° C., the cells pelleted by centrifugation at 500 g, and the cell-free supernatant was assayed for cytokines as described above. Cell viability was assayed by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, Wis.) according to the manufacturer's instructions. Cytotoxicity was calculated by taking the luciferase signal of a given well, subtracting the luciferase signal of the T cell only wells, and normalizing to the luciferase signal of the wells without scDb.
Co-cultures with other target cell lines were set up similarly, with target cells and human T cells combined as specified in the figure legends. For Luminex assays, IFNγ, IL-2, TNFα, granzyme B, and perforin were measured using MILLIPLEX panels (MilliporeSigma, Burlington, Mass.).
COS-7 cells were transfected as above and harvested after 24 hours. Cells were pelleted, snap frozen, and re-suspended in radioimmunoprecipitation assay (RIPA) buffer (Thermo Fisher Scientific). 25 μg of cell lysate or approximately 0.5-1.5 μg of recombinant protein was run on mini-PROTEAN TGX stain-free gels (Bio-Rad, Hercules, Calif.). Protein was transferred from the stain-free gel to a Trans-Blot Turbo Mini 0.2 μm PVDF membrane (Bio-Rad) using the Trans-Blot Turbo Transfer System (Bio-Rad). Membrane was blocked with blocking buffer (5% Bio-Rad Blotting-Grade Blocker Bio-Rad in 1×TBST) on an orbital shaker (VWR) for 1 hour at RT. All antibody incubations were also performed using this blocking buffer. All incubations with primary antibodies were done at 4° C. overnight (except for β-Actin as noted below). All incubations with secondary antibodies were done for 1 hour at RT. All washes were performed with 1×TBST. KRAS was detected with mouse monoclonal antibody (mAb) F234 (Santa Cruz Biotechnology, Dallas, Tex., at 1:500), followed by anti-mouse-HRP (Thermo Fisher Scientific, at 1:10 k). HLA-A3 was detected with mouse mAb 7g7h8 (Abcam, at 1:500), followed by anti-mouse-HRP (Thermo Fisher Scientific, at 1:10 k). Rab-7b was detected with rabbit anti-Rab7b mAb ab193360 (Abcam, at 1:500), followed by anti-rabbit-HRP (Thermo Fisher Scientific, at 1:10 k). β-Actin was detected with rabbit mAb 13E5 conjugated to HRP (Cell Signaling Technology, Danvers, Mass., at 1:3000) for 1 hour at RT. Recombinant proteins with 6×HIS tags were detected with anti-6×HIS tag rabbit polyclonal antibody ab9108 (Abcam, 1:1000), followed by anti-rabbit-HRP (Thermo Fisher Scientific, at 1:10 k). Prior to imaging, the membranes were washed with ddH2O, then imaged using SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific) on a ChemiDoc XRS+ imager (Bio-Rad).
To generate luciferase-expressing NCI-H358 and HL-60 cell lines for in vivo experiments, NCI-H358 and HL-60 cells were transduced with RediFect Red-Fluc-GFP lentiviral particles (Perkin Elmer, Waltham, Mass.). Non-tissue culture-treated 48-well plates were coated with 250 μL of 20 μg/mL RetroNectin (Clontech) per well for 2 hours at RT, then blocked with 10% FBS for 1 hour at RT. Viral particles and 2×105 target cells were added to each well in a total volume of 275 μL of cell culture media and subjected to centrifugation at 1200×g for 1 hour at 20° C. Cell culture volumes were brought up to 500 μL with complete media. Cells were then incubated at 37° C. for 3 days before exchanging media. Transduced cells were sorted based on the presence of GFP using FACSAria Fusion (BD Biosciences) 18 days after transduction.
Female NOD.Cg-PrkdcscidIl2rgtmlWjl/SzJ (NSG) mice at 6-10 weeks were acquired from the Johns Hopkins Sidney Kimmel Comprehensive Cancer Center Animal Resources Core and treated in compliance with a research protocol approved by the Johns Hopkins University Animal Care and Use Committee. Mice were maintained on an irradiated Uniprim rodent diet (Envigo, Indianapolis, Ind.). Littermate controls were used for all experiments.
For the NCI-H358 intrasplenic model, 5×105 luciferase-expressing NCI-H358 cells (CRISPR KRAS G12V-KI clone 1) or isogenic parental NCI-H358 clone (KRAS G12C/WT) were inoculated into mouse spleens on day zero using sterile surgical techniques. Intraoperatively, two-week micro-osmotic pumps (model 1002, ALZET, Cupertino, Calif.) filled with V2-U scDb or isotype control (L2-U scDb) were placed intraperitoneally. Successful tumor inoculation was ensured by bioluminescence imaging one day later, followed by intravenous injection of 1×107 human T cells via lateral tail vein. Bioluminescent imaging (IVIS imaging system) was performed using RediJect D-luciferin Ultra (Perkin Elmer) according to the manufacturer's instructions (Perkin Elmer). Image analysis was performed using Living Image software. Individual luminescence measurements were normalized to the average fluorescence of the injection marker dye (745ex/800em) in the thoracic region.
For the HL-60 leukemia cell model, mice were inoculated intravenously with 1×107 human T cells and 5×105 luciferase-expressing parental HL-60 (NRAS WT/Q61L) or isogenic control HL-60 (NRAS Q61H/Q61H) via lateral tail vein injection. On day 1, mice were randomized based on luminescence to ensure similar pretreatment tumor burden. Two-week micro-osmotic pumps filled with L2-U scDb or isotype control scDb (V2-U scDb) that had been primed in 1 mL PBS overnight at 37° C. were then placed intraperitoneally using sterile surgical techniques. Tumor growth was serially monitored by bioluminescent imaging.
Statistical analyses were performed with Prism 8 (GraphPad Software, La Jolla, Calif.). Unless otherwise indicated, error bars represent the standard deviation of three technical replicates that were independently assembled. Error bars smaller than the symbols used to represent the mean of these replicates are not shown. Percent cytotoxicity of target cells for in vitro experiments was calculated as described above. For in vivo experiments, statistical significance was performed with an unpaired, two-tailed t-test with Bonferroni-Dunn correction for multiple comparisons.
This Example describes an immunotherapeutic agent that targets a common TP53 mutation. Results from Example 2 were reanalyzed with additional samples and included in the following.
The p53R175H Neoantigen is Presented on the Surface of Cancer Cells
The p53R175H (aa 168-176, HMTEVVRHC; SEQ ID NO:1) and p53WT(HMTEVVRRC; SEQ ID NO:135) peptides were predicted on the NetMHCpan 4.0 server to bind HLA-A*02:01 at 5177.6 nM (rank 9.7%) and 7121.5 nM (11.6%), respectively. To provide experimental evidence of, and to quantify such presentation, peptides eluted from HLA molecules were analyzed in four different cell culture systems using a mass spectrometry (MS)-based method. First, the human HLA-A*02:01 and either full-length p53R175H or p53WT were co-expressed in monkey COS-7 cells. MS analysis of the peptides immunopurified with an anti-HLA antibody detected the p53R175H peptide at approximately 700 copies per cell (
Identification of scFv-Expressing Phage Clones Specific for the HLA-A*02:01-Restricted p53R175H Peptide and Conversion to scDb Format
To identify TCRm single-chain variable fragments (scFvs) selectively targeting mutant pHLA complexes, an scFv-displaying phage library was screened with an estimated complexity >1×1010. Positive selection against HLA-A*02:01 pHLA monomers containing the p53R175H peptide were combined with negative selection against pHLA monomers containing the p53WT and irrelevant peptides. Selected phage clones were amplified and assessed for binding to T2 cells presenting the mutant or wild-type (WT) peptide via flow cytometry (
Twenty-three phage clones with median fluorescence intensity (MFI) ratios of p53R175H to p53WT>4 were then converted to T cell-retargeting bispecific antibodies. This was achieved through linking each individual scFv to an anti-CD3E scFv (UCHT1) in a single-chain diabody (scDb) format (
Next, it was examined whether anti-CD3 arms of the scDb other than the original UCHT1, could influence the ability of H2 to induce T-cell activation. The H2-scFv was linked to a panel of commonly used anti-CD3E scFvs, including UCHT1, UCHT1v9, L2K-07, OKT3, and hXR32 (
H2-scDb Specifically Recognizes Cancer Cells Expressing the p53R175H Neoantigen
The ability of H2-scDb to recognize cancer cell lines expressing various levels of HLA-A*02:01 and having different p53 mutation status was next evaluated. H2-scDb elicited T-cell responses in a dose-dependent manner when T cells were co-cultured with three cell lines that expressed moderate to high levels of HLA-A*02:01 and harboring p53R175H (KMS26, KLE, TYK-nu, as well as the cisplatin-resistant variant of TYK-nu) (
The specificity of H2-scDb was validated using nine pairs of isogenic cell lines that differed with respect to HLA-A*02:01 expression or p53R175H mutation (
To understand the structural basis for the high specificity of the H2 clone for p53R175H/HLA-A*02:01, H2 was converted into full-length IgG (H2-IgG) and confirmed that binding specificity was preserved in this format (
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Binding of the p53R175H Peptide to HLA-A*02:01
The p53R175H peptide (HMTEVVRHC; SEQ ID NO:1) occupied the binding cleft al-α2 of HLA-A*02:01, burying a solvent accessible surface area of 870 Å2, slightly larger than other peptide/HLA-A*02:01 complexes (
Structural Basis for the Recognition of p53R175H HLA-A*02:01 by the H2-Fab
The recognition of the HLA-A*02:01 by the H2-Fab was mediated by all six CDRs. There were a total of 79 contacts, with a cutoff of 4 Å, between the H2-Fab CDRs and the α1 and α2 of HLA-A*02:01, with the light chain contributing to 61% of those contacts (Table 22). The H2-Fab buried a solvent accessible surface area of 818 Å2 within the HLA, of which 427 Å2 were contributed by the light chain and 391 Å2 by the heavy chain (Table 22). In contrast, only four of the six H2-Fab CDRs (H1, H2, H3 and L3) interacted with the p53R175H peptide. Overall, the H2-Fab made 36 contacts with the p53R175H neoantigen, including five hydrogen bonds and numerous van der Waals interactions. His175 made 47% of all direct contacts with the H2-Fab. The CDR-H1, H2, and H3 of the heavy chain and CDR-L3 of the light chain formed a cage-like configuration around the C-terminus of the p53R175H peptide, trapping Arg174 and His175 into position by providing a stable interaction (
Viewed from the axis of the C-terminus to the N-terminus of the p53R175H peptide, the CDRs were arranged in the order H2, H1, L3, H3, L1, L2 (
One of the major challenges confronting new immunotherapeutic antibodies is off-target binding, which can result in toxicity to normal cells. Scanning mutagenesis was employed to identify peptides in the human proteome to which H2-scDb might cross-react. A peptide library was generated by systemically substituting amino acids at each position of the target p53R175H peptide (HMTEVVRHC; SEQ ID NO:1) with each of the remaining 19 common amino acids. T2 cells loaded with each of the 171 variant peptides were then used to assess T-cell activation by measuring IFN-γ release following incubation with T cells and H2-scDb (
Next, a nonamer binding motif, x-[AILMVNQTC]-[ST]-[DE]-[IV]-[IMVST]-R-H-[AILVGHSTYC] (SEQ ID NO:197), was generated using 20% target peptide reactivity as a cutoff for permissive amino acids at each position (
To determine whether H2-scDb could control tumor growth in vivo, KMS26 multiple myeloma cells were engrafted into NOD-SCID-Il2rg−/− (NSG) mice through intravenous injection, establishing widespread, actively growing cancers throughout the body. Two models were used to assess the effects of the H2-scDb in combination with human T cells engrafted in these mice (
Together these results demonstrate that highly specific bispecific antibodies can be generated against pHLA complexes resulting from common mutations occurring in cancer cells. The format and configuration of the bispecific antibodies developed here are highly specific and sensitive scDbs against protein products containing mutations occurring in cancer cells.
COS-7, RPMI 6666, T2 (174×CEM.T2), Raji, HH, AU565, SK-BR-3, KLE, HCT116, SW480, NCI-H441, Saos-2, and CCRF-CEM cells were purchased from American Type Culture Collection (ATCC, Manassas, Va.). KMS26, TYK-nu, and HuCCT1 were purchased from Japanese Collection of Research Bioresources Cell Bank (JCRB, Osaka, Japan). SigM5 was obtained from DSMZ (Braunschweig, Germany). HEK293FT was obtained from Invitrogen (Thermo Fisher Scientific, Waltham, Mass.). T2, Raji, Jurkat, HH, AU565, NCI-H441, TOV-112D, CCRF-CEM, KMS26, TYK-nu, TYK-nu.CP-r and HuCCT1 were cultured in RPMI-1640 (ATCC, 30-2001) with 10% FBS (GE Healthcare, SH30070.03) and 1% Penicillin-Streptomycin (Thermo Fisher Scientific, 15140163). RPMI 6666 was cultured in RPMI-1640 with 20% FBS and 1% Penicillin-Streptomycin. COS-7, SK-BR-3, HCT116, SW480, and Saos-2 were cultured in McCoy's 5A modified media (Thermo Fisher Scientific, 16600108) with 10% FBS and 1% Penicillin-Streptomycin. SigM5 was cultured in IMDM (Thermo Fisher Scientific, 12440061) with 20% FBS and 1% Penicillin-Streptomycin. HEK293FT was cultured in DMEM (high glucose, pyruvate, Thermo Fisher Scientific, 11995065) with 10% FBS, additional 2 mM GlutaMAX (Thermo Fisher Scientific, 35050061), 0.1 mM MEM non-essential amino acids (Thermo Fisher Scientific, 11140050), 1% Penicillin-Streptomycin, and 500 μg/mL Geneticin (Thermo Fisher Scientific, 10131027). PBMCs were isolated from leukapheresis samples (Stem Cell Technologies, Vancouver, BC) by standard density gradient centrifugation with Ficoll Paque Plus (GE Healthcare, 17-1440-03). T cells were expanded from PBMCs with addition of the anti-human CD3 antibody (OKT3, BioLegend, San Diego, 317347) at 15 ng/mL for three days. T cells were cultured in RPMI-1640 with 10% FBS, 1% Penicillin-Streptomycin, 100 IU/mL recombinant human IL-2 (aldesleukin, Prometheus Therapeutics and Diagnostics, San Diego, Calif.), and 5 ng/mL recombinant human IL-7 (BioLegend, 581908). In general, T cells from at least two different donors were tested in in vitro assays. All cells were grown at 37° C. in 5% C02 with humidification.
HLA-A*02:01 restricted p53R175H peptide was directly detected and quantified in human cancer cells carrying p53R175H mutations through MANA-SRM in COS-7 cells transfected with HLA-A*02:01 and p53R175H and in human cancer cells carrying p53R175H mutations and expressing HLA-A*02:01. In particular, the dual-reduction approach described in MANA-SRM was critical for this detection because a cysteine and a methionine coexist in the p53R175H peptide. One hundred femtomole heavy-isotope labeled p53R175H peptide HMTEVVRHC (SEQ ID NO:1) and p53WT peptide HMTEVVRRC (SEQ ID NO:135; New England Peptide Inc, Gardner, Mass.) were spiked into each sample before the assay. The MANA-SRM assays were performed at Complete Omics Inc. (Baltimore, Md.).
All peptides were synthesized at a purity of >90% by Peptide 2.0 (Chantilly, Va.) or ELIM Biopharm (Hayward, Calif.), except for the positional scanning library, where crude peptides were used. Peptides were resuspended in dimethylformamide at 10 mg/mL and stored at −20° C. Biotinylated pHLA monomers were synthesized by Fred Hutchinson Cancer Research Center Immune Monitoring Lab (Seattle, Wash.). Monomers were confirmed to be folded prior to selection by performing an ELISA using W6/32 antibody (BioLegend, 311402), which recognizes only folded HLA.
The scFv-bearing phage library used in this study has been described elsewhere (see, e.g., Miller et al., J. Biol. Chem. 294:19322-19334 (2019)). Briefly, oligonucleotides were synthesized by GeneArt (Thermo Fisher Scientific) using trinucleotide mutagenesis (TRIM) technology to diversify complementarity-determining region (CDR)-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3. A FLAG (DYKDDDDK; SEQ ID NO:190) epitope tag was placed immediately downstream of the scFv, which was followed in-frame by the full-length M13 pIII coat protein sequence. The total number of transformants obtained was determined to be 3.6×1010.
Selection of Mutant pHLA Specific Phage Clone
Phage clones bearing scFvs specific to p53R175H/HLA-A*02:01 pHLA were identified using an approach described elsewhere (see, e.g., Skora et al, 2015 PNAS. 112:9967-72). One g of biotinylated HLA-A*02:01 pHLA monomer complexes were conjugated to 25 μL of M-280 streptavidin magnetic Dynabeads (Thermo Fisher Scientific, 11206D). During the enrichment phase (Round 1), phages were negatively selected with a mixture of unconjugated Dynabeads and free streptavidin protein (RayBiotech, Norcross, Ga., 228-11469). After negative selection, supernatant containing unbound phages were transferred for positive selection using 1 μg of p53R175H/HLA-A*02:01 pHLA. Beads were then washed and phages were eluted to infect mid-log-phase SS320 bacteria, with the addition of M13K07 helper phages (multiplicity of infection of 4). Bacteria were then grown overnight at 30° C. for phage production and the phages were precipitated the next morning with PEG/NaCl.
During the selection phase (Rounds 2-5), phages from the previous round were subjected to two stages of negative selection: 1) against cell lines without p53R175H/HLA-A*02:01 (RPMI 6666, Jurkat, Raji, SigM5, HH, T2, and NCI-H441) and 2) against p53WT/HLA-A*02:01 pHLA, unrelated HLA-A*02:01 pHLA, and free streptavidin. For negative selection using cell lines, phages were incubated with a total number of 0.5-1×107 of cells at 4° C. overnight. After negative selection, beads were isolated and unbound phages were transferred for positive selection by incubating with 1 μg (Round 2), 0.5 μg (Round 3), or 0.25 μg (Round 4, 5) of p53R175H/HLA-A*02:01 pHLA. Phages were then eluted and amplified by infecting SS320 as described above.
After five rounds of selection, SS320 cells were infected with a limiting dilution of the enriched phages. A total of 190 individual colonies of SS320 were picked and phage DNA was PCR amplified by primers flanking the CDRs (Forward: GGCCATGGCAGATATTCAGA (SEQ ID NO:198), Reverse: CCGGGCCTTTATCATCATC (SEQ ID NO:199)) using Q5 Hot Start High-Fidelity 2× Master Mix (New England BioLabs, M0494L) and Sanger sequenced by GENEWIZ (South Plainfield, N.J.). Sequences flanking the CDRs were trimmed using DNA Baser Sequence Assembler v4 (Arges, Romania) and the sequences spanning the CDRs were clustered using the CD-HIT Suite. Colonies containing unique phage clones were selected and grown overnight in 400 μL of media in deep 96-well plates (Thermo Fisher Scientific, 278743) with the addition of M13K07 helper phages. Bacteria were pelleted the next day and the phage-laden supernatants were used for downstream analysis.
For peptide pulsing, T2 cells were washed with serum-free RPMI-1640 media before incubation at 0.5-1×106 cells per mL in serum-free RPMI-1640 containing peptides at the specified concentration of 2 hours at 37° C. For experiments assessed using flow cytometry, human β2M (ProSpec, East Brunswick, N.J., PRO-337) at 10 μg/mL was added with the peptides and specified in the figure legends of such experiments.
Phage staining of peptide-pulsed T2 cells was performed with 50 μL phage supernatant on ice for 1 hour, followed by staining with 1 μg of rabbit anti-M13 antibody (Novus Biologicals, NB100-1633), and anti-rabbit-PE (BioLegend, 406421). HLA-A*02 staining was performed by staining cells with fluorescently labeled anti-human HLA-A*02 (BB7.2, BioLegend, 343308) or mouse isotype IgG2b, K (BioLegend, 402206). Stained cells were analyzed using an LSRII flow cytometer (Becton Dickinson, Mansfield, Mass.) or an iQue Screener (IntelliCyt, Albuquerque, N. Mex.).
Streptavidin-coated, 96-well plates (R&D Systems, Minneapolis, Minn., CP004) were coated with 50 ng of biotinylated HLA-A*02:01 pHLA monomers in 50 μL of blocking buffer (PBS with 0.5% BSA, 2 mM EDTA, and 0.1% sodium azide) or 25 ng of recombinant human CD3/6 (Acro Biosystems, DE, CDD-H52W4) at 4° C. overnight. Plates were washed with 1×TBST (TBS+0.05% Tween-20) using a BioTek 405 TS plate washer (BioTek, Winooski, Vt.). Serial dilutions of scDb or IgG was incubated on the plate for 1 hour at RT and washed. For scDbs, the plate was then incubated with 1 μg/mL recombinant protein L (Thermo Fisher Scientific, 77679) for 1 hour at RT, washed, followed by incubation with anti-protein L HRP (1:10000, Abcam, ab63506) for 1 hour at RT. For IgG, the plate was incubated with anti-human IgG HRP (1:1000, Thermo Fisher Scientific 62-8420) for 1 hour at RT. Plates were washed, 50 μL of 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate (BioLegend, 4211101) was added to each well, and the reaction was quenched with 50 μl 2N sulfuric acid (Thermo Fisher Scientific). Absorbance at 450 nm was measured with a Synergy H1 Multi-Mode Reader (BioTek).
scDb Production
scDbs were produced by cloning gBlocks (IDT, Coralville, Iowa) encoding each of the variants in the format (from N- to C-terminus): IL-2 signal sequence, anti-pHLA variable light chain (VL), GGGGS (SEQ ID NO:200) short linker, anti-CD3 variable heavy chain (VH), (GGGGS)3 (SEQ ID NO:201) long linker, anti-CD3 VL, GGGGS (SEQ ID NO:200) short linker, anti-pHLA VH, and 6×HIS tag into linearized pcDNA3.4 vector (Thermo Fisher Scientific, A14697). The proteins were expressed by the Eukaryotic Tissue Culture Core Facility of Johns Hopkins University. Briefly, 1 mg of plasmid DNA was transfected with polyethylenimine (PEI) at a ratio of 1:3 into 1 L of FreeStyle 293-F cells at a concentration of 2-2.5×106 cells per mL and the transfected cells incubated at 37° C. Five days after transfection, culture media was collected and filtered through a 0.22-μm unit. The scDbs were purified using HisPur Ni-NTA Resin (Thermo Fisher Scientific, 88222) and desalted into PBS pH 7.4 or 20 mM Tris pH 9.0, 150 mM NaCl using 7 k MWCO Zeba Spin desalting columns (Thermo Fisher Scientific, 89890). Proteins were quantified using a 4-15% Mini-PROTEAN TGX gel (Bio-Rad, Hercules, Calif., 4568085) and/or NanoDrop (Thermo Fisher Scientific). Alternatively, the scDb proteins were produced by GeneArt (Thermo Fisher Scientific) in Expi293s, purified with a HisTrap column (GE Healthcare, 17-5255-01) followed by size exclusion chromatography with a HiLoad Superdex 200 26/600 column (GE Healthcare, 28989335). Analytic chromatography was performed using TSKgel G3000SWxl column (TOSOH Bioscience, Tokyo, Japan) using a running buffer of 50 mM sodium phosphate and 300 mM sodium chloride at pH 7, at a flow rate of 1.0 mL/minute.
Surface Plasmon Resonance Affinity Measurements of p53R175H/HLA-A*02:01 and H2-scDb Interaction
Biotinylated p53R175H/HLA-A*02:01, p53WT/HLA-A*02:01, and H2-scDb binding experiments were performed at 25° C. using a Biacore T200 SPR instrument (GE Healthcare). Approximately 100-110 response units (RU) of biotinylated p53R175H/HLA-A*02:01 and p53WT/HLA-A*02:01 were captured in flow cells (Fc) 2 and 4, respectively, using a streptavidin chip. Single-cycle kinetics were performed by injecting increasing concentrations (3, 12, 50, 200, and 800 nM) of purified H2-scDb which was flowed over Fc 1-4. Binding responses for kinetic analysis were both blank- and reference-subtracted. Both binding curves were fit with a 1:1 binding model using Biacore Insight evaluation software.
Thermal stability of the H2-scDb was evaluated by a differential scanning fluorimetry (DSF) assay which monitor the fluorescence of a dye that binds to the hydrophobic region of a protein as it becomes exposed upon temperature induced denaturation. Reaction mixture (20 μL) was set up in a white low-profile 96-well, unskirted polymerase chain reaction plate (BioRad, MLL9651) by mixing 2 μL of purified H2-scDb at a concentration of 1 mg/mL (final concentration ˜2 μg) with 2 μL of 50×SYPRO orange dye (Invitrogen, S6650, 5× final concentration) in PBS, pH 7.4. The plate was sealed with an optical transparent film and centrifuged for 1,000×g for 30 seconds. Thermal scanning was performed from 25 to 100° C. (1° C./minute temperature gradient) using a CFX9 Connect real-time polymerase chain reaction instrument (BioRad). Protein unfolding/melting temperature Tm was calculated from the maximum value of the negative first derivative of the melt curve using CFX Manager software (BioRad).
The Alt-R CRISPR system (IDT) was used to knock out the TP53 gene from KMS26, TYK-nu, and KLE cell lines. CRISPR-Cas9 crRNAs targeting TP53 exon 3 (p53-5: CCCCGGACGATATTGAACAA (SEQ ID NO:191) or p53-6: CCCCTTGCCGTCCCAAGCAA (SEQ ID NO:202)) as well as CRISPR-Cas9 tracrRNA were resuspended at 100 μM with Nuclease-Free Duplex Buffer. The crRNAs and tracrRNA were duplexed at a 1:1 molar ratio for 5 minutes at 95° C. followed by cooling down slowly to RT according to the manufacturer's instructions. The duplexed RNA was then mixed with Cas9 Nuclease at a 1.2:1 molar ratio for 15 minutes. A total of 40 μmols of the Cas9 RNP complexed with TP53 gRNA were mixed with 2×105 cells in 20 μL of OptiMEM. This mixture was loaded into a 0.1 cm cuvette (Bio-Rad, 1652089) and electroporated at 120V and 16 ms using an ECM 2001 (BTX, Holliston, Mass.). Cells were transferred to complete growth medium and cultured for 7 days. Single cell clones were established by limiting dilution and genomic DNA was harvested using a Quick-DNA 96 Kit (Zymo Research, Irvine, Calif., D3012). A region flanking the CRISPR cut site was PCR amplified (forward primer: GCTGCCCTGGTAGGTTTTCT (SEQ ID NO:203), reverse primer: GAGACCTGTGGGAAGCGAAA (SEQ ID NO:204)) and Sanger sequenced to select for clones with the desired TP53 status.
Cells were lysed in cold RIPA buffer (Thermo Fisher Scientific, 89901) supplemented with protease inhibitor cocktail (Thermo Fisher Scientific, 87785). Protein concentration was determined using a BCA assay (Thermo Fisher Scientific, 23227). Equal amounts of total protein (20-50 μg) were loaded in each lane of a 4-15% Mini-PROTEAN TGX gel (Bio-Rad, 4568085) and transferred to polyvinylidene difluoride membranes after electrophoresis. The membranes were incubated with appropriate primary antibodies (anti-6×His tag, 1:2000, Abcam, ab9108; p53 [DO-1], 1:1000, Santa Cruz, sc-126; STAT2, 1:1000, Thermo Fisher Scientific, 44-362G; ZFP3, 1:1000, Thermo Fisher Scientific, PA5-62726; β-actin [13E5], 1:1000, Cell Signaling Technology, 5125S; β-actin [8H10D10], 1:1000, Cell Signaling Technology, 3700S) and species-specific HRP-conjugated secondary antibodies (1:5000-10000). Signal was detected by a ChemiDoc MP chemiluminescence system (Bio-Rad).
gBlocks (IDT) encoding HLA and target proteins were cloned into pcDNA3.1 or pcDNA3.4 vectors (Thermo Fisher Scientific, V79020, A14697). COS-7, HEK293FT, and Saos-2 cells were transfected at 70-80% confluency using Lipofectamine 3000 (Thermo Fisher Scientific, L3000015) and incubated at 37° C. overnight. A total of 15 μg and 30 μg plasmid (1:1 ratio of HLA plasmid/target protein plasmid in co-transfections) was used for T25 and T75 flasks, respectively.
HLA-A*02:01-encoding retrovirus was produced using the MSCV retroviral expression system (Clontech, Mountain View, Calif., 634401). In brief, a gBlock encoding HLA-A*02:01-T2A-GFP (IDT) was cloned into the pMSCVpuro retroviral vector by HiFi DNA assembly (New England Biolabs, Ipswich, Mass., E2621L). The pMSCVpuro-HLA-A*02:01-T2A-GFP plasmid was then co-transfected with a pVSV-G envelope vector into the GP2-293 packaging cell line. Viral supernatant was harvested 48 hours after transfection and concentrated 20-fold using Retro-X Concentrator (Clontech, 631456). RediFect Red-Fluc-GFP lentivirus particles (Perkin Elmer, Waltham, Mass., CLS960003) was used for generating luciferase-expressing cell lines. NucLight green lentivirus (Essen Bioscience, Ann Arbor, Mich., 4624) was used to generate TYK-nu cell lines with nuclear GFP expression.
For transduction, non-tissue culture-treated 48-well plates were coated with 200 μL of 10 μg/mL RetroNectin (Clontech, T100B) per well overnight at 4° C. and blocked with 10% FBS for 1 hour at RT. Viral particles and 2×105 target cells were added to each well in a total volume of 500 μL cell culture media and spun at 2000×g for 1 hour then incubated at 37° C. Selection with 1 μg/mL puromycin (Thermo Fisher Scientific, A1113803) began three days later. Transduced cells were sorted based on presence of GFP using FACSAria Fusion (BD Biosciences, San Jose, Calif.) 10-14 days after transduction.
In Vitro scDb Co-Incubation Assays
To each well of a 96-well flat-bottom plate, the following components were combined in a final volume of 100 μL RPMI-1640 with 10% FBS, 1% Penicillin-Streptomycin, and 100 IU/mL IL-2: scDb diluted to the specified concentration, 5×104 human T cells, and 1-5×104 target cells (COS-7, T2, or other tumor cell lines). The effector to target cell ratio is specified in the figure legend for each experiment. The co-culture plate was incubated for 20 hour at 37° C. and conditioned media was assayed for cytokine and cytotoxic granule protein secretion using the Human IFN-γ Quantikine Kit (R&D Systems, Minneapolis, Minn., SIF50), Human IFN-γ Flex Set Cytometric Bead Array (BD, 558269), or the MILLIPLEX Luminex assays (Millipore Sigma, HSTCMAG28SPMX13, HCD8MAG-15K) read on the Bioplex 200 platform (Bio-Rad). Cytotoxicity was assayed by CellTiter-Glo Luminescent Cell Viability Assay (Promega, Madison, Wis., G7571), Bio-Glo Luciferase Assay (Promega, G7941), or Steady-Glo Luciferase Assay (Promega, E2510) per manufacturer's instructions. For CellTiter-Glo assays, percent cytotoxicity was calculated by subtracting the luminescence signal from the average of the T cell only wells and normalizing to the no scDb condition: 1−(scDb well−T cell only)/(no scDb well−T cell only)×100. For Bio-Glo assays, percent cytotoxicity was calculated by normalizing luminescence signal to the no scDb condition: 1−(scDb well)/(no scDb well)×100.
A total of 1×104 NucLight Green-labeled target cells were plated in each well of a 96-well flat bottom plate and allowed to attach for 4 hours before adding 2×104 T cells and scDb at the indicated concentrations. Each condition was plated in triplicate. Plates were imaged every 6 hours using the IncuCyte ZOOM Live-Cell analysis system (Essen Bioscience) for a total of 120 hours. Four images per well at 10× zoom were collected at each time point. The number of GFP positive objects per mm2 in each well was quantified using the green fluorescence channel.
Expression, purification and refolding of p53R175H HLA-A*02:01
Plasmids for HLA-A*02:01 and β2M were received from the NIH Tetramer Facility (Atlanta, Ga.) and separately transformed into BL21(DE3) cells. Each was expressed in inclusion bodies using auto-induction media. Purification of the HLA-A*02:01 and β2M inclusion bodies was achieved with a series of detergent washes followed by solubilization with 8 M urea. Refolding of the HLA-A*02:01, β2M, and mutant p53R175H peptide was performed. Briefly, solubilized HLA-A*02:01 and β2M were combined in a refolding buffer containing 100 mM Tris pH 8.3, 400 mM L-arginine, 2 mM EDTA, 5 mM reduced glutathione, 0.5 mM oxidized glutathione, 2 mM PMSF, and 30 mg of the mutant p53R175Hpeptide (aa 168-176, HMTEVVRHC; SEQ ID NO:1) dissolved in 1 mL of DMSO. The resultant solution was stirred at 4° C. for 2 days, with two further additions of HLA-A*02:01 on day 2, concentrated to 10 mL and purified by size exclusion chromatography on a HiLoad 26/60 Superdex 75 Prep grade column (GE Healthcare, 28989334). For incubation with the H2-Fab, purified pHLA-A*02:01 was concentrated to ˜1-3 mg/mL and stored at −80° C. until use.
The light chain (LC) and heavy chain (HC) variable region sequences of H2 scFv were grafted onto the respective constant chains of trastuzumab and separately cloned into a pcDNA3.4 vector (Thermo Fisher Scientific, A14697). Both chains were preceded by a mouse IgKVIII signal peptide. Before large-scale expression of full-length antibody, optimization of the LC:HC DNA ratio for transfection was performed to determine optimal recombinant protein yields. For a 1 L expression, a total of 50 μg of purified plasmids (1:1 LC:HC ratio) were transfected with PEI at a ratio of 1:3 into Freestyle 293-F cells at a concentration of 2-2.5×106 cells per mL and incubated at 37° C. for 7 days. The media was harvested via centrifugation, filtered through a 0.22-μm unit and the full-length antibody was purified via protein A affinity chromatography on a HiTrap MabSelect™ SuRe™ column (GE Healthcare, 29-0491-04). Full-length antibody was eluted using a linear gradient of 0-100 mM sodium citrate, pH 3.5. The protein A fractions containing pure H2 antibody were pooled, quantified by SDS-PAGE gel electrophoresis and dialyzed into 20 mM sodium phosphate buffer, pH 7.0, 10 mM EDTA.
For generation of H2-Fab fragments, ˜1-3 mg of full-length antibody was mixed with 0.5 mL of a 50% Immobilized Papain slurry (Thermo Fisher Scientific, 20341) pre-activated with digestion buffer (20 mM sodium phosphate buffer, pH 7.0, 10 mM EDTA) containing 20 mM cysteine-HCl. The mixture was incubated at 37° C. overnight with constant shaking at 200 rpm. The H2 antibody digest was separated from the immobilized resin by a gravity resin separator and washed with 10 mM Tris-HCl, pH 7.5. Newly generated H2-Fab fragments were further purified by cation-exchange chromatography using a Mono-S column (GE Healthcare, 17516801) and eluted using a linear gradient of 0-500 mM NaCl.
The H2-Fab fragments were concentrated, mixed with equimolar p53R175H/HLA-A*02:01 and incubated at 4° C. overnight. The H2-Fab-p53R175H/HLA-A*02:01 mixture was evaluated by size exclusion chromatography on a Superdex™ 200 Increase 10/300 column (GE Healthcare, 28990944). The fractions of ˜98% pure pHLA-A*02:01-H2-Fab complex were pooled, concentrated to 12.6 mg/mL and exchanged into a buffer containing 25 mM HEPES, pH 7.0, 200 mM NaCl.
Crystals of the ternary complex H2-Fab-p53R175H/HLA-A*02:01 were grown by vapor diffusion in hanging drops set up with a TTP mosquito robot with a reservoir solution of 0.2 M ammonium chloride and 20% (w/v) PEG 3350 MME. Crystals were flash-cooled in mother liquor. Data were collected at National Synchrotron Light Source-II at beamlines 17-ID-1(AMX) on a Dectris EIGER X 16M detector. The dataset was indexed, integrated and scaled using fastdp, XDS, and aimless. Monoclinic crystals of H2-Fab-p53R175H/HLA-A*02:01 diffracted to 3.5 Å. The structure for the H2-Fab-p53R175H/HLA-A*02:01 complex was determined by molecular replacement with PHASER using PDB ID 604Y and 6UJ9 as the search models. The data were refined to a final resolution of 3.5 Å using iterative rounds of refinement with REFMAC5 and manual rebuilding in Coot. Structures were validated using Coot and PDB Deposition tools. The model has 95.2% of the residues in preferred and 3.8% in allowed regions according to Ramachandran statistics (Table 20). Figures were rendered in PyMOL (v2.2.3, Schrödinger, LLC, New York, N.Y.). Buried areas were calculated with PDBePISA. The docking angle that determines the relative orientation between the pHLA and the Fab/TCR was calculated by the web server TCR3d.
Female NOD.Cg-PrkdcscidIl2rgtmlWjl/SzJ (NSG) mice at 6-10 weeks were acquired from the Jackson Laboratory (Bar Harbor, Me., 005557) and treated in compliance with the institutional Animal Care and Use Committee approved protocol. In the early treatment model, mice were inoculated intravenously with 1×106 luciferase-expressing KMS26 or KMS26-TP53 KO cells and 1×107 in vitro expanded human T cells via lateral tail vein injection on day 0. On day 1, mice were randomized based on luminescence quantification using the IVIS imaging system and Living Image software (Perkin Elmer) to ensure similar pretreatment tumor burden. Prior to imaging, mice received intraperitoneal injection of luciferin (150 μl, RediJect D-Luciferin Ultra Bioluminescent Substrate, PerkinElmer, 770505) were anesthetized using inhaled isoflurane in an induction chamber for 5 minutes. After randomization, two-week micro-osmotic pumps (ALZET, Cupertino, Calif., 1002) filled with H2-scDb, isotype control scDb (scFv against an irrelevant pHLA linked with UCHT1 scFv), or vehicle only that had been primed in 1 mL PBS overnight at 37° C. were placed intraperitoneally using sterile surgical technique. Tumor growth was serially monitored by bioluminescent imaging. In the established tumor model, mice were inoculated with 3.5×105 or 5×105 luciferase-expressing KMS26 cells and 1×107 human T cells via lateral tail vein injection on day 0. On day 6, H2-scDb or isotype control scDb was administered similarly as in the early treatment model.
For mouse blood-based analysis, 200 μL blood was collected in EDTA-treated microvettes (Sarstedt, Nümbrecht, Germany, 20.1278.100) by cheek bleed, followed by centrifugation at 1000×g for 3 minutes. Plasma was collected and stored at −80° C. until analysis. The blood cell pellet was resuspended with 100 μL PBS, followed by two 5-minute incubations with 1 mL ACK lysis buffer (Thermo Fisher Scientific, A1049201) with one PBS wash in between, and resuspended in flow stain buffer with TruStain FcX (anti-mouse CD16/32) antibody (BioLegend, 101320) and cell-surface staining antibodies. For scDb quantification, plasma was thawed and incubated in biotinylated recombinant human CD3ε/δ coated streptavidin plate and detected as described in “ELISA.”
Data are presented as means±SD unless otherwise specified. Statistical analyses were carried out using specific tests indicated in the figure legends. A P value of <0.05 was used to denote statistical significance. All analyses were performed using Prism version 8.0 (GraphPad, San Diego, Calif.). In all figures, NS, P>0.05; * P<0.05; ** P<0.01, ***P<0.001, **** P<0.0001.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Patent Application Ser. No. 62/949,220, filed on Dec. 17, 2019 and U.S. Patent Application Ser. No. 63/059,638, filed on Jul. 31, 2020. The disclosure of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.
This invention was made with government support under grant CA062924 awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/065617 | 12/17/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 62949220 | Dec 2019 | US | |
| 63059638 | Jul 2020 | US |
