The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jun. 4, 2020, is named 52426-711_601_SL.txt and is 116,335 bytes in size.
Protein-based therapies, such as modified T-cell engagers, have proven effective as treatments for a variety of diseases. As with any therapeutic class, there is a need to improve toxicity and side effects of such treatments, along with improving the half-life of the therapeutic molecules.
Modified T-cell engagers can be used for selective destruction of an individual cell or cell type such as cancer cells of a tumor. Such modified T-cell engagers induce an immune response against the tumor to clear the tumor. However, current therapies using modified T-cell engagers can be toxic and inefficacious. Further, such modified T-cell engagers can have poor pharmacokinetic properties (PK). Provided herein are modified T-cell engagers that reduce toxicity in healthy tissue and thus improving safety while having improved PK properties and efficacy in eliminating the tumor. In some embodiments, the modified T-cell engagers described herein are linked to a peptide that blocks interactions of the T-cell engager with its target in healthy tissue thereby reducing target mediated drug disposition (TMDD). The modified T-cell engagers as described herein are also linked to half-life extending molecule, such as single-domain antibody, which improves the PK profile of the modified T-cell engager as compared to an unmodified T-cell engager.
Disclosed herein, in certain embodiments, are polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in certain embodiments, are pharmaceutical compositions comprising: (i) the polypeptide complex described herein; and (ii) a pharmaceutically acceptable excipient.
Disclosed herein, in certain embodiments, are isolated recombinant nucleic acid molecules encoding the polypeptide or polypeptide complex described herein.
The novel features of the disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings of which:
The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including”, “includes”, “having”, “has”, “with”, or variants thereof are used in either the detailed description and/or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”
The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” should be assumed to mean an acceptable error range for the particular value.
“Transmembrane domain”, as used herein, refers to the region of a receptor which crosses the plasma membrane. Examples include the transmembrane region of a transmembrane protein (for example a Type 1 transmembrane protein), an artificial hydrophobic sequence, and a combination thereof.
“Fragment” as used herein refers to a peptide or a polypeptide that comprises less than the full length amino acid sequence.
“Antigen-binding site” as used herein refers to the region of a polypeptide that interacts with an antigen. The antigen binding site includes amino acid residues that interact directly with an antigen and those amino acid residues that are within proximity to the antigen but that may not interact directly with the antigen.
Native TCRs are transmembrane receptors expressed on the surface of T cells that recognize antigens bound to major histocompatibility complex molecules (MHC). Native TCRs are heterodimeric and comprise an alpha polypeptide chain and a beta polypeptide chain linked through a disulfide bond (
In native TCRs, the alpha polypeptide chain and the beta polypeptide chain comprise an extracellular domain, a transmembrane domain, and a cytoplasmic domain. Each extracellular domain comprises a variable region (V), a joining region (J), and a constant region (C). The constant region is N-terminal to the transmembrane domain, and the transmembrane domain is N-terminal to the cytoplasmic domain. The variable regions of both the alpha polypeptide chain and the beta polypeptide chain comprise three hypervariable or complementarity determining regions (CDRs). The beta polypeptide chain usually contains a short diversity region between the variable and joining regions. The three CDRs are embedded into a framework sequence, with one CDR being the hypervariable region named CDR3. The alpha chain variable region (Vα) and the beta chain variable region (Vβ) are of several types that are distinguished by their framework sequences, CDR1 and CDR2 sequences, and a partly defined CDR3 sequence.
TCRs are described using the International Immunogenetics (IMGT) TCR nomenclature. The Vα in IMGT nomenclature is referred to by a unique “TRAV” number. In the same way, Vβ is referred to by a unique “TRBV” number. The corresponding joining and constant regions are referred to as TRAJ and TRAC, respectively for the α joining and constant regions, and TRBJ and TRBC, respectively for the β joining and constant regions. The sequences defined by the IMGT nomenclature are known in the art and are contained within the online IMGT public database.
Disclosed herein, in some embodiments, are modified T cell engager polypeptides or polypeptide complexes comprising a half-life extending molecule. In some embodiments, the polypeptides or polypeptide complexes comprise a T cell receptor (TCR). In some embodiments, the polypeptides or polypeptide complexes comprise an antibody or an antibody fragment. In some embodiments, the polypeptides or polypeptide complexes comprise a T cell receptor (TCR) and an antibody or an antibody fragment.
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes according to Formula I:
wherein A1 comprises a first antigen recognizing molecule that binds to a first target antigen; P1 comprises a peptide that binds to A1; L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 comprises a half-life extending molecule; and A2 comprises a second antigen recognizing molecule that binds to a second target antigen. Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising Formula I:
wherein A1 comprises a first antigen recognizing molecule that binds to a first target antigen; P1 comprises a peptide that binds to A1; L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 comprises a half-life extending molecule; and A2 comprises a second antigen recognizing molecule that binds to a second target antigen. Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising Formula I:
wherein A1 is a first antigen recognizing molecule that binds to a first target antigen; P1 is a peptide that binds to A1; L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 is a half-life extending molecule; and A2 is a second antigen recognizing molecule that binds to a second target antigen. Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes according to Formula I:
wherein A1 is a first antigen recognizing molecule that binds to a first target antigen; P1 is a peptide that binds to A1; L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 is a half-life extending molecule; and A2 is a second antigen recognizing molecule that binds to a second target antigen. In some embodiments, the first target antigen comprises a tumor cell antigen and the second target antigen comprises an effector cell antigen. In some embodiments, the first target antigen comprises an effector cell antigen and the second target antigen comprises a tumor cell antigen. In some embodiments, the polypeptide or polypeptide complex of formula I binds to a target cell when L1 is cleaved by the tumor specific protease. In some embodiments, the polypeptide of formula I binds to an effector cell when L1 is cleaved by the tumor specific protease.
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes according to Formula Ia:
wherein: A1 comprises a first antigen recognizing molecule that binds to a first target antigen; P1 comprises a peptide that binds to A1; L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 comprises a half-life extending molecule; A2 comprises a second antigen recognizing molecule that binds to a second target antigen; P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease.
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes according to Formula Ia:
wherein: A1 is a first antigen recognizing molecule that binds to a first target antigen; P1 is a peptide that binds to A1; L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 is a half-life extending molecule; A2 is a second antigen recognizing molecule that binds to a second target antigen; P2 is a peptide that binds to A2; and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease.
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising Formula Ia:
wherein: A1 comprises a first antigen recognizing molecule that binds to a first target antigen; P1 comprises a peptide that binds to A1; L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 comprises a half-life extending molecule; A2 comprises a second antigen recognizing molecule that binds to a second target antigen; P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease.
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising Formula Ia:
wherein: A1 is a first antigen recognizing molecule that binds to a first target antigen; P1 is a peptide that binds to A1; L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 is a half-life extending molecule; A2 is a second antigen recognizing molecule that binds to a second target antigen P2 is a peptide that binds to A2; and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease.
In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay as compared to the EC50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 10X higher than the EC50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 20X higher than the EC50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 30X higher than the EC50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 40X higher than the EC50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 50X higher than the EC50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 60X higher than the EC50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 70X higher than the EC50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 80X higher than the EC50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 90X higher than the EC50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 100X higher than the EC50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have P1 or L1.
In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay as compared to the EC50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 10X higher than the EC50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 20X higher than the EC50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 30X higher than the EC50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 40X higher than the EC50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 50X higher than the EC50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 60X higher than the EC50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 70X higher than the EC50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 80X higher than the EC50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 90X higher than the EC50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 100X higher than the EC50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease.
In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay as compared to the EC50 in an IFNγ release T-cell activation assay of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 10X higher than the EC50 in an IFNγ release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 20X higher than the EC50 in an IFNγ release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 30X higher than the EC50 in an IFNγ release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 40X higher than the EC50 in an IFNγ release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 50X higher than the EC50 in an IFNγ release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 60X higher than the EC50 in an IFNγ release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 70X higher than the EC50 in an IFNγ release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 80X higher than the EC50 in an IFNγ release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 90X higher than the EC50 in an IFNγ release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 100X higher than the EC50 in an IFNγ release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1.
In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay as compared to the EC50 in an IFNγ release T-cell activation assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 10X higher than the EC50 in an IFNγ release T-cell activation assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 20X higher than the EC50 in an IFNγ release T-cell activation assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 30X higher than the EC50 in an IFNγ release T-cell activation assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 40X higher than the EC50 in an IFNγ release T-cell activation assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 50X higher than the EC50 in an IFNγ release T-cell activation assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 60X higher than the EC50 in an IFNγ release T-cell activation assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 70X higher than the EC50 in an IFNγ release T-cell activation assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 80X higher than the EC50 in an IFNγ release T-cell activation assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 90X higher than the EC50 in an IFNγ release T-cell activation assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 100X higher than the EC50 in an IFNγ release T-cell activation assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease.
In some embodiments, the polypeptide or polypeptide complex comprises a modified amino acid, a non-natural amino acid, a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or modified non-natural amino acid comprises a post-translational modification.
Further disclosed herein, in some embodiments, are polypeptides or polypeptide complexes according to Formula II:
wherein: L1a comprises a tumor specific protease-cleaved linking moiety that when uncleaved connects P1a to an antigen recognizing molecule that binds to a target antigen and; P1a comprises a peptide that binds to the antigen recognizing molecule when L1a is uncleaved; and H1a comprises a half-life extending molecule. Further disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising Formula II:
wherein: L1a comprises a tumor specific protease-cleaved linking moiety that when uncleaved connects P1a to an antigen recognizing molecule that binds to a target antigen and; P1a comprises a peptide that binds to the antigen recognizing molecule when L1a is uncleaved; and H1a comprises a half-life extending molecule. Further disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising Formula II:
wherein: L1a is a tumor specific protease-cleaved linking moiety that when uncleaved connects P1a to an antigen recognizing molecule that binds to a target antigen and; P1a is a peptide that binds to the antigen recognizing molecule when L1a is uncleaved; and H1a is a half-life extending molecule. Further disclosed herein, in some embodiments, are polypeptides or polypeptide complexes according to Formula II:
wherein: L1a is a tumor specific protease-cleaved linking moiety that when uncleaved connects P1a to an antigen recognizing molecule that binds to a target antigen and; P1a is a peptide that binds to the antigen recognizing molecule when L1a is uncleaved; and H1a is a half-life extending molecule. In some embodiments, the antigen recognizing molecule comprises a soluble TCR that comprises an alpha TCR polypeptide comprising a TCR alpha extracellular domain and a beta TCR polypeptide comprising a TCR beta extracellular domain. In some embodiments, the antigen recognizing molecule comprises an antibody or antibody fragment. In some embodiments, the target antigen is an anti-CD3 effector cell antigen.
In some embodiments, A1 is a soluble T cell receptor (TCR). In some embodiments, the soluble TCR is a single chain TCR comprising a variable region of a TCR alpha extracellular domain, or fragment thereof, and a variable region of a TCR beta extracellular domain, or fragment thereof. In some embodiments, the soluble TCR comprises an alpha TCR polypeptide comprising a TCR alpha extracellular domain and a beta TCR polypeptide comprising a TCR beta extracellular domain. In some embodiments, L1 is bound to N-terminus of the alpha TCR polypeptide. In some embodiments, L1 is bound to N-terminus of the beta TCR polypeptide. In some embodiments, A2 is bound to C-terminus of the alpha TCR polypeptide. In some embodiments, A2 is bound to N-terminus of the alpha TCR polypeptide. In some embodiments, A2 is bound to C-terminus of the beta TCR polypeptide. In some embodiments, A2 is bound to N-terminus of the beta TCR polypeptide. In some embodiments, L1 is bound to N-terminus of the alpha TCR polypeptide and A2 is bound to N-terminus of the beta TCR polypeptide. In some embodiments, L1 is bound to N-terminus of the alpha TCR polypeptide and A2 is bound to C-terminus of the beta TCR polypeptide. In some embodiments, L1 is bound to N-terminus of the alpha TCR polypeptide and A2 is bound to C-terminus of the alpha TCR polypeptide. In some embodiments, L1 is bound to N-terminus of the beta TCR polypeptide and A2 is bound to N-terminus of the alpha TCR polypeptide. In some embodiments, L1 is bound to N-terminus of the beta TCR polypeptide and A2 is bound to C-terminus of the beta TCR polypeptide. In some embodiments, L1 is bound to N-terminus of the beta TCR polypeptide and A2 is bound to C-terminus of the alpha TCR polypeptide.
In some embodiments, A1 comprises a MAGEA3 binding TCR alpha domain. In some embodiments, A1 comprises a MAGEA3 binding TCR beta domain. In some embodiments, A1 comprises a MART1 binding TCR alpha domain. In some embodiments, A1 comprises a MART1 binding TCR beta domain. In some embodiments, the tumor cell antigen comprises MAGEA3. In some embodiments, the tumor cell antigen comprises MART1.
In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence according to SEQ ID NO: 1. In some embodiments, the polypeptide or polypeptide complex comprises an amino acid sequence according to SEQ ID NO: 7. In some embodiments, the TCR alpha extracellular domain comprises three hypervariable complementarity determining regions (CDRs). In some embodiments, at least one CDR comprises a mutation to increase binding affinity or binding specificity to the first target antigen. In some embodiments, at least one CDR comprises a mutation to increase binding affinity and binding specificity to the first target antigen. In some embodiments, the TCR beta extracellular domain comprises three hypervariable complementarity determining regions (CDRs). In some embodiments, at least one CDR comprises a mutation to increase binding affinity or binding specificity to the first target antigen. In some embodiments, at least one CDR comprises a mutation to increase binding affinity and binding specificity to the first target antigen. In some embodiments, there are 2-20, 3-15, 4-12, or 4-10 mutation in one or two CDRs. In some embodiments, the TCR alpha extracellular domain, or fragment thereof, and the TCR beta extracellular domain, or fragment thereof, are connected by a disulfide bond.
In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC as compared to the binding affinity for the pMHC of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 5X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 8X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 10X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 20X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 25X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 30X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 35X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 40X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 45 higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 50X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 55X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 60X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 65X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 70X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 75X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 80X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 85X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 90X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 95X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 100X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 120X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 150X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1.
In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC as compared to the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 5X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 8X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 10X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 15X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 20X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 25X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 30X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 35X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 40X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 45X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 50X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 55X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 60X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 65X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 70X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 75X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 80X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 85X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 90X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 95X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 100X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease.
In some embodiments, A1 is an antibody or an antibody fragment. In some embodiments, the antibody or the antibody fragment thereof comprises a single chain variable fragment, a single domain antibody, or a Fab. In some embodiments, wherein the antibody or antibody fragment thereof comprises a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), or a variable domain (VHH) of a camelid derived single domain antibody. In some embodiments, the antibody or antibody fragment thereof comprises a single-chain variable fragment. In some embodiments, the antibody or antibody fragment thereof is humanized or human. In some embodiments, L1 is bound to N-terminus of antibody or antibody fragment. In some embodiments, A2 is bound to C-terminus of antibody or antibody fragment. In some embodiments, L1 is bound to N-terminus of antibody or antibody fragment and A2 is bound to C-terminus of antibody or antibody fragment.
In some embodiments, A1 is the Fab. In some embodiments, the Fab comprises (a) a Fab light chain polypeptide comprising a light chain variable domain and a constant domain; and (b) a Fab heavy chain polypeptide comprising a heavy chain variable domain and a constant domain. In some embodiments, L1 is bound to N-terminus of the Fab light chain polypeptide. In some embodiments, L1 is bound to N-terminus of the Fab heavy chain polypeptide. In some embodiments, A2 is bound to C-terminus of the Fab light chain polypeptide. In some embodiments, A2 is bound to N-terminus of the Fab light chain polypeptide. In some embodiments, A2 is bound to C-terminus of the Fab heavy chain polypeptide. In some embodiments, A2 is bound to N-terminus of the Fab heavy chain polypeptide. In some embodiments, L1 is bound to N-terminus of the Fab light chain polypeptide and A2 is bound to N-terminus of the Fab heavy chain polypeptide. In some embodiments, L1 is bound to N-terminus of the Fab light chain polypeptide and A2 is bound to C-terminus of the Fab heavy chain polypeptide. In some embodiments, L1 is bound to N-terminus of the Fab light chain polypeptide and A2 is bound to C-terminus of the Fab light chain polypeptide. In some embodiments, L1 is bound to N-terminus of the Fab heavy chain polypeptide and A2 is bound to N-terminus of the Fab light chain polypeptide. In some embodiments, L1 is bound to N-terminus of the Fab heavy chain polypeptide and A2 is bound to C-terminus of the Fab heavy chain polypeptide. In some embodiments, L1 is bound to N-terminus of the Fab heavy chain polypeptide and A2 is bound to C-terminus of the Fab light chain polypeptide. In some embodiments, A2 is bound to the N-terminus of the Fab heavy chain polypeptide and A2 further comprises P2 and L2, wherein P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease.
In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen as compared to the binding affinity for the tumor cell antigen of a polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 5X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 8X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 10X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 15X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 20X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 25X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 30X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 35X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 40X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 45X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 50X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 55X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 60X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 65X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 70X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 75X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 80X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 85X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 90X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 95X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 100X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 120X higher than the binding affinity for the tumor cell antigen of a form of the polypeptide or polypeptide complex that does not have P1 or L1.
In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen as compared to the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 5X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 8X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 10X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 15X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 20X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 25X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 30X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 35X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 40X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 45X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 50X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 55X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 60X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 65X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 70X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 75X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 80X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 85X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 90X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 95X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 100X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease. In some embodiments, the polypeptide or polypeptide complex has weaker binding affinity for the tumor cell antigen that is at least 120X higher than the binding affinity for the tumor cell antigen of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease.
In some embodiments, the polypeptide or polypeptide complex of formula I binds to a target cell when L1 is cleaved by the tumor specific protease and A2 binds to an effector cell. In some embodiments, the effector cell is a T cell. In some embodiments, A2 binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell. In some embodiments, the polypeptide that is part of the TCR-CD3 complex is human CD3ε. In some embodiments, A1 comprises an anti-CD3e single-chain variable fragment. In some embodiments, A1 comprises an anti-CD3e single-chain variable fragment that has a KD binding of 1 µM or less to CD3 on CD3 expressing cells. In some embodiments, A1 comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3. In some embodiments, A1 comprises complementary determining regions (CDRs) selected from the group consisting of muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16, and 15865v19. In some embodiments, the polypeptide or polypeptide complex of formula I binds to an effector cell when L1 is cleaved by the tumor specific protease and A1 binds to the effector cell. In some embodiments, the effector cell is a T cell. In some embodiments, A1 binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell. In some embodiments, the polypeptide that is part of the TCR-CD3 complex is human CD3ε. In some embodiments, the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 86 or 8.
In some embodiments, A2 comprises an antibody or antibody fragment. In some embodiments, A2 comprises an antibody or antibody fragment that is human or humanized. In some embodiments, A2 comprises a single chain variable fragment, a single domain antibody, or a Fab. In some embodiments, A2 comprises a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), or a variable domain (VHH) of a camelid derived single domain antibody. In some embodiments, A2 comprises an anti-CD3e single-chain variable fragment. In some embodiments, A2 comprises an anti-CD3e single-chain variable fragment that has a KD binding of 1 µM or less to CD3 on CD3 expressing cells. In some embodiments, A2 comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3. In some embodiments, A2 comprises complementary determining regions (CDRs) selected from the group consisting of muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16, and 15865v19. In some embodiments, A1 is bound to the N-terminus of A2.
In some embodiments, A2 is a soluble T cell receptor (TCR). In some embodiments, the soluble TCR is a single chain TCR comprising a variable region of a TCR alpha extracellular domain, or fragment thereof, and a variable region of a TCR beta extracellular domain, or fragment thereof. In some embodiments, the soluble TCR comprises an alpha TCR polypeptide comprising a TCR alpha extracellular domain and a beta TCR polypeptide comprising a TCR beta extracellular domain. In some embodiments, A1 is bound to C-terminus of the alpha TCR polypeptide. In some embodiments, A1 is bound to C-terminus of the beta TCR polypeptide. In some embodiments, A1 is bound to N-terminus of the beta TCR polypeptide. In some embodiments, the alpha TCR polypeptide further comprises P2 and L2, wherein P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease. In some embodiments, A1 is bound to N-terminus of the alpha TCR polypeptide. In some embodiments, the beta TCR polypeptide further comprises P2 and L2, wherein P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease. In some embodiments, A2 comprises a MAGEA3 binding TCR alpha domain. In some embodiments, A2 comprises a MAGEA3 binding TCR beta domain. In some embodiments, A2 comprises a MART1 binding TCR alpha domain. In some embodiments, A2 comprises a MART1 binding TCR beta domain. In some embodiments, the tumor cell antigen comprises MAGEA3 or MART1. In some embodiments, A2 comprises an amino acid sequence according to SEQ ID NO: 5. In some embodiments, A2 comprises an amino acid sequence according to SEQ ID NO: 9. In some embodiments, the TCR alpha extracellular domain comprises three hypervariable complementarity determining regions (CDRs). In some embodiments, at least one CDR comprises a mutation to increase binding affinity or binding specificity to the first target antigen. In some embodiments, at least one CDR comprises a mutation to increase binding affinity and binding specificity to the first target antigen. In some embodiments, the TCR beta extracellular domain comprises three hypervariable complementarity determining regions (CDRs). In some embodiments, at least one CDR comprises a mutation to increase binding affinity or binding specificity to the first target antigen. In some embodiments, at least one CDR comprises a mutation to increase binding affinity and binding specificity to the first target antigen. In some embodiments, the TCR alpha extracellular domain, or fragment thereof, and the TCR beta extracellular domain, or fragment thereof, are connected by a disulfide bond. In some embodiments, the tumor cell antigen comprises MAGEA3, and the alpha TCR polypeptide comprises an alpha chain of TCR-1, TCR-2, TCR-3, TCR-4, TCR-5, TCR-6, TCR-7, TCR-8, TCR-9, TCR-10, TCR-11, TCR-12, TCR-13, TCR-14, TCR-15, TCR-16, TCR-17, TCR-18, TCR-19, TCR-20, TCR-21, or TCR-22. In some embodiments, the tumor cell antigen comprises MAGEA3, and the beta TCR polypeptide comprises a beta chain of TCR-1, TCR-2, TCR-3, TCR-4, TCR-5, TCR-6, TCR-7, TCR-8, TCR-9, TCR-10, TCR-11, TCR-12, TCR-13, TCR-14, TCR-15, TCR-16, TCR-17, TCR-18, TCR-19, TCR-20, TCR-21, or TCR-22. In some embodiments, the tumor cell antigen comprises MAGEA3, and the alpha TCR polypeptide comprises an amino acid sequence according to SEQ ID NOs: 1, 5, 73, 75, 76, 79, 80, 85, 91, 92, 95, 96, 97, or 98. In some embodiments, the tumor cell antigen comprises MAGEA3, and the beta TCR polypeptide comprises an amino acid sequence according to SEQ ID NOs: 7, 9, 74, 77, 78, 81, 82, 83, 84, 87, 88, 89, 90, 93, or 94.
In some embodiments, P1 impairs binding of A1 to the first target antigen. In some embodiments, P1 is bound to A1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P1 is bound to A1 at or near an antigen binding site. In some embodiments, P1 becomes unbound from A1 when L1 is cleaved by the tumor specific protease thereby exposing A1 to the first target antigen. In some embodiments, P1 has less than 70% sequence homology to the first target antigen. In some embodiments, P1 has less than 75% sequence homology to the first target antigen. In some embodiments, P1 has less than 80% sequence homology to the first target antigen. In some embodiments, P1 has less than 85% sequence homology to the first target antigen. In some embodiments, P1 has less than 90% sequence homology to the first target antigen. In some embodiments, P1 has less than 95% sequence homology to the first target antigen. In some embodiments, P1 has less than 98% sequence homology to the first target antigen. In some embodiments, P1 has less than 99% sequence homology to the first target antigen.
In some embodiments, P2 impairs binding of A2 to the second target antigen. In some embodiments, P2 is bound to A2 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof. In some embodiments, P2 is bound to A2 at or near an antigen binding site. In some embodiments, P2 becomes unbound from A2 when L2 is cleaved by the tumor specific protease thereby exposing A2 to the second target antigen. In some embodiments, P2 has less than 70% sequence homology to the second target antigen. In some embodiments, P2 has less than 75% sequence homology to the second target antigen. In some embodiments, P2 has less than 80% sequence homology to the second target antigen. In some embodiments, P2 has less than 85% sequence homology to the second target antigen. In some embodiments, P2 has less than 90% sequence homology to the second target antigen. In some embodiments, P2 has less than 95% sequence homology to the second target antigen. In some embodiments, P2 has less than 98% sequence homology to the second target antigen. In some embodiments, P2 has less than 99% sequence homology to the second target antigen.
In some embodiments, P1a when L1a is uncleaved impairs binding of the antigen recognizing molecule to the target antigen. In some embodiments, P1a has less than 70% sequence homology to the target antigen. In some embodiments, P1a has less than 75% sequence homology to the target antigen. In some embodiments, P1a has less than 80% sequence homology to the target antigen. In some embodiments, P1a has less than 85% sequence homology to the target antigen. In some embodiments, P1a has less than 90% sequence homology to the target antigen. In some embodiments, P1a has less than 95% sequence homology to the target antigen. In some embodiments, P1a has less than 98% sequence homology to the target antigen. In some embodiments, P1a has less than 99% sequence homology to the target antigen.
In some embodiments, P1, P2, or P1a comprises a peptide sequence of at least 5 amino acids in length. In some embodiments, P1, P2, or P1a comprises a peptide sequence of at least 6 amino acids in length. In some embodiments, P1, P2, or P1a comprises a peptide sequence of at least 10 amino acids in length. In some embodiments, P1, P2, or P1a comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length. In some embodiments, P1, P2, or P1a comprises a peptide sequence of at least 16 amino acids in length. In some embodiments, P1, P2, or P1a comprises a peptide sequence of no more than 40 amino acids in length. In some embodiments, P1, P2, or P1a comprises at least two cysteine amino acid residues. In some embodiments, P1, P2, or P1a comprises an amino acid sequence YDXXF, wherein X is any amino acid. In some embodiments, P1, P2, or P1a comprises an amino acid sequence YDXXF, wherein X is any amino acid except for cysteine. In some embodiments, P1, P2, or P1a comprises an amino acid sequence DVYDEAF (SEQ ID NO: 11). In some embodiments, P1, P2, or P1a comprises an amino sequence according to GGVSCKDVYDEAFCWT (SEQ ID NO: 12) (Peptide-5). In some embodiments, P1, P2, or P1a comprises a cyclic peptide or a linear peptide. In some embodiments, P1, P2, or P1a comprises a cyclic peptide. In some embodiments, P1, P2, or P1a comprises a linear peptide. In some embodiments, the tumor cell antigen comprises MAGEA3, and the and the P1 or P2 comprises Peptide-1, Peptide-2, Peptide-3, Peptide-4, Peptide-5, Peptide-6, Peptide-7, Peptide-8, Peptide-9, Peptide-10, Peptide-11, Peptide-12, Peptide-13, Peptide-14, Peptide-15, Peptide-16, Peptide-17, Peptide-18, Peptide-19, Peptide-20, Peptide-21, Peptide-22, Peptide-23, Peptide-24, Peptide-25, Peptide-26, Peptide-27, Peptide-28, Peptide-29, Peptide-30, Peptide-31, Peptide-32, Peptide-33, Peptide-34, Peptide-35, or Peptide-36. In some embodiments, the tumor cell antigen comprises MAGEA3, and the and the P1 or P2 comprises an amino acid sequence selected from the group consisting of GGESCQSVYDSSFCYD (SEQ ID NO: 13), GGNACEMTYDHTFCDP (SEQ ID NO: 14), GGRICEEVYDWIFCES (SEQ ID NO: 15), GGRRCVDVYDNAFCLI (SEQ ID NO: 16), GGVSCKDVYDEAFCWT (SEQ ID NO: 12), GGTSCAQIYDFEFCYS (SEQ ID NO: 17), GGSLCSLVYDQDFCES (SEQ ID NO: 18), GGNSCSLVYDKAFCLF (SEQ ID NO: 19), GGNQCWEVYDQEFCSL (SEQ ID NO: 20), GGSACSRIYDFAFCHT (SEQ ID NO: 21), GGTFCYFDHGLVNCQW (SEQ ID NO: 22), GGHCFVSPASGEWWCV (SEQ ID NO: 23), GGCSWIFDGLRYFSKC (SEQ ID NO: 24), VRTWFEKFPELV (SEQ ID NO: 25), LVWGCIWDDMCS (SEQ ID NO: 26), WHWEPSMVWGML (SEQ ID NO: 27), GGGCFVSPATGFTWCV (SEQ ID NO: 28), GGDCQPDSVWSYWYCR (SEQ ID NO: 29), GGCTFVDWWVLGSPYC (SEQ ID NO: 30), GGCLMNDYYYLWGGHC (SEQ ID NO: 31), GGASCKDVYDEAFCWT (SEQ ID NO: 32), GGVACKDVYDEAFCWT (SEQ ID NO: 33), GGVSAKDVYDEAFCWT (SEQ ID NO: 34), GGVSCADVYDEAFCWT (SEQ ID NO: 35), GGVSCKAVYDEAFCWT (SEQ ID NO: 36), GGVSCKDAYDEAFCWT (SEQ ID NO: 37), GGVSCKDVADEAFCWT (SEQ ID NO: 38), GGVSCKDVYAEAFCWT (SEQ ID NO: 39), GGVSCKDVYDAAFCWT (SEQ ID NO: 40), GGVSCKDVYDEAACWT (SEQ ID NO: 41), GGVSCKDVYDEAFAWT (SEQ ID NO: 42), GGVSCKDVYDEAFCAT (SEQ ID NO: 43), GGVSCKDVYDEAFCWA (SEQ ID NO: 44), EVDPIGHLY (SEQ ID NO: 45), ESDPIVAQY (SEQ ID NO: 46), and GGASCAASASAAACAS (SEQ ID NO: 47).
In some embodiments, P1, P2, or P1a or P1, P2, and P1a comprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or a modified non-natural amino acid comprises a post-translational modification. In some embodiments P1, P2, or P1a or P1, P2, and P1a comprise a modification including, but not limited to acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Modifications are made anywhere to P1, P2, or P1a or P1, P2, and P1a including the peptide backbone, the amino acid side chains, and the terminus.
In some embodiments, P1, P2, or P1a does not comprise albumin or an albumin fragment. In some embodiments, P1, P2, or P1a does not comprise an albumin binding domain.
In some embodiments, L1, L2, or L3 is a peptide sequence having at least 5 to no more than 50 amino acids. In some embodiments, L1, L2, or L3 is a peptide sequence having at least 10 to no more than 30 amino acids. In some embodiments, L1, L2, or L3 is a peptide sequence having at least 10 amino acids. In some embodiments, L1, L2, or L3 is a peptide sequence having at least 18 amino acids. In some embodiments, L1, L2, or L3 is a peptide sequence having at least 26 amino acids. In some embodiments, L1, L2, or L3 has a formula comprising (G2S)n, wherein n is an integer from 1 to 3 (SEQ ID NO: 48). In some embodiments, L1, L2, or L3 has a formula comprising (G2S)n, wherein n is an integer of at least 1. In some embodiments, L1, L2, or L3 has a formula selected from the group consisting of (G2S)n, (GS)n, (GSGGS)n (SEQ ID NO: 49), (GGGS)n (SEQ ID NO: 50), (GGGGS)n (SEQ ID NO: 51), and (GSSGGS)n (SEQ ID NO: 52), wherein n is an integer of at least 1. In some embodiments, the tumor specific protease is selected from the group consisting of metalloprotease, serine protease, cysteine protease, threonine protease, and aspartic protease. In some embodiments, L1, L2, or L3 comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, or a legumain cleavable amino acid sequence. In some embodiments, L1, L2, or L3 comprises an amino acid sequence selected from the group consisting of GGGGSLSGRSDNHGSSGT (SEQ ID NO: 53), GGGGSSGGSGGSGLSGRSDNHGSSGT (SEQ ID NO: 54), ASGRSDNH (SEQ ID NO: 55), LAGRSDNH (SEQ ID NO: 56), ISSGLASGRSDNH (SEQ ID NO: 57), ISSGLLAGRSDNH (SEQ ID NO: 58), LSGRSDNH (SEQ ID NO: 4), ISSGLLSGRSDNP (SEQ ID NO: 59), ISSGLLSGRSDNH (SEQ ID NO: 60), LSGRSDNHSPLGLAGS (SEQ ID NO: 61), SPLGLAGSLSGRSDNH (SEQ ID NO: 62), SPLGLSGRSDNH (SEQ ID NO: 63), LAGRSDNHSPLGLAGS (SEQ ID NO: 64), LSGRSDNHVPLSLKMG (SEQ ID NO: 65), and LSGRSDNHVPLSLSMG (SEQ ID NO: 66). In some embodiments, L1, L2, or L3 comprises an amino acid sequence ASGRSDNH (SEQ ID NO: 55), LAGRSDNH (SEQ ID NO: 56), ISSGLASGRSDNH (SEQ ID NO: 57), and ISSGLLAGRSDNH (SEQ ID NO: 58). In some embodiments, L1, L2, or L3 comprises an amino acid sequence SSGGGGSGGGS (SEQ ID NO: 67). In some embodiments, L1, L2, or L3 is Linker-1, Linker-2, Linker-3, Linker-4, Linker-5, Linker-6, Linker-7, Linker-8, Linker-9, Linker-10, Linker-11, Linker-12, Linker-13, Linker-14, Linker-15, Linker-16, Linker-17, Linker-18, or Linker-19. In some embodiments, L1, L2, or L3 comprises an amino acid sequence GGGGSLSGRSDNHGSSGT (SEQ ID NO: 53), GGGGSSGGSGGSGLSGRSDNHGSSGT (SEQ ID NO: 54), ASGRSDNH (SEQ ID NO: 55), LAGRSDNH (SEQ ID NO: 56), ISSGLASGRSDNH (SEQ ID NO: 57), ISSGLLAGRSDNH (SEQ ID NO: 58), LSGRSDNH (SEQ ID NO: 4), ISSGLLSGRSDNP (SEQ ID NO: 59), ISSGLLSGRSDNH (SEQ ID NO: 60), LSGRSDNHSPLGLAGS (SEQ ID NO: 61), SPLGLAGSLSGRSDNH (SEQ ID NO: 62), SPLGLSGRSDNH (SEQ ID NO: 63), LAGRSDNHSPLGLAGS (SEQ ID NO: 64), LSGRSDNHVPLSLKMG (SEQ ID NO: 65), LSGRSDNHVPLSLSMG (SEQ ID NO: 66), GSSGGSGGSGGSGLSGRSDNHGSSGT (SEQ ID NO: 68), GSSGGSGGSGGSGGGSGGGSGGSSGT (SEQ ID NO: 69), GGGGSGGSGGGSGGSSGT (SEQ ID NO: 70), and GGGGSGGGS (SEQ ID NO: 71).
In some embodiments, L1, L2, or L3 or L1, L2, and L3 comprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or a modified non-natural amino acid comprises a post-translational modification. In some embodiments, L1, L2, or L3 or L1, L2, and L3 comprise a modification including, but not limited, to acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Modifications are made anywhere to L1, L2, or L3 or L1, L2, and L3 including the peptide backbone, or the amino acid side chains.
In some embodiments, H1 does not block A1 binding to the first target antigen. In some embodiments, H1a does not block antigen recognizing molecule binding to the target antigen. In some embodiments, half-life extending molecule (H1 or H1a) does not have binding affinity to antigen recognizing molecule. In some embodiments, half-life extending molecule (H1 or H1a) does not have binding affinity to the target antigen. In some embodiments, half-life extending molecule (H1 or H1a) does not shield antigen recognizing molecule from the target antigen. In some embodiments, half-life extending molecule (H1 or H1a) is not directly linked to antigen recognizing molecule.
In some embodiments, H1 or H1a comprise an amino acid sequence that has repetitive sequence motifs. In some embodiments, H1 or H1a comprises an amino acid sequence that has highly ordered secondary structure. “Highly ordered secondary structure,” as used in this context, means that at least about 50%, or about 70%, or about 80%, or about 90%, of amino acid residues of H1 or H1a contribute to secondary structure, as measured or determined by means, including, but not limited to, spectrophotometry (e.g. by circular dichroism spectroscopy in the “far-UV” spectral region (190-250 nm), and computer programs or algorithms, such as the Chou-Fasman algorithm and the Garnier-Osguthorpe-Robson (“GOR”) algorithm.
In some embodiments, H1 or H1a comprises a polymer. In some embodiments, the polymer is polyethylene glycol (PEG). In some embodiments, H1 or H1a comprises albumin. In some embodiments, H1 or H1a comprises an Fc domain. In some embodiments, the albumin is serum albumin. In some embodiments, the albumin is human serum albumin. In some embodiments, H1 or H1a comprises a polypeptide, a ligand, or a small molecule. In some embodiments, the polypeptide, the ligand or the small molecule binds serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1. In some embodiments, the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin. In some embodiments, the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM or IgD. In some embodiments, the serum protein is albumin. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody comprises a single domain antibody, a single chain variable fragment or a Fab. In some embodiments, the antibody is a human or humanized antibody. In some embodiments, the antibody is selected from the group consisting of 645gH1gL1, 645dsgH5gL4, 23-13-A01 -sc02, A10m3 or a fragment thereof, DOM7r-31, DOM7h-11-15, Alb-1, Alb-8, Alb-23, 10G, 10GE, and SA21. In some embodiments, the single domain antibody is 10G, and the single domain antibody comprises an amino acid sequence
In some embodiments, the single domain antibody is 10G, and the single domain antibody comprises an amino acid sequence
In some embodiments, H1 or H1a or H1 and H1a comprise a modified amino acid or non-natural amino acid, or a modified non-natural amino acid, or a combination thereof. In some embodiments, the modified amino acid or a modified non-natural amino acid comprises a post-translational modification. In some embodiments H1 or H1a or H1 and H1a comprise a modification including, but not limited to acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination. Modifications are made anywhere to H1 or H1a or H1 and H1a including the peptide backbone, the amino acid side chains, and the terminus.
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes as disclosed herein. In some embodiments, the polypeptides or polypeptide complexes comprise a T cell receptor (TCR). In some embodiments, the polypeptides or polypeptide complexes comprise an antibody or an antibody fragment. In some embodiments, the polypeptides or polypeptide complexes comprise a T cell receptor (TCR) and an antibody or an antibody fragment.
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula I:
wherein A1 comprises a first antigen recognizing molecule that binds to a first target antigen; P1 comprises a peptide that binds to A1; L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 comprises a half-life extending molecule; and A2 comprises a second antigen recognizing molecule that binds to a second target antigen. Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula I:
wherein A1 comprises a first antigen recognizing molecule that binds to a first target antigen; P1 comprises a peptide that binds to A1; L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 comprises a half-life extending molecule; and A2 comprises a second antigen recognizing molecule that binds to a second target antigen. Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula I:
wherein A1 is a first antigen recognizing molecule that binds to a first target antigen; P1 is a peptide that binds to A1; L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 is a half-life extending molecule; and A2 is a second antigen recognizing molecule that binds to a second target antigen. Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula I:
wherein A1 is a first antigen recognizing molecule that binds to a first target antigen; P1 is a peptide that binds to A1; L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 is a half-life extending molecule; and A2 is a second antigen recognizing molecule that binds to a second target antigen.
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula Ia:
wherein: A1 is a first antigen recognizing molecule that binds to a first target antigen; P1 is a peptide that binds to A1; L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 is a half-life extending molecule; A2 is a second antigen recognizing molecule that binds to a second target antigen; P2 is a peptide that binds to A2; and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease.
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula Ia:
wherein: A1 comprises a first antigen recognizing molecule that binds to a first target antigen; P1 comprises a peptide that binds to A1; L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 comprises a half-life extending molecule; A2 comprises a second antigen recognizing molecule that binds to a second target antigen; P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease.
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula Ia:
wherein: A1 is a first antigen recognizing molecule that binds to a first target antigen; P1 is a peptide that binds to A1; L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 is a half-life extending molecule; A2 is a second antigen recognizing molecule that binds to a second target antigen P2 is a peptide that binds to A2; and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease.
In some embodiments, L1 is bound to N-terminus of the alpha TCR polypeptide. In some embodiments, L1 is bound to N-terminus of the beta TCR polypeptide. In some embodiments, A2 is bound to C-terminus of the alpha TCR polypeptide. In some embodiments, A2 is bound to N-terminus of the alpha TCR polypeptide. In some embodiments, A2 is bound to C-terminus of the beta TCR polypeptide. In some embodiments, A2 is bound to N-terminus of the beta TCR polypeptide. In some embodiments, L1 is bound to N-terminus of the alpha TCR polypeptide and A2 is bound to N-terminus of the beta TCR polypeptide. In some embodiments, L1 is bound to N-terminus of the alpha TCR polypeptide and A2 is bound to C-terminus of the beta TCR polypeptide. In some embodiments, L1 is bound to N-terminus of the alpha TCR polypeptide and A2 is bound to C-terminus of the alpha TCR polypeptide. In some embodiments, L1 is bound to N-terminus of the beta TCR polypeptide and A2 is bound to N-terminus of the alpha TCR polypeptide. In some embodiments, L1 is bound to N-terminus of the beta TCR polypeptide and A2 is bound to C-terminus of the beta TCR polypeptide. In some embodiments, L1 is bound to N-terminus of the beta TCR polypeptide and A2 is bound to C-terminus of the alpha TCR polypeptide. In some embodiments, the alpha TCR polypeptide of A1 is bound to a C-terminus of the single chain variable fragment (scFv) of A2. In some embodiments, the beta TCR polypeptide of A1 is bound to a C-terminus of the single chain variable fragment (scFv) A2. In some embodiments, the alpha TCR polypeptide of A1 is bound to a N-terminus of the single chain variable fragment (scFv) of A2. In some embodiments, the beta TCR polypeptide of A1 is bound to a N-terminus of the single chain variable fragment (scFv) A2. In some embodiments, the beta TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 and L1 is bound to the alpha TCR polypeptide of A1. In some embodiments, the beta TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide. In some embodiments, the beta TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide and the polypeptide complex comprises amino acid sequences of (TCR-20-alpha and TCR-20 -beta. In some embodiments, the beta TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the N-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide. In some embodiments, the alpha TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 and L1 is bound to the beta TCR polypeptide of A1 In some embodiments, the alpha TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the beta TCR polypeptide of A1 at the N-terminus of the beta TCR polypeptide. In some embodiments, the alpha TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the N-terminus of the scFv and L1 is bound to the beta TCR polypeptide of A1 at the N-terminus of the beta TCR polypeptide. In some embodiments, the beta TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 and L1 is bound to the alpha TCR polypeptide of A1 In some embodiments, the beta TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide. In some embodiments, the beta TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 at the N-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide In some embodiments, the alpha TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 and L1 is bound to the beta TCR polypeptide of A1. In some embodiments, the alpha TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the beta TCR polypeptide of A1 at the N-terminus of the beta TCR polypeptide. In some embodiments, the alpha TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 at the N-terminus of the scFv and L1 is bound to the beta TCR polypeptide of A1 at the N-terminus of the beta TCR polypeptide. In some embodiments, the alpha TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the N-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide. In some embodiments, the beta TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the N-terminus of the scFv and L1 is bound to the beta TCR polypeptide of A1 at the N-terminus of the beta TCR polypeptide. In some embodiments, the alpha TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 at the N-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide. In some embodiments, the beta TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 at the N-terminus of the scFv and L1 is bound to the beta TCR polypeptide of A1 at the N-terminus of the beta TCR polypeptide. In some embodiments, A2 further comprises P2 and L2, wherein P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease.
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes according to Formula Ia:
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising Formula Ia:
In some embodiments, the beta TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 and L1 is bound to the alpha TCR polypeptide of A1 and L2 is bound to the scFv light chain polypeptide of A2. In some embodiments, the beta TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide and L2 is bound to the scFv light chain polypeptide of A2 at the N-terminus of the scFv. In some embodiments, the alpha TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 and L1 is bound to the beta TCR polypeptide of A1 and L2 is bound to the scFv light chain polypeptide of A2. In some embodiments, the alpha TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the beta TCR polypeptide of A1 at the N-terminus of the beta TCR polypeptide and L2 is bound to the scFv light chain polypeptide of A2 at the N-terminus of the scFv. In some embodiments, the beta TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 and L1 is bound to the alpha TCR polypeptide of A1 and L2 is bound to the scFv heavy chain polypeptide of A2. In some embodiments, the beta TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide and L2 is bound to the scFv heavy chain polypeptide of A2 at the N-terminus of the scFv. In some embodiments, the alpha TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 and L1 is bound to the beta TCR polypeptide of A1 and L2 is bound to the scFv heavy chain polypeptide of A2. In some embodiments, the alpha TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the beta TCR polypeptide of A1 at the N-terminus of the beta TCR polypeptide and L2 is bound to the scFv heavy chain polypeptide of A2 at the N-terminus of the scFv.
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are isolated recombinant nucleic acid molecules encoding polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, are pharmaceutical compositions comprising: (a) the polypeptides or polypeptide complexes as disclosed herein; and (b) a pharmaceutically acceptable excipient.
In some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula I:
wherein A1 comprises a first antigen recognizing molecule that binds to a first target antigen; P1 comprises a peptide that binds to A1; L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 comprises a half-life extending molecule; and A2 comprises a second antigen recognizing molecule that binds to a second target antigen; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising Formula I:
wherein A1 comprises a first antigen recognizing molecule that binds to a first target antigen; P1 comprises a peptide that binds to A1; L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 comprises a half-life extending molecule; and A2 comprises a second antigen recognizing molecule that binds to a second target antigen; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising Formula I:
wherein A1 is a first antigen recognizing molecule that binds to a first target antigen; P1 is a peptide that binds to A1; L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 is a half-life extending molecule; and A2 is a second antigen recognizing molecule that binds to a second target antigen; and (b) a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula I:
wherein A1 is a first antigen recognizing molecule that binds to a first target antigen; P1 is a peptide that binds to A1; L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 is a half-life extending molecule; and A2 is a second antigen recognizing molecule that binds to a second target antigen; and (b) a pharmaceutically acceptable excipient.
In some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula Ia:
wherein: A1 comprises a first antigen recognizing molecule that binds to a first target antigen; P1 comprises a peptide that binds to A1; L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 comprises a half-life extending molecule; A2 comprises a second antigen recognizing molecule that binds to a second target antigen; P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease; and (b) a pharmaceutically acceptable excipient.
In some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes according to Formula Ia:
wherein: A1 is a first antigen recognizing molecule that binds to a first target antigen; P1 is a peptide that binds to A1; L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 is a half-life extending molecule; A2 is a second antigen recognizing molecule that binds to a second target antigen; P2 is a peptide that binds to A2; and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease; and (b) a pharmaceutically acceptable excipient.
In some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising Formula Ia:
wherein: A1 comprises a first antigen recognizing molecule that binds to a first target antigen; P1 comprises a peptide that binds to A1; L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 comprises a half-life extending molecule; A2 comprises a second antigen recognizing molecule that binds to a second target antigen; P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease; and (b) a pharmaceutically acceptable excipient.
In some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising Formula Ia:
wherein: A1 is a first antigen recognizing molecule that binds to a first target antigen; P1 is a peptide that binds to A1; L1 is a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 is a half-life extending molecule; A2 is a second antigen recognizing molecule that binds to a second target antigen P2 is a peptide that binds to A2; and L2 is a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease; and (b) a pharmaceutically acceptable excipient.
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
Disclosed herein, in some embodiments, the pharmaceutical composition comprises (a) polypeptides or polypeptide complexes comprising a structural arrangement according to the configuration shown in
In some embodiments, the polypeptide or polypeptide complex further comprises a detectable label, a therapeutic agent, or a pharmacokinetic modifying moiety. In some embodiments, the detectable label comprises a fluorescent label, a radiolabel, an enzyme, a nucleic acid probe, or a contrast agent.
For administration to a subject, the polypeptide or polypeptide complex as disclosed herein, may be provided in a pharmaceutical composition together with one or more pharmaceutically acceptable carriers or excipients. The term “pharmaceutically acceptable carrier” includes, but is not limited to, any carrier that does not interfere with the effectiveness of the biological activity of the ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservative, emulsifying agents and dispersing agents. Prevention of the action of microorganisms may be ensured by the inclusion of various antibacterial and antifungal agents.
The pharmaceutical composition may be in any suitable form, (depending upon the desired method of administration). It may be provided in unit dosage form, may be provided in a sealed container and may be provided as part of a kit. Such a kit may include instructions for use. It may include a plurality of said unit dosage forms.
The pharmaceutical composition may be adapted for administration by any appropriate route, including a parenteral (e.g., subcutaneous, intramuscular, or intravenous) route. Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
Dosages of the substances of the present disclosure can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.
Table 1 provides the amino acid sequences of constructs described herein.
Polypeptides or polypeptide complexes, in some embodiments, comprise a sequence set forth in Table 1. In some embodiments, the sequence comprises at least or about 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98. In some instances, the sequence comprises at least or about 95% homology to SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98. In some instances, the sequence comprises at least or about 97% homology to SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98. In some instances, the sequence comprises at least or about 99% homology to SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98. In some instances, the sequence comprises at least or about 100% homology to SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98. In some instances, the sequence comprises at least a portion having at least or about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, or more than 400 amino acids of SEQ ID NOs: 1, 2, 4, 5, 7, 8, 9, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, or 98.
The term “sequence identity” means that two polynucleotide sequences are identical (i.e., on a nucleotide-by-nucleotide basis) over the window of comparison. The term “percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g., A, T, C, G, U, or I) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. Typically, techniques for determining sequence identity include comparing two nucleotide or amino acid sequences and the determining their percent identity. Sequence comparisons, such as for the purpose of assessing identities, may be performed by any suitable alignment algorithm, including but not limited to the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available at www.ebi.ac.uk/Tools/psa/emboss_needle/, optionally with default settings), the BLAST algorithm (see, e.g., the BLAST alignment tool available at blast.ncbi.nlm.nih.gov/Blast.cgi, optionally with default settings), and the Smith-Waterman algorithm (see, e.g., the EMBOSS Water aligner available at www.ebi.ac.uk/Tools/psa/emboss_water/, optionally with default settings). Optimal alignment may be assessed using any suitable parameters of a chosen algorithm, including default parameters. The “percent identity”, also referred to as “percent homology”, between two sequences may be calculated as the number of exact matches between two optimally aligned sequences divided by the length of the reference sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul, et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the sequences being compared. Default parameters are provided to optimize searches with short query sequences, for example, with the blastp program. The program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). High sequence identity generally includes ranges of sequence identity of approximately 80% to 100% and integer values there between.
Embodiment 1 comprises a polypeptide or polypeptide complex according to Formula I:
wherein: A1 comprises a first antigen recognizing molecule that binds to a first target antigen; P1 comprises a peptide that binds to A1; L1 comprises a linking moiety that connects A1 to P1 and is a substrate for a tumor specific protease; H1 comprises a half-life extending molecule; and A2 comprises a second antigen recognizing molecule that binds to a second target antigen.
Embodiment 2 comprises a polypeptide or polypeptide complex of embodiment 1, wherein the first target antigen comprises an effector cell antigen and the second target antigen comprises a tumor cell antigen.
Embodiment 3 comprises a polypeptide or polypeptide complex of any one of embodiments 1-2, wherein the effector cell antigen comprises CD3.
Embodiment 4 comprises a polypeptide or polypeptide complex of any one of embodiments 1-3, wherein the tumor cell antigen comprises MAGEA3 or MART1.
Embodiment 5 comprises a polypeptide or polypeptide complex of any one of embodiments 1-4, wherein A1 comprises an antibody or antibody fragment.
Embodiment 6 comprises a polypeptide or polypeptide complex of any one of embodiments 1-5, wherein A1 comprises an antibody or antibody fragment that is human or humanized.
Embodiment 7 comprises a polypeptide or polypeptide complex of any one of embodiments 1-6, wherein L1 is bound to N-terminus of the antibody or antibody fragment.
Embodiment 8 comprises a polypeptide or polypeptide complex of any one of embodiments 1-7, wherein A2 is bound to C-terminus of the antibody or antibody fragment.
Embodiment 9 comprises a polypeptide or polypeptide complex of any one of embodiments 1-8, wherein L1 is bound to C-terminus of the antibody or antibody fragment.
Embodiment 10 comprises a polypeptide or polypeptide complex of any one of embodiments 1-9, wherein A2 is bound to N-terminus of the antibody or antibody fragment.
Embodiment 11 comprises a polypeptide or polypeptide complex of any one of embodiments 1-10, wherein the antibody or antibody fragment comprises a single chain variable fragment, a single domain antibody, or a Fab fragment.
Embodiment 12 comprises a polypeptide or polypeptide complex of any one of embodiments 1-11, wherein A1 is the single chain variable fragment (scFv).
Embodiment 13 comprises a polypeptide or polypeptide complex of any one of embodiments 1-12, wherein the scFv comprises a scFv heavy chain polypeptide and a scFv light chain polypeptide.
Embodiment 14 comprises a polypeptide or polypeptide complex of any one of embodiments 1-13, wherein A1 is the single domain antibody.
Embodiment 15 comprises a polypeptide or polypeptide complex of any one of embodiments 1-14, wherein the single domain antibody comprises a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), or a variable domain (VHH) of a camelid derived single domain antibody.
Embodiment 16 comprises a polypeptide or polypeptide complex of any one of embodiments 1-15, wherein A1 comprises an anti-CD3e single chain variable fragment.
Embodiment 17 comprises a polypeptide or polypeptide complex of any one of embodiments 1-16, wherein A1 comprises an anti-CD3e single chain variable fragment that has a KD binding of 1 µM or less to CD3 on CD3 expressing cells.
Embodiment 18 comprises a polypeptide or polypeptide complex of any one of embodiments 1-17, wherein A1 comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3.
Embodiment 19 comprises a polypeptide or polypeptide complex of any one of embodiments 1-18, wherein A1 comprises complementary determining regions (CDRs) selected from the group consisting of muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16, UCHT1, and 15865v19.
Embodiment 20 comprises a polypeptide or polypeptide complex of any one of embodiments 1-19, wherein the polypeptide or polypeptide complex of formula I binds to an effector cell when L1 is cleaved by the tumor specific protease.
Embodiment 21 comprises a polypeptide or polypeptide complex of any one of embodiments 1-20, wherein the polypeptide or polypeptide complex of formula I binds to an effector cell when L1 is cleaved by the tumor specific protease and A1binds to the effector cell.
Embodiment 22 comprises a polypeptide or polypeptide complex of any one of embodiments 1-21, wherein the effector cell is a T cell.
Embodiment 23 comprises a polypeptide or polypeptide complex of any one of embodiments 1-22, wherein A1 binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell.
Embodiment 24 comprises a polypeptide or polypeptide complex of any one of embodiments 1-23, wherein the polypeptide that is part of the TCR-CD3 complex is human CD3ε.
Embodiment 25 comprises a polypeptide or polypeptide complex of any one of embodiments 1-24, wherein the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 86 or 8.
Embodiment 26 comprises a polypeptide or polypeptide complex of any one of embodiments 1-25, wherein A2 is a soluble T cell receptor (TCR).
Embodiment 27 comprises a polypeptide or polypeptide complex of any one of embodiments 1-26, wherein the soluble TCR is a single chain TCR comprising a variable region of a TCR alpha extracellular domain, or fragment thereof, and a variable region of a TCR beta extracellular domain, or fragment thereof.
Embodiment 28 comprises a polypeptide or polypeptide complex of any one of embodiments 1-27, wherein the soluble TCR comprises an alpha TCR polypeptide comprising a TCR alpha extracellular domain and a beta TCR polypeptide comprising a TCR beta extracellular domain.
Embodiment 29 comprises a polypeptide or polypeptide complex of any one of embodiments 1-28, wherein A1 is bound to C-terminus of the alpha TCR polypeptide.
Embodiment 30 comprises a polypeptide or polypeptide complex of any one of embodiments 1-29, wherein A1 is bound to C-terminus of the beta TCR polypeptide.
Embodiment 31 comprises a polypeptide or polypeptide complex of any one of embodiments 1-30, wherein A1 is bound to N-terminus of the beta TCR polypeptide.
Embodiment 32 comprises a polypeptide or polypeptide complex of any one of embodiments 1-31, wherein the TCR alpha extracellular domain comprises three hypervariable complementarity determining regions (CDRs).
Embodiment 33 comprises a polypeptide or polypeptide complex of any one of embodiments 1-32, wherein at least one CDR comprises a mutation to increase binding affinity or binding specificity to the tumor cell antigen.
Embodiment 34 comprises a polypeptide or polypeptide complex of any one of embodiments 1-33, wherein the TCR beta extracellular domain comprises three hypervariable complementarity determining regions (CDRs).
Embodiment 35 comprises a polypeptide or polypeptide complex of any one of embodiments 1-34, wherein at least one CDR comprises a mutation to increase binding affinity or binding specificity to the tumor cell antigen.
Embodiment 36 comprises a polypeptide or polypeptide complex of any one of embodiments 1-35, wherein the TCR alpha extracellular domain, or fragment thereof, and the TCR beta extracellular domain, or fragment thereof, are connected by a disulfide bond.
Embodiment 37 comprises a polypeptide or polypeptide complex of any one of embodiments 1-36, wherein A2 comprises a MAGEA3 binding TCR alpha domain.
Embodiment 38 comprises a polypeptide or polypeptide complex of any one of embodiments 1-37, wherein A2 comprises a MAGEA3 binding TCR beta domain.
Embodiment 39 comprises a polypeptide or polypeptide complex of any one of embodiments 1-38, wherein A2 comprises a MART1 binding TCR alpha domain.
Embodiment 40 comprises a polypeptide or polypeptide complex of any one of embodiments 1-39, wherein A2 comprises a MART1 binding TCR beta domain.
Embodiment 41 comprises a polypeptide or polypeptide complex of any one of embodiments 1-40, wherein the tumor cell antigen comprises MAGEA3 or MART1.
Embodiment 42 comprises a polypeptide or polypeptide complex of any one of embodiments 1-41, wherein the tumor cell antigen comprises MAGEA3, and the alpha TCR polypeptide comprises an amino acid sequence according to SEQ ID NOs: 1, 5, 73, 75, 76, 79, 80, 85, 91, 92, 95, 96, 97, or 98.
Embodiment 43 comprises a polypeptide or polypeptide complex of any one of embodiments 1-42, wherein the tumor cell antigen comprises MAGEA3, and the beta TCR polypeptide an amino acid sequence according to SEQ ID NOs: 7, 9, 74, 77, 78, 81, 82, 83, 84, 87, 88, 89, 90, 93, or 94.
Embodiment 44 comprises a polypeptide or polypeptide complex of any one of embodiments 1-43, wherein the alpha TCR polypeptide of A2 is bound to a C-terminus of the single chain variable fragment (scFv) of A1.
Embodiment 45 comprises a polypeptide or polypeptide complex of any one of embodiments 1-44, wherein the beta TCR polypeptide of A2 is bound to a C-terminus of the single chain variable fragment (scFv) A1.
Embodiment 46 comprises a polypeptide or polypeptide complex of any one of embodiments 1-45, wherein the alpha TCR polypeptide of A2 is bound to a N-terminus of the single chain variable fragment (scFv) of A1.
Embodiment 47 comprises a polypeptide or polypeptide complex of any one of embodiments 1-46, wherein the beta TCR polypeptide of A2 is bound to a N-terminus of the single chain variable fragment (scFv) A1.
Embodiment 48 comprises a polypeptide or polypeptide complex of any one of embodiments 1-47, wherein the beta TCR polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1.
Embodiment 49 comprises a polypeptide or polypeptide complex of any one of embodiments 1-48, wherein the beta TCR polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1 at the C-terminus of the scFv.
Embodiment 50 comprises a polypeptide or polypeptide complex of any one of embodiments 1-49, wherein the alpha TCR polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1.
Embodiment 51 comprises a polypeptide or polypeptide complex of any one of embodiments 1-50, wherein the alpha TCR polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1 at the C-terminus of the scFv.
Embodiment 52 comprises a polypeptide or polypeptide complex of any one of embodiments 1-51, wherein the beta TCR polypeptide of A2 is bound to the scFv light chain polypeptide of A1.
Embodiment 53 comprises a polypeptide or polypeptide complex of any one of embodiments 1-52, wherein the alpha TCR polypeptide of A2 is bound to the scFv light chain polypeptide of A1 at the C-terminus of the scFv.
Embodiment 54 comprises a polypeptide or polypeptide complex of any one of embodiments 1-53, wherein A2 further comprises P2 and L2, wherein P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease.
Embodiment 55 comprises the polypeptide or polypeptide complex according to any one of embodiments 1-54 wherein the polypeptide or polypeptide complex is according to Formula Ia:
Embodiment 56 comprises a polypeptide or polypeptide complex of any one of embodiments 1-55, wherein the beta TCR polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1 and L2 is bound to the alpha TCR polypeptide of A2.
Embodiment 57 comprises a polypeptide or polypeptide complex of any one of embodiments 1-56, wherein the beta TCR polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1 at the C-terminus of the scFv and L2 is bound to the alpha TCR polypeptide of A2 at the N-terminus of the alpha TCR polypeptide.
Embodiment 58 comprises a polypeptide or polypeptide complex of any one of embodiments 1-57, wherein the alpha TCR polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1 and L2 is bound to the beta TCR polypeptide of A2.
Embodiment 59 comprises a polypeptide or polypeptide complex of any one of embodiments 1-58, wherein the alpha TCR polypeptide of A2 is bound to the scFv heavy chain polypeptide of A1 at the C-terminus of the scFv and L2 is bound to the beta TCR polypeptide of A2 at the N-terminus of the beta TCR polypeptide.
Embodiment 60 comprises a polypeptide or polypeptide complex of any one of embodiments 1-59, wherein the beta TCR polypeptide of A2 is bound to the scFv light chain polypeptide of A1 and L2 is bound to the alpha TCR polypeptide of A2.
Embodiment 61 comprises a polypeptide or polypeptide complex of any one of embodiments 1-60, wherein the beta TCR polypeptide of A2 is bound to the scFv light chain polypeptide of A1 at the C-terminus of the scFv and L2 is bound to the alpha TCR polypeptide of A2 at the N-terminus of the alpha TCR polypeptide.
Embodiment 62 comprises a polypeptide or polypeptide complex of any one of embodiments 1-61, wherein the alpha TCR polypeptide of A2 is bound to the scFv light chain polypeptide of A1 and L2 is bound to the beta TCR polypeptide of A2.
Embodiment 63 comprises a polypeptide or polypeptide complex of any one of embodiments 1-62, wherein the alpha TCR polypeptide of A2 is bound to the scFv light chain polypeptide of A1 at the C-terminus of the scFv and L2 is bound to the beta TCR polypeptide of A2 at the N-terminus of the beta TCR polypeptide.
Embodiment 64 comprises a polypeptide or polypeptide complex of any one of embodiments 1-63, wherein the first target antigen comprises a tumor cell antigen and the second target antigen comprises an effector cell antigen.
Embodiment 65 comprises a polypeptide or polypeptide complex of any one of embodiments 1-64, wherein the tumor cell antigen comprises MAGEA3 or MART1.
Embodiment 66 comprises a polypeptide or polypeptide complex of any one of embodiments 1-65, wherein the effector cell antigen comprises CD3.
Embodiment 67 comprises a polypeptide or polypeptide complex of any one of embodiments 1-66, wherein A1 is a soluble T cell receptor (TCR).
Embodiment 68 comprises a polypeptide or polypeptide complex of any one of embodiments 1-67, wherein the soluble TCR is a single chain TCR comprising a variable region of a TCR alpha extracellular domain, or fragment thereof, and a variable region of a TCR beta extracellular domain, or fragment thereof.
Embodiment 69 comprises a polypeptide or polypeptide complex of any one of embodiments 1-68, wherein the soluble TCR comprises an alpha TCR polypeptide comprising a TCR alpha extracellular domain and a beta TCR polypeptide comprising a TCR beta extracellular domain.
Embodiment 70 comprises a polypeptide or polypeptide complex of any one of embodiments 1-69, wherein the tumor cell antigen comprises MAGEA3, and the alpha TCR polypeptide comprises an amino acid sequence according to SEQ ID NOs: 1, 5, 73, 75, 76, 79, 80, 85, 91, 92, 95, 96, 97, or 98.
Embodiment 71 comprises a polypeptide or polypeptide complex of any one of embodiments 1-70, wherein the tumor cell antigen comprises MAGEA3, and the beta TCR polypeptide comprises an amino acid sequence according to SEQ ID NOs: 7, 9, 74, 77, 78, 81, 82, 83, 84, 87, 88, 89, 90, 93, or 94.
Embodiment 72 comprises a polypeptide or polypeptide complex of any one of embodiments 1-71, wherein A2 comprises an antibody or antibody fragment.
Embodiment 73 comprises a polypeptide or polypeptide complex of any one of embodiments 1-72, wherein A2 comprises an antibody or antibody fragment that is human or humanized.
Embodiment 74 comprises a polypeptide or polypeptide complex of any one of embodiments 1-73, wherein the antibody or antibody fragment comprises a single chain variable fragment, a single domain antibody, or a Fab fragment.
Embodiment 75 comprises a polypeptide or polypeptide complex of any one of embodiments 1-74, wherein A2 is the single chain variable fragment (scFv).
Embodiment 76 comprises a polypeptide or polypeptide complex of any one of embodiments 1-75, wherein the scFv comprises a scFv heavy chain polypeptide and a scFv light chain polypeptide.
Embodiment 77 comprises a polypeptide or polypeptide complex of any one of embodiments 1-76, wherein A2 is the single domain antibody.
Embodiment 78 comprises a polypeptide or polypeptide complex of any one of embodiments 1-77, wherein the single domain antibody comprises a single chain variable fragment (scFv), a heavy chain variable domain (VH domain), a light chain variable domain (VL domain), or a variable domain (VHH) of a camelid derived single domain antibody.
Embodiment 79 comprises a polypeptide or polypeptide complex of any one of embodiments 1-78, wherein A2 comprises an anti-CD3e single chain variable fragment.
Embodiment 80 comprises a polypeptide or polypeptide complex of any one of embodiments 1-79, wherein A2 comprises an anti-CD3e single chain variable fragment that has a KD binding of 1 µM or less to CD3 on CD3 expressing cells.
Embodiment 81 comprises a polypeptide or polypeptide complex of any one of embodiments 1-80, wherein A2 comprises a variable light chain and variable heavy chain each of which is capable of specifically binding to human CD3.
Embodiment 82 comprises a polypeptide or polypeptide complex of any one of embodiments 1-81,wherein A2 comprises complementary determining regions (CDRs) selected from the group consisting of muromonab-CD3 (OKT3), otelixizumab (TRX4), teplizumab (MGA031), visilizumab (Nuvion), SP34, X35, VIT3, BMA030 (BW264/56), CLB-T3/3, CRIS7, YTH12.5, F111-409, CLB-T3.4.2, TR-66, WT32, SPv-T3b, 11D8, XIII-141, XIII-46, XIII-87, 12F6, T3/RW2-8C8, T3/RW2-4B6, OKT3D, M-T301, SMC2, F101.01, UCHT-1, WT-31, 15865, 15865v12, 15865v16, and 15865v19.
Embodiment 83 comprises a polypeptide or polypeptide complex of any one of embodiments 1-82, wherein the polypeptide or polypeptide complex of formula I binds to an effector cell.
Embodiment 84 comprises a polypeptide or polypeptide complex of any one of embodiments 1-83, wherein the effector cell is a T cell.
Embodiment 85 comprises a polypeptide or polypeptide complex of any one of embodiments 1-84,wherein A2 binds to a polypeptide that is part of a TCR-CD3 complex on the effector cell.
Embodiment 86 comprises a polypeptide or polypeptide complex of any one of embodiments 1-85, wherein the polypeptide that is part of the TCR-CD3 complex is human CD3ε.
Embodiment 87 comprises a polypeptide or polypeptide complex of any one of embodiments 1-86, wherein the effector cell antigen comprises CD3, and the scFv comprises an amino acid sequence according to SEQ ID NO: 86 or 8.
Embodiment 88 comprises a polypeptide or polypeptide complex of any one of embodiments 1-87, wherein L1 is bound to N-terminus of the alpha TCR polypeptide.
Embodiment 89 comprises a polypeptide or polypeptide complex of any one of embodiments 1-88, wherein L1 is bound to N-terminus of the beta TCR polypeptide.
Embodiment 90 comprises a polypeptide or polypeptide complex of any one of embodiments 1-89, wherein A2 is bound to C-terminus of the alpha TCR polypeptide.
Embodiment 91 comprises a polypeptide or polypeptide complex of any one of embodiments 1-90, wherein A2 is bound to N-terminus of the alpha TCR polypeptide.
Embodiment 92 comprises a polypeptide or polypeptide complex of any one of embodiments 1-91, wherein A2 is bound to C-terminus of the beta TCR polypeptide.
Embodiment 93 comprises a polypeptide or polypeptide complex of any one of embodiments 1-92, wherein A2 is bound to N-terminus of the beta TCR polypeptide.
Embodiment 94 comprises a polypeptide or polypeptide complex of any one of embodiments 1-93, wherein L1 is bound to N-terminus of the alpha TCR polypeptide and A2 is bound to N-terminus of the beta TCR polypeptide.
Embodiment 95 comprises a polypeptide or polypeptide complex of any one of embodiments 1-94, wherein L1 is bound to N-terminus of the alpha TCR polypeptide and A2 is bound to C-terminus of the beta TCR polypeptide.
Embodiment 96 comprises a polypeptide or polypeptide complex of any one of embodiments 1-95, wherein L1 is bound to N-terminus of the alpha TCR polypeptide and A2 is bound to C-terminus of the alpha TCR polypeptide.
Embodiment 97 comprises a polypeptide or polypeptide complex of any one of embodiments 1-96, wherein L1 is bound to N-terminus of the beta TCR polypeptide and A2 is bound to N-terminus of the alpha TCR polypeptide.
Embodiment 98 comprises a polypeptide or polypeptide complex of any one of embodiments 1-97, wherein L1 is bound to N-terminus of the beta TCR polypeptide and A2 is bound to C-terminus of the beta TCR polypeptide.
Embodiment 99 comprises a polypeptide or polypeptide complex of any one of embodiments 1-98, wherein L1 is bound to N-terminus of the beta TCR polypeptide and A2 is bound to C-terminus of the alpha TCR polypeptide.
Embodiment 100 comprises a polypeptide or polypeptide complex of any one of embodiments 1-99, wherein the alpha TCR polypeptide of A1 is bound to a C-terminus of the single chain variable fragment (scFv) of A2.
Embodiment 101 comprises a polypeptide or polypeptide complex of any one of embodiments 1-100, wherein the beta TCR polypeptide of A1 is bound to a C-terminus of the single chain variable fragment (scFv) A2.
Embodiment 102 comprises a polypeptide or polypeptide complex of any one of embodiments 1-101, wherein the alpha TCR polypeptide of A1 is bound to a N-terminus of the single chain variable fragment (scFv) of A2.
Embodiment 103 comprises a polypeptide or polypeptide complex of any one of embodiments 1-102, wherein the beta TCR polypeptide of A1 is bound to a N-terminus of the single chain variable fragment (scFv) A2.
Embodiment 104 comprises a polypeptide or polypeptide complex of any one of embodiments 1-103, wherein the beta TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 and L1 is bound to the alpha TCR polypeptide of A1.
Embodiment 105 comprises a polypeptide or polypeptide complex of any one of embodiments 1-104, wherein the beta TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide.
Embodiment 106 comprises a polypeptide or polypeptide complex of any one of embodiments 1-105, wherein the beta TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide and the polypeptide complex comprises amino acid sequences of TCR-20alpha and TCR-20-beta.
Embodiment 107 comprises a polypeptide or polypeptide complex of any one of embodiments 1-106, wherein the beta TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the N-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide.
Embodiment 108 comprises a polypeptide or polypeptide complex of any one of embodiments 1-107, wherein the alpha TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 and L1 is bound to the beta TCR polypeptide of A1.
Embodiment 109 comprises a polypeptide or polypeptide complex of any one of embodiments 1-108, wherein the alpha TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the beta TCR polypeptide of A1 at the N-terminus of the beta TCR polypeptide.
Embodiment 110 comprises a polypeptide or polypeptide complex of any one of embodiments 1-109, wherein the alpha TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the N-terminus of the scFv and L1 is bound to the beta TCR polypeptide of A1 at the N-terminus of the beta TCR polypeptide.
Embodiment 111 comprises a polypeptide or polypeptide complex of any one of embodiments 1-110, wherein the beta TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 and L1 is bound to the alpha TCR polypeptide of A1.
Embodiment 112 comprises a polypeptide or polypeptide complex of any one of embodiments 1-111, wherein the beta TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide.
Embodiment 113 comprises a polypeptide or polypeptide complex of any one of embodiments 1-112, wherein the beta TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 at the N-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide.
Embodiment 114 comprises a polypeptide or polypeptide complex of any one of embodiments 1-113, wherein the alpha TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 and L1 is bound to the beta TCR polypeptide of A1.
Embodiment 115 comprises a polypeptide or polypeptide complex of any one of embodiments 1-114, wherein the alpha TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the beta TCR polypeptide of A1 at the N-terminus of the beta TCR polypeptide.
Embodiment 116 comprises a polypeptide or polypeptide complex of any one of embodiments 1-115, wherein the alpha TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 at the N-terminus of the scFv and L1 is bound to the beta TCR polypeptide of A1 at the N-terminus of the beta TCR polypeptide.
Embodiment 117 comprises a polypeptide or polypeptide complex of any one of embodiments 1-116, wherein the alpha TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the N-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide.
Embodiment 118 comprises a polypeptide or polypeptide complex of any one of embodiments 1-117, wherein the beta TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the N-terminus of the scFv and L1 is bound to the beta TCR polypeptide of A1 at the N-terminus of the beta TCR polypeptide.
Embodiment 119 comprises a polypeptide or polypeptide complex of any one of embodiments 1-118, wherein the alpha TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 at the N-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide.
Embodiment 120 comprises a polypeptide or polypeptide complex of any one of embodiments 1-119, wherein the beta TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 at the N-terminus of the scFv and L1 is bound to the beta TCR polypeptide of A1 at the N-terminus of the beta TCR polypeptide.
Embodiment 121 comprises a polypeptide or polypeptide complex of any one of embodiments 1-120, wherein A2 further comprises P2 and L2, wherein P2 comprises a peptide that binds to A2; and L2 comprises a linking moiety that connects A2 to P2 and is a substrate for a tumor specific protease.
Embodiment 122 comprises the polypeptide or polypeptide complex according to any one of embodiments 1-121 wherein the polypeptide or polypeptide complex is according to Formula Ia
Embodiment 123 comprises a polypeptide or polypeptide complex of any one of embodiments 1-122, wherein the beta TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 and L1 is bound to the alpha TCR polypeptide of A1 and L2 is bound to the scFv light chain polypeptide of A2.
Embodiment 124 comprises a polypeptide or polypeptide complex of any one of embodiments 1-123, wherein the beta TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide and L2 is bound to the scFv light chain polypeptide of A2 at the N-terminus of the scFv.
Embodiment 125 comprises a polypeptide or polypeptide complex of any one of embodiments 1-124, wherein the alpha TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 and L1 is bound to the beta TCR polypeptide of A1 and L2 is bound to the scFv light chain polypeptide of A2.
Embodiment 126 comprises a polypeptide or polypeptide complex of any one of embodiments 1-125, wherein the alpha TCR polypeptide of A1 is bound to the scFv heavy chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the beta TCR polypeptide of A1 at the N-terminus of the beta TCR polypeptide and L2 is bound to the scFv light chain polypeptide of A2 at the N-terminus of the scFv.
Embodiment 127 comprises a polypeptide or polypeptide complex of any one of embodiments 1-126, wherein the beta TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 and L1 is bound to the alpha TCR polypeptide of A1 and L2 is bound to the scFv heavy chain polypeptide of A2.
Embodiment 128 comprises a polypeptide or polypeptide complex of any one of embodiments 1-127, wherein the beta TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the alpha TCR polypeptide of A1 at the N-terminus of the alpha TCR polypeptide and L2 is bound to the scFv heavy chain polypeptide of A2 at the N-terminus of the scFv.
Embodiment 129 comprises a polypeptide or polypeptide complex of any one of embodiments 1-128,wherein the alpha TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 and L1 is bound to the beta TCR polypeptide of A1 and L2 is bound to the scFv heavy chain polypeptide of A2.
Embodiment 130 comprises a polypeptide or polypeptide complex of any one of embodiments 1-129, wherein the alpha TCR polypeptide of A1 is bound to the scFv light chain polypeptide of A2 at the C-terminus of the scFv and L1 is bound to the beta TCR polypeptide of A1 at the N-terminus of the beta TCR polypeptide and L2 is bound to the scFv heavy chain polypeptide of A2 at the N-terminus of the scFv.
Embodiment 131 comprises a polypeptide or polypeptide complex of any one of embodiments 1-130,wherein P1 impairs binding of A1 to the first target antigen.
Embodiment 132 comprises a polypeptide or polypeptide complex of any one of embodiments 1-131, wherein P1 is bound to A1 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.
Embodiment 133 comprises a polypeptide or polypeptide complex of any one of embodiments 1-132, wherein P1 has less than 70% sequence homology to the first target antigen.
Embodiment 134 comprises a polypeptide or polypeptide complex of any one of embodiments 1-133, wherein P2 impairs binding of A2 to the second target antigen.
Embodiment 135 comprises a polypeptide or polypeptide complex of any one of embodiments 1-134, wherein P2 is bound to A2 through ionic interactions, electrostatic interactions, hydrophobic interactions, Pi-stacking interactions, and H-bonding interactions, or a combination thereof.
Embodiment 136 comprises a polypeptide or polypeptide complex of any one of embodiments 1-135, wherein P2 is bound to A2 at or near an antigen binding site.
Embodiment 137 comprises a polypeptide or polypeptide complex of any one of embodiments 1-136, wherein P2 has less than 70% sequence homology to the second target antigen.
Embodiment 138 comprises a polypeptide or polypeptide complex of any one of embodiments 1-137, wherein P1 or P2 comprises a peptide sequence of at least 10 amino acids in length.
Embodiment 139 comprises a polypeptide or polypeptide complex of any one of embodiments 1-138, wherein P1 or P2 comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.
Embodiment 140 comprises a polypeptide or polypeptide complex of any one of embodiments 1-139, wherein P1 or P2 comprises a peptide sequence of at least 16 amino acids in length.
Embodiment 141 comprises a polypeptide or polypeptide complex of any one of embodiments 1-140, wherein P1 or P2 comprises a peptide sequence of no more than 40 amino acids in length.
Embodiment 142 comprises a polypeptide or polypeptide complex of any one of embodiments 1-141, wherein P1 or P2 comprises at least two cysteine amino acid residues.
Embodiment 143 comprises a polypeptide or polypeptide complex of any one of embodiments 1-142, wherein P1 or P2 comprises a cyclic peptide or a linear peptide.
Embodiment 144 comprises a polypeptide or polypeptide complex of any one of embodiments 1-143, wherein P1 or P2 comprises a cyclic peptide.
Embodiment 145 comprises a polypeptide or polypeptide complex of any one of embodiments 1-144, wherein P1 or P2 comprises a linear peptide.
Embodiment 146 comprises a polypeptide or polypeptide complex of any one of embodiments 1-145, wherein the tumor cell antigen comprises MAGEA3, and the and the P1 or P2 comprises an amino acid sequence selected from the group consisting of GGESCQSVYDSSFCYD (SEQ ID NO: 13), GGNACEMTYDHTFCDP (SEQ ID NO: 14), GGRICEEVYDWIFCES (SEQ ID NO: 15), GGRRCVDVYDNAFCLI (SEQ ID NO: 16), GGVSCKDVYDEAFCWT (SEQ ID NO: 12), GGTSCAQIYDFEFCYS (SEQ ID NO: 17), GGSLCSLVYDQDFCES (SEQ ID NO: 18), GGNSCSLVYDKAFCLF (SEQ ID NO: 19), GGNQCWEVYDQEFCSL (SEQ ID NO: 20), GGSACSRIYDFAFCHT (SEQ ID NO: 21), GGTFCYFDHGLVNCQW (SEQ ID NO: 22), GGHCFVSPASGEWWCV (SEQ ID NO: 23), GGCSWIFDGLRYFSKC (SEQ ID NO: 24), VRTWFEKFPELV (SEQ ID NO: 25), LVWGCIWDDMCS (SEQ ID NO: 26), WHWEPSMVWGML (SEQ ID NO: 27), GGGCFVSPATGFTWCV (SEQ ID NO: 28), GGDCQPDSVWSYWYCR (SEQ ID NO: 29), GGCTFVDWWVLGSPYC (SEQ ID NO: 30), GGCLMNDYYYLWGGHC (SEQ ID NO: 31), GGASCKDVYDEAFCWT (SEQ ID NO: 32), GGVACKDVYDEAFCWT (SEQ ID NO: 33), GGVSAKDVYDEAFCWT (SEQ ID NO: 34), GGVSCADVYDEAFCWT (SEQ ID NO: 35), GGVSCKAVYDEAFCWT (SEQ ID NO: 36), GGVSCKDAYDEAFCWT (SEQ ID NO: 37), GGVSCKDVADEAFCWT (SEQ ID NO: 38), GGVSCKDVYAEAFCWT (SEQ ID NO: 39), GGVSCKDVYDAAFCWT (SEQ ID NO: 40), GGVSCKDVYDEAACWT (SEQ ID NO: 41), GGVSCKDVYDEAFAWT (SEQ ID NO: 42), GGVSCKDVYDEAFCAT (SEQ ID NO: 43), GGVSCKDVYDEAFCWA (SEQ ID NO: 44), EVDPIGHLY (SEQ ID NO: 45), ESDPIVAQY (SEQ ID NO: 46), and GGASCAASASAAACAS (SEQ ID NO: 47).
Embodiment 147 comprises a polypeptide or polypeptide complex of any one of embodiments 1-146, wherein L1 is bound to N-terminus of A1.
Embodiment 148 comprises a polypeptide or polypeptide complex of any one of embodiments 1-147, wherein L1 is bound to C-terminus of A1.
Embodiment 149 comprises a polypeptide or polypeptide complex of any one of embodiments 1-148, wherein L2 is bound to N-terminus of A2.
Embodiment 150 comprises a polypeptide or polypeptide complex of any one of embodiments 1-149, wherein L2 is bound to C-terminus of A2.
Embodiment 151 comprises a polypeptide or polypeptide complex of any one of embodiments 1-150, wherein L1 or L2 is a peptide sequence having at least 5 to no more than 50 amino acids.
Embodiment 152 comprises a polypeptide or polypeptide complex of any one of embodiments 1-151, wherein L1 or L2 is a peptide sequence having at least 10 to no more than 30 amino acids.
Embodiment 153 comprises a polypeptide or polypeptide complex of any one of embodiments 1-152, wherein L1 or L2 is a peptide sequence having at least 10 amino acids.
Embodiment 154 comprises a polypeptide or polypeptide complex of any one of embodiments 1-153, wherein L1 or L2 is a peptide sequence having at least 18 amino acids.
Embodiment 155 comprises a polypeptide or polypeptide complex of any one of embodiments 1-154, wherein L1 or L2 is a peptide sequence having at least 26 amino acids.
Embodiment 156 comprises a polypeptide or polypeptide complex of any one of embodiments 1-155, wherein L1 or L2 has a formula comprising (G2S)n, wherein n is an integer from 1 to 3 (SEQ ID NO: 48).
Embodiment 157 comprises a polypeptide or polypeptide complex of any one of embodiments 1-156, wherein L1 has a formula selected from the group consisting of (G2S)n, (GS)n, (GSGGS)n (SEQ ID NO: 49), (GGGS)n (SEQ ID NO: 50), (GGGGS)n (SEQ ID NO: 51), and (GSSGGS)n (SEQ ID NO: 52), wherein n is an integer of at least 1.
Embodiment 158 comprises a polypeptide or polypeptide complex of any one of embodiments 1-157, wherein P1 becomes unbound from A1 when L1 is cleaved by the tumor specific protease thereby exposing A1 to the first target antigen.
Embodiment 159 comprises a polypeptide or polypeptide complex of any one of embodiments 1-158, wherein P2 becomes unbound from A2 when L2 is cleaved by the tumor specific protease thereby exposing A2 to the second target antigen.
Embodiment 160 comprises a polypeptide or polypeptide complex of any one of embodiments 1-159, wherein the tumor specific protease is selected from the group consisting of metalloprotease, serine protease, cysteine protease, threonine protease, and aspartic protease.
Embodiment 161 comprises a polypeptide or polypeptide complex of any one of embodiments 1-160, wherein L1 or L2 comprises a urokinase cleavable amino acid sequence, a matriptase cleavable amino acid sequence, matrix metalloprotease cleavable amino acid sequence, or a legumain cleavable amino acid sequence.
Embodiment 162 comprises a polypeptide or polypeptide complex of any one of embodiments 1-161, wherein L1 or L2 comprises an amino acid sequence selected from the group consisting of GGGGSLSGRSDNHGSSGT (SEQ ID NO: 53), GGGGSSGGSGGSGLSGRSDNHGSSGT (SEQ ID NO: 54), ASGRSDNH (SEQ ID NO: 55), LAGRSDNH (SEQ ID NO: 56), ISSGLASGRSDNH (SEQ ID NO: 57), ISSGLLAGRSDNH (SEQ ID NO: 58), LSGRSDNH (SEQ ID NO: 4), ISSGLLSGRSDNP (SEQ ID NO: 59), ISSGLLSGRSDNH (SEQ ID NO: 60), LSGRSDNHSPLGLAGS (SEQ ID NO: 61), SPLGLAGSLSGRSDNH (SEQ ID NO: 62), SPLGLSGRSDNH (SEQ ID NO: 63), LAGRSDNHSPLGLAGS (SEQ ID NO: 64), LSGRSDNHVPLSLKMG (SEQ ID NO: 65), LSGRSDNHVPLSLSMG (SEQ ID NO: 66), GGGGSLSGRSDNHGSSGT (SEQ ID NO: 53), GGGGSSGGSGGSGLSGRSDNHGSSGT (SEQ ID NO: 54), ASGRSDNH (SEQ ID NO: 55), LAGRSDNH (SEQ ID NO: 56), ISSGLASGRSDNH (SEQ ID NO: 57), ISSGLLAGRSDNH (SEQ ID NO: 58), LSGRSDNH (SEQ ID NO: 4), ISSGLLSGRSDNP (SEQ ID NO: 59), ISSGLLSGRSDNH (SEQ ID NO: 60), LSGRSDNHSPLGLAGS (SEQ ID NO: 61), SPLGLAGSLSGRSDNH (SEQ ID NO: 62), SPLGLSGRSDNH (SEQ ID NO: 63), LAGRSDNHSPLGLAGS (SEQ ID NO: 64), LSGRSDNHVPLSLKMG (SEQ ID NO: 65), LSGRSDNHVPLSLSMG (SEQ ID NO: 66), GSSGGSGGSGGSGLSGRSDNHGSSGT (SEQ ID NO: 68), GSSGGSGGSGGSGGGSGGGSGGSSGT (SEQ ID NO: 69), GGGGSGGSGGGSGGSSGT (SEQ ID NO: 70), and GGGGSGGGS (SEQ ID NO: 71).
Embodiment 163 comprises a polypeptide or polypeptide complex of any one of embodiments 1-162, wherein L1 or L2 comprises an amino acid sequence ASGRSDNH (SEQ ID NO: 55), LAGRSDNH (SEQ ID NO: 56), ISSGLASGRSDNH (SEQ ID NO: 57), and ISSGLLAGRSDNH (SEQ ID NO: 58).
Embodiment 164 comprises a polypeptide or polypeptide complex of any one of embodiments 1-163, wherein H1 comprises a polymer.
Embodiment 165 comprises a polypeptide or polypeptide complex of any one of embodiments 1-164, wherein the polymer is polyethylene glycol (PEG).
Embodiment 166 comprises a polypeptide or polypeptide complex of any one of embodiments 1-165, wherein H1 comprises albumin.
Embodiment 167 comprises a polypeptide or polypeptide complex of any one of embodiments 1-166, wherein H1 comprises an Fc domain.
Embodiment 168 comprises a polypeptide or polypeptide complex of any one of embodiments 1-167, wherein the albumin is serum albumin.
Embodiment 169 comprises a polypeptide or polypeptide complex of any one of embodiments 1-168,wherein the albumin is human serum albumin.
Embodiment 170 comprises a polypeptide or polypeptide complex of any one of embodiments 1-169, wherein H1 comprises a polypeptide, a ligand, or a small molecule.
Embodiment 171 comprises a polypeptide or polypeptide complex of any one of embodiments 1-170, wherein the polypeptide, the ligand or the small molecule binds serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1.
Embodiment 172 comprises a polypeptide or polypeptide complex of any one of embodiments 1-171, wherein the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin.
Embodiment 173 comprises a polypeptide or polypeptide complex of any one of embodiments 1-172, wherein the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM or IgD.
Embodiment 174 comprises a polypeptide or polypeptide complex of any one of embodiments 1-173, wherein the serum protein is albumin.
Embodiment 175 comprises a polypeptide or polypeptide complex of any one of embodiments 1-174, wherein the polypeptide is an antibody.
Embodiment 176 comprises a polypeptide or polypeptide complex of any one of embodiments 1-175, wherein the single domain antibody comprises a single domain antibody, a single chain variable fragment or a Fab.
Embodiment 177 comprises a polypeptide or polypeptide complex of any one of embodiments 1-176, wherein the single domain antibody comprises a single domain antibody that binds to albumin.
Embodiment 178 comprises a polypeptide or polypeptide complex of any one of embodiments 1-177, wherein the single domain antibody is a human or humanized antibody.
Embodiment 179 comprises a polypeptide or polypeptide complex of any one of embodiments 1-178, wherein the single domain antibody is 645gH1gL1.
Embodiment 180 comprises a polypeptide or polypeptide complex of any one of embodiments 1-179, wherein the single domain antibody is 645dsgH5gL4.
Embodiment 181 comprises a polypeptide or polypeptide complex of any one of embodiments 1-180, wherein the single domain antibody is 23-13-A01 -sc02.
Embodiment 182 comprises a polypeptide or polypeptide complex of any one of embodiments 1-181, wherein the single domain antibody is A10m3 or a fragment thereof.
Embodiment 183 comprises a polypeptide or polypeptide complex of any one of embodiments 1-182, wherein the single domain antibody is DOM7r-31.
Embodiment 184 comprises a polypeptide or polypeptide complex of any one of embodiments 1-183, wherein the single domain antibody is DOM7h-11-15.
Embodiment 185 comprises a polypeptide or polypeptide complex of any one of embodiments 1-184, wherein the single domain antibody is Alb-1, Alb-8, or Alb-23.
Embodiment 186 comprises a polypeptide or polypeptide complex of any one of embodiments 1-185, wherein the single domain antibody is 10G or 10GE.
Embodiment 187 comprises a polypeptide or polypeptide complex of any one of embodiments 1-186, wherein the single domain antibody is 10G, and the single domain antibody comprises an amino acid sequence
or
Embodiment 188 comprises a polypeptide or polypeptide complex of any one of embodiments 1-187, wherein the single domain antibody is SA21.
Embodiment 189 comprises a polypeptide or polypeptide complex of any one of embodiments 1-188, wherein the polypeptide or polypeptide complex comprises a modified amino acid, a non-natural amino acid, a modified non-natural amino acid, or a combination thereof.
Embodiment 190 comprises a polypeptide or polypeptide complex of any one of embodiments 1-189, wherein the modified amino acid or modified non-natural amino acid comprises a post-translational modification.
Embodiment 191 comprises a polypeptide or polypeptide complex of any one of embodiments 1-190, wherein H1 comprises a linking moiety (L3) that connects H1 to P1.
Embodiment 192 comprises a polypeptide or polypeptide complex of any one of embodiments 1-191, wherein L3 is a peptide sequence having at least 5 to no more than 50 amino acids.
Embodiment 193 comprises a polypeptide or polypeptide complex of any one of embodiments 1-192, wherein L3 is a peptide sequence having at least 10 to no more than 30 amino acids.
Embodiment 194 comprises a polypeptide or polypeptide complex of any one of embodiments 1-193, wherein L3 is a peptide sequence having at least 10 amino acids.
Embodiment 195 comprises a polypeptide or polypeptide complex of any one of embodiments 1-194, wherein L3 is a peptide sequence having at least 18 amino acids.
Embodiment 196 comprises a polypeptide or polypeptide complex of any one of embodiments 1-195, wherein L3 is a peptide sequence having at least 26 amino acids.
Embodiment 197 comprises a polypeptide or polypeptide complex of any one of embodiments 1-196, wherein L3 has a formula selected from the group consisting of (G2S)n, (GS)n, (GSGGS)n (SEQ ID NO: 49), (GGGS)n (SEQ ID NO: 50), (GGGGS)n (SEQ ID NO: 51), and (GSSGGS)n (SEQ ID NO: 52), wherein n is an integer of at least 1.
Embodiment 198 comprises a polypeptide or polypeptide complex of any one of embodiments 1-197, wherein L3 comprises an amino acid sequence of SSGGGGSGGGS (SEQ ID NO: 67).
Embodiment 199 comprises a polypeptide or polypeptide complex of any one of embodiments 1-198, wherein the polypeptide or polypeptide complex has weaker binding affinity for its pMHC as compared to the binding affinity for the pMHC of a polypeptide or polypeptide complex that does not have P1 or L1.
Embodiment 200 comprises a polypeptide or polypeptide complex of any one of embodiments 1-199, wherein the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 10X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1.
Embodiment 201 comprises a polypeptide or polypeptide complex of any one of embodiments 1-200, wherein the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 100X higher than the binding affinity for the pMHC of a form of the polypeptide or polypeptide complex that does not have P1 or L1.
Embodiment 202 comprises a polypeptide or polypeptide complex of any one of embodiments 1-201, wherein the polypeptide or polypeptide complex has weaker binding affinity for its pMHC as compared to the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease.
Embodiment 203 comprises a polypeptide or polypeptide complex of any one of embodiments 1-202, wherein the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 10X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease.
Embodiment 204 comprises a polypeptide or polypeptide complex of any one of embodiments 1-203, wherein the polypeptide or polypeptide complex has weaker binding affinity for its pMHC that is at least 100X higher than the binding affinity for the pMHC of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease.
Embodiment 205 comprises a polypeptide or polypeptide complex of any one of embodiments 1-204, wherein the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay as compared to the EC50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have P1 or L1.
Embodiment 206 comprises a polypeptide or polypeptide complex of any one of embodiments 1-205, wherein the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 10X higher than the EC50 in a T-cell cytolysis assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1.
Embodiment 207 comprises a polypeptide or polypeptide complex of any one of embodiments 1-206, wherein the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 100X higher than the EC50 in a T-cell cytolysis assay of a polypeptide or polypeptide complex that does not have P1 or L1.
Embodiment 208 comprises a polypeptide or polypeptide complex of any one of embodiments 1-207, wherein the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay as compared to the EC50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease.
Embodiment 209 comprises a polypeptide or polypeptide complex of any one of embodiments 1-208, wherein the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 10X higher than the EC50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease.
Embodiment 210 comprises a polypeptide or polypeptide complex of any one of embodiments 1-209, wherein the polypeptide or polypeptide complex has an increased EC50 in a T-cell cytolysis assay that is at least 100X higher than the EC50 in a T-cell cytolysis assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease.
Embodiment 211 comprises a polypeptide or polypeptide complex of any one of embodiments 1-210, wherein the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay as compared to the EC50 in an IFNγ release T-cell activation assay of a polypeptide or polypeptide complex that does not have P1 or L1.
Embodiment 212 comprises a polypeptide or polypeptide complex of any one of embodiments 1-211, wherein the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 10X higher than the EC50 in an IFNγ release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1.
Embodiment 213 comprises a polypeptide or polypeptide complex of any one of embodiments 1-212, wherein the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 100X higher than the EC50 in an IFNγ release T-cell activation assay of a form of the polypeptide or polypeptide complex that does not have P1 or L1.
Embodiment 214 comprises a polypeptide or polypeptide complex of any one of embodiments 1-213, wherein the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay as compared to the EC50 in an IFNγ release T-cell activation assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease.
Embodiment 215 comprises a polypeptide or polypeptide complex of any one of embodiments 1-214, wherein the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 10X higher than the EC50 in an IFNγ release T-cell activation assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease.
Embodiment 216 comprises a polypeptide or polypeptide complex of any one of embodiments 1-215, wherein the polypeptide or polypeptide complex has an increased EC50 in an IFNγ release T-cell activation assay that is at least 100X higher than the EC50 in an IFNγ release T-cell activation assay of the polypeptide or polypeptide complex in which L1 has been cleaved by the tumor specific protease.
Embodiment 217 comprises a pharmaceutical composition comprising: the polypeptide or polypeptide complex of any one of embodiments 1-216; and a pharmaceutically acceptable excipient.
Embodiment 218 comprises an isolated recombinant nucleic acid molecule encoding the polypeptide or polypeptide complex of any one of embodiments 1-217.
Embodiment 219 comprises a polypeptide or polypeptide complex according to Formula II:
wherein: L1a comprises a tumor specific protease-cleaved linking moiety that when uncleaved connects P1a to an antigen recognizing molecule that binds to a target antigen and; P1a comprises a peptide that binds to the antigen recognizing molecule when L1a is uncleaved; and H1a comprises a half-life extending molecule.
Embodiment 220 comprises a polypeptide or polypeptide complex of any one of embodiments 1-219, wherein P1a when L1 is uncleaved impairs binding of the antigen recognizing molecule to the target antigen.
Embodiment 221 comprises a polypeptide or polypeptide complex of any one of embodiments 1-220, wherein the antigen recognizing molecule comprises an antibody or antibody fragment.
Embodiment 222 comprises a polypeptide or polypeptide complex of any one of embodiments 1-221, wherein the target antigen is an anti-CD3 effector cell antigen.
Embodiment 223 comprises a polypeptide or polypeptide complex of any one of embodiments 1-222, wherein the target antigen is a tumor cell antigen.
Embodiment 224 comprises a polypeptide or polypeptide complex of any one of embodiments 1-223, wherein the tumor cell antigen MAGEA3 or MART1.
Embodiment 225 comprises a polypeptide or polypeptide complex of any one of embodiments 1-224, wherein P1a has less than 70% sequence homology to the target antigen.
Embodiment 226 comprises a polypeptide or polypeptide complex of any one of embodiments 1-225, wherein P1a comprises a peptide sequence of at least 10 amino acids in length.
Embodiment 227 comprises a polypeptide or polypeptide complex of any one of embodiments 1-226, wherein P1a comprises a peptide sequence of at least 10 amino acids in length and no more than 20 amino acids in length.
Embodiment 228 comprises a polypeptide or polypeptide complex of any one of embodiments 1-227, wherein P1a comprises a peptide sequence of at least 16 amino acids in length.
Embodiment 229 comprises a polypeptide or polypeptide complex of any one of embodiments 1-228, wherein P1a comprises a peptide sequence of no more than 40 amino acids in length.
Embodiment 230 comprises a polypeptide or polypeptide complex of any one of embodiments 1-229, wherein P1a comprises at least two cysteine amino acid residues.
Embodiment 231 comprises a polypeptide or polypeptide complex of any one of embodiments 1-230, wherein P1a comprises a cyclic peptide or a linear peptide.
Embodiment 232 comprises a polypeptide or polypeptide complex of any one of embodiments 1-231, wherein P1a comprises a cyclic peptide.
Embodiment 233 comprises a polypeptide or polypeptide complex of any one of embodiments 1-232, wherein P1a comprises a linear peptide.
Embodiment 234 comprises a polypeptide or polypeptide complex of any one of embodiments 1-233, wherein the wherein the target antigen comprises MAGEA3, and the and the P1 or P2 comprises an amino acid sequence selected from the group consisting of GGESCQSVYDSSFCYD (SEQ ID NO: 13), GGNACEMTYDHTFCDP (SEQ ID NO: 14), GGRICEEVYDWIFCES (SEQ ID NO: 15), GGRRCVDVYDNAFCLI (SEQ ID NO: 16), GGVSCKDVYDEAFCWT (SEQ ID NO: 12), GGTSCAQIYDFEFCYS (SEQ ID NO: 17), GGSLCSLVYDQDFCES (SEQ ID NO: 18), GGNSCSLVYDKAFCLF (SEQ ID NO: 19), GGNQCWEVYDQEFCSL (SEQ ID NO: 20), GGSACSRIYDFAFCHT (SEQ ID NO: 21), GGTFCYFDHGLVNCQW (SEQ ID NO: 22), GGHCFVSPASGEWWCV (SEQ ID NO: 23), GGCSWIFDGLRYFSKC (SEQ ID NO: 24), VRTWFEKFPELV (SEQ ID NO: 25), LVWGCIWDDMCS (SEQ ID NO: 26), WHWEPSMVWGML (SEQ ID NO: 27), GGGCFVSPATGFTWCV (SEQ ID NO: 28), GGDCQPDSVWSYWYCR (SEQ ID NO: 29), GGCTFVDWWVLGSPYC (SEQ ID NO: 30), GGCLMNDYYYLWGGHC (SEQ ID NO: 31), GGASCKDVYDEAFCWT (SEQ ID NO: 32), GGVACKDVYDEAFCWT (SEQ ID NO: 33), GGVSAKDVYDEAFCWT (SEQ ID NO: 34), GGVSCADVYDEAFCWT (SEQ ID NO: 35), GGVSCKAVYDEAFCWT (SEQ ID NO: 36), GGVSCKDAYDEAFCWT (SEQ ID NO: 37), GGVSCKDVADEAFCWT (SEQ ID NO: 38), GGVSCKDVYAEAFCWT (SEQ ID NO: 39), GGVSCKDVYDAAFCWT (SEQ ID NO: 40), GGVSCKDVYDEAACWT (SEQ ID NO: 41), GGVSCKDVYDEAFAWT (SEQ ID NO: 42), GGVSCKDVYDEAFCAT (SEQ ID NO: 43), GGVSCKDVYDEAFCWA (SEQ ID NO: 44), EVDPIGHLY (SEQ ID NO: 45), ESDPIVAQY (SEQ ID NO: 46), and GGASCAASASAAACAS (SEQ ID NO: 47).
Embodiment 235 comprises a polypeptide or polypeptide complex of any one of embodiments 1-234, wherein H1a comprises a polymer.
Embodiment 236 comprises a polypeptide or polypeptide complex of any one of embodiments 1-235, wherein the polymer is polyethylene glycol (PEG).
Embodiment 237 comprises a polypeptide or polypeptide complex of any one of embodiments 1-236, wherein H1a comprises albumin.
Embodiment 238 comprises a polypeptide or polypeptide complex of any one of embodiments 1-237, wherein H1a comprises an Fc domain.
Embodiment 239 comprises a polypeptide or polypeptide complex of any one of embodiments 1-238, wherein the albumin is serum albumin.
Embodiment 240 comprises a polypeptide or polypeptide complex of any one of embodiments 1-239, wherein the albumin is human serum albumin.
Embodiment 241 comprises a polypeptide or polypeptide complex of any one of embodiments 1-240, wherein H1a comprises a polypeptide, a ligand, or a small molecule.
Embodiment 242 comprises a polypeptide or polypeptide complex of any one of embodiments 1-241, wherein the polypeptide, the ligand or the small molecule binds a serum protein or a fragment thereof, a circulating immunoglobulin or a fragment thereof, or CD35/CR1.
Embodiment 243 comprises a polypeptide or polypeptide complex of any one of embodiments 1-242, wherein the serum protein comprises a thyroxine-binding protein, a transthyretin, a 1-acid glycoprotein, a transferrin, transferrin receptor or a transferrin-binding portion thereof, a fibrinogen, or an albumin.
Embodiment 244 comprises a polypeptide or polypeptide complex of any one of embodiments 1-243, wherein the circulating immunoglobulin molecule comprises IgG1, IgG2, IgG3, IgG4, slgA, IgM or IgD.
Embodiment 245 comprises a polypeptide or polypeptide complex of any one of embodiments 1-244, wherein the serum protein is albumin.
Embodiment 246 comprises a polypeptide or polypeptide complex of any one of embodiments 1-245, wherein the polypeptide is an antibody.
Embodiment 247 comprises a polypeptide or polypeptide complex of any one of embodiments 1-246, wherein the antibody comprises a single domain antibody, a single chain variable fragment or a Fab.
Embodiment 248 comprises a polypeptide or polypeptide complex of any one of embodiments 1-247, wherein the antibody comprises a single domain antibody that binds to albumin.
Embodiment 249 comprises a polypeptide or polypeptide complex of any one of embodiments 1-248, wherein the antibody is a human or humanized antibody.
Embodiment 250 comprises a polypeptide or polypeptide complex of any one of embodiments 1-249, wherein the single domain antibody is 645gH1gL1.
Embodiment 251 comprises a polypeptide or polypeptide complex of any one of embodiments 1-250, wherein the single domain antibody is 645dsgH5gL4.
Embodiment 252 comprises a polypeptide or polypeptide complex of any one of embodiments 1-251, wherein the single domain antibody is 23-13-A01 -sc02.
Embodiment 253 comprises a polypeptide or polypeptide complex of any one of embodiments 1-252, wherein the single domain antibody is A10m3 or a fragment thereof.
Embodiment 254 comprises a polypeptide or polypeptide complex of any one of embodiments 1-253, wherein the single domain antibody is DOM7r-31.
Embodiment 255 comprises a polypeptide or polypeptide complex of any one of embodiments 1-254, wherein the single domain antibody is DOM7h-11-15.
Embodiment 256 comprises a polypeptide or polypeptide complex of any one of embodiments 1-255, wherein the single domain antibody is Alb-1, Alb-8, or Alb-23.
Embodiment 257 comprises a polypeptide or polypeptide complex of any one of embodiments 1-256, wherein the single domain antibody is 10G or 10GE.
Embodiment 258 comprises a polypeptide or polypeptide complex of any one of embodiments 1-257, wherein the single domain antibody is 10G, and the single domain antibody comprises an amino acid sequence
or
Embodiment 259 comprises a polypeptide or polypeptide complex of any one of embodiments 1-258, wherein the single domain antibody is SA21.
Embodiment 260 comprises a polypeptide or polypeptide complex of any one of embodiments 1-259, wherein H1a comprises a linking moiety (L3a) that connects H1a to P1a.
Embodiment 261 comprises a polypeptide or polypeptide complex of any one of embodiments 1-260, wherein L1a is a peptide sequence having at least 5 to no more than 50 amino acids.
Embodiment 262 comprises a polypeptide or polypeptide complex of any one of embodiments 1-261, wherein L1a is a peptide sequence having at least 10 to no more than 30 amino acids.
Embodiment 263 comprises a polypeptide or polypeptide complex of any one of embodiments 1-262, wherein L1a is a peptide sequence having at least 10 amino acids.
Embodiment 264 comprises a polypeptide or polypeptide complex of any one of embodiments 1-263, wherein L1a is a peptide sequence having at least 18 amino acids.
Embodiment 265 comprises a polypeptide or polypeptide complex of any one of embodiments 1-264, wherein L1a is a peptide sequence having at least 26 amino acids.
Embodiment 266 comprises a polypeptide or polypeptide complex of any one of embodiments 1-265, wherein L1a has a formula selected from the group consisting of (G2S)n, (GS)n, (GSGGS)n (SEQ ID NO: 49), (GGGS)n (SEQ ID NO: 50), (GGGGS)n (SEQ ID NO: 51), and (GSSGGS)n (SEQ ID NO: 52), wherein n is an integer of at least 1.
Embodiment 267 comprises a polypeptide or polypeptide complex of any one of embodiments 1-266,wherein L3a comprises an amino acid sequence of SSGGGGSGGGS (SEQ ID NO: 67).
Embodiment 268 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 269 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 270 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 271 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 272 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 273 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 274 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 275 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 276 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 277 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 278 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 279 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 280 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 281 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 282 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 283 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 284 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 285 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 286 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 287 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 288 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 289 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 290 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Embodiment 291 comprises a polypeptide or polypeptide complex comprising a structural arrangement according to the configuration shown in
Expression plasmids encoding the TCR alpha and beta chains are produced using standard molecular biology techniques. Plasmids are transformed into chemically-competent cells and grown overnight at 37° C. Protein expression is induced by the addition of Isopropyl β-D-1 -thiogalactopyranoside (IPTG) to 1 mM and bacteria are grown for a further 3 hours at 37° C. Bacteria are harvested by centrifugation at 4000 × g for 15 minutes and lysed in a protein extraction reagent containing DNAse. Lysis proceeds for 1 hour at room temperature with agitation before inclusion bodies are harvested by centrifugation at 10000 × g for 5 minutes. Pellets are washed twice with a detergent buffer containing 1% Triton X100 and resuspended in a buffered saline solution.
Soluble TCRs are prepared by dissolving alpha and beta inclusion bodies in 6 M guanidine-HCI containing 10 mM dithiothreitol and incubating at 37°Cfor 30 minutes. Samples are diluted into 50 ml urea folding buffer (5 M urea; 0.4 M L-arginine; 0.1 M Tris-CI, pH 8.1; 2 mM EDTA; 6.5 mM β-mercapthoethylamine; 1.9 mM cystamine) and dialyzed against eight volumes of water overnight at 4° C., followed by dialysis for a further 24 hours in eight volumes of 10 mM Tris (8.1), with one buffer change. The resultant TCR complexes will be concentrated and purified using Ni-NTA, and size-exclusion chromatography. Isolated proteins were characterized using standard size exclusion chromatography, SDS PAGE, and LC-MS procedures. TCR fusion constructs can also be produced in mammalian cells, insect cells, or yeast cells according to known methods.
A modified TCR is tested for its ability to recognize antigens when separately expressed in CD8+ T cells and CD4+ T cells. PBMC from a subject is transfected as described in Zhao et al. (2006), et al., Mol. Ther. 13: 151-159 (2006) with (i) RNA encoding the WT alpha chain of the TCR and (ii) RNA encoding the WT beta chain of the TCR, or DNA encoding Green Fluorescence Protein (GFP).
Transfected cells are washed and stimulated with or without (T alone) one of the following cells: T2+ pulsed with antigen. Responder cells (1 × 105 electroporated PBLs) and 1 × 105 stimulator cells are incubated in a 0.2-ml culture volume in individual wells of 96-well plates. Stimulator cells and responder cells are co-cultured for 16 to 24 h. Cytokine secretion of culture supernatants diluted to the linear range of the assay is measured using commercially available ELISA kits (IFN-γ Endogen, Cambridge, Mass.).The amount of IFN-γ (pg/ml) produced by transfected CD8+ T cells is determined, while the amount of IFN-γ (pg/ml) produced by transfected CD4+ T cells is determined.
This example outlines an exemplary way to reformat peptides and scFv into bispecific recombinant TCR fusions. T cell receptors are comprised of an alpha chain complexed with a beta chain. Each alpha and beta chains include the entire extracellular domain and lack the membrane spanning and intracellular domains. The individual T cell receptor chains were overexpressed in E. coli and recovered from inclusion bodies. Specifically, genes encoding the alpha or beta subunits with or without additional peptide or protein fusions added to either the amino or carboxy-termini were synthesized using E. coli codon optimization. Additionally, the C-terminus of the alpha subunit has appended a poly histidine epitope for protein purification purposes and to the C-terminus of the beta subunit a BirA biotinylation substrate (“Avitag”) has been appended for enzymatic site specific biotin conjugation. Following protein expression inclusion bodies were isolated and then dissolved in solubilization buffer (8 M urea, 25 mM MES pH 6.0, 10 mM EDTA, 0.1 mM DTT), while TCR was dissolved in the solubilization buffer containing 6 M guanidine hydrochloride (GnHCl). Ninety milligrams total of TCR alpha and TCR beta were diluted into 500 mL refolding buffer [3 M urea, 0.2 M Arg-HCl, 150 mM Tris-HCl pH 8.0, 1.5 mM reduced glutathione, 0.15 mM oxidized glutathione and stirred at 4° C. for 72 h. The subunits with CD3 scFv fusions were added in a two-fold excess by weight compared to chains lacking scFv fusions. Specifically, sixty milligrams of each of the CD3 scFv containing TCR chains were combined with thirty milligrams of the complementary TCR chain to complete heterodimeric TCR. Refolded TCR was dialyzed at 4° C. for 24 h in 4 L dialysis buffer (10 mM Tris pH 8.5, 50 mM NaCl) and then for an additional 24 h in fresh 4 L dialysis buffer. The resultant TCR complexes are concentrated and purified using Ni-NTA, and size-exclusion chromatography. Isolated proteins were characterized using standard size exclusion chromatography, SDS PAGE, and LC-MS procedures.
Biopanning with m13 phagemid p8 or p3 displayed peptide libraries was performed with biotin-conjugated target immobilized on streptavidin coated paramagnetic beads. The targets were either recombinant proteins enzymatically biotinylated by a birA directed process onto an engineered substrate (Avitag) or through chemical biotin conjugation, typically through random labeling of primary amines. Following binding to bead bound target and washing steps, specifically bound phage were recovered by elution at pH 2.2. Enrichment of specific binding clones was generally accomplished by 2-4 rounds of successive biopanning and amplification. After 2 rounds of biopanning the resulting phage pools were infected into TG1 cells and plated out on LB-ampicillin/agar plates for clonal isolation and subsequent characterization.
For hit identification, individual colonies were grown in 96-deep well plates for 2-4 hours and infected with helper phage and induced to produce peptide displayed phagemid following an overnight growth. The next day the deep well plates were centrifuged to separate the soluble phagemid from the E. coli cells. The phagemid containing supernatants were then combined with PBS-Tween 20 (0.05%) + BSA (1%) blocking buffer and incubated in ELISA wells containing Neutravidin captured biotin-conjugated target or Neutravidin alone. After binding at 4 degrees the plates were washed, and specifically bound phage were detected by anti-m13 HRP conjugated antibodies using standard TMB-based chromogenic ELISA procedures. Daughter plates or individual wells were subjected to standard DNA sequencing for clonal peptide sequence identification.
Outbred llamas were immunized with purified human serum albumin first in Freund’s Complete Adjuvant (FCA) and subsequently boosted with Incomplete Freund’s Adjuvant. Upon completion of the immunization regimen immune Vhh p3 phagemid display libraries were generated from cDNA prepared using total RNA isolated from llama PBMCs. The phage display libraries were biopanned against human serum albumin immobilized on paramagnetic beads. Random clones isolated following round 2 and subsequent rounds were grown in 96-deep well culture were tested by either phagemid or soluble single domain ELISA for specific reactivity to human serum albumin, cynomolgus serum albumin, and mouse serum albumin.
Single domain antibodies derived from clones with desired specificity profiles were purified from either scaled up production in E. coli and purification from periplasmic fractions and Ni-NTA chromatography or mammalian transient expression in HEK293 cells. Resulting protein was analyzed by SDS-PAGE for relative purity and protein concentrations quantitated by A280 analysis. The resulting proteins were available for subsequent quantitative binding assessments by either ELISA-based methods or kinetic-based methods.
Llama single domain antibodies (SDAs) that bind serum albumin or their humanized variants were cloned as recombinant constructs with carboxy terminal fusions comprising peptides of specific binding profiles from the example above. The fusions can either be recombinantly fused to the SDA terminus or with an intervening peptide linker. The resulting recombinant constructs were then produced from periplasmic production in E.coli as follows. A single colony or small portion of frozen starter was inoculated into 10 ml LB/Ampicillin (100 mcg/mL) + 2% glucose and grown overnight at 37 degrees. Next day 500 mL of LB/Ampicillin (100 mcg/mL) + 0.1% glucose was inoculated with 5 mL of the preculture and grown at 37 degrees until reaching OD 600 nm ~0.7. Next protein expression was induced by the addition of IPTG (final concentration 1 mM) and shifted to growth overnight at 28 degrees, or alternatively induction was grown at for 4 hours at 37 degrees. Bacteria were harvested by centrifugation. The cell pellet was next resuspended in 7.5 mL TES buffer (TES buffer 24.22 g Tris, 0.19 g EDTA and 171.15 g sucrose in 1 liter ddH2O. Adjust to pH 8.0 with HCl.) and incubate for at least 1 hr on ice on an orbital shaking platform. Add 30 ml of TES/4 to the resuspended pellet and shake for 45 min on ice on an orbital shaking platform. Centrifuge 30 min at 10,000 g at 4° C. and recover the supernatant as the periplasmic extract. Purify the protein through a nickel chelate support (Ni/NTA), followed by size exclusion chromatography on Superdex200 columns. Resulting protein was analyzed by SDS-PAGE for relative purity and protein concentrations quantitated by A280 analysis.
The following procedure was used to reformat peptides found above into recombinant TCR fusions represented in
Each of the individual T cell receptor chains necessary for the above constructs were overexpressed in E. coli and recovered from inclusion bodies. As described in
The following procedure was used to reformat peptides found above into recombinant TCR fusions represented in
Each of the individual T cell receptor chains necessary for the above constructs were overexpressed in E. coli and recovered from inclusion bodies. As described in
The following procedure was used to reformat peptides found above into recombinant TCR fusions. Notably, T cell receptors are comprised of an alpha chain complexed with a beta chain. Each alpha and beta chains include the entire extracellular domain and lack the membrane spanning and intracellular domains. Bispecific soluble T cell receptors (TCRs) were generated with fusions of anti-CD3 binding modules to engender simultaneous binding of cells presenting corresponding pMHC and CD3 positive cells with the goal of forming a cytolytic response against the pMHC targeted cell. To generate these desired bispecific proteins either anti-CD3 antibody fragments or anti-CD3 single domain antibodies were fused to either of the heteromeric TCR amino-termini. Dual control was provided by masking both TCR and the anti-CD3 antibody. TCR peptide masks identified above are appended as recombinant fusions to either the alpha or beta N-termini, and secondly recombinantly fused with anti-CD3 peptide masks to the free amino termini of the anti-CD3 antibody. Finally to assess the combined effects of dual masking in single constructs the peptide masked TCRs and peptide masked anti-CD3 antibodies were integrated into single heterodimeric protein constructs. In each of the masked descriptions protein linkers of defined proteolytic lability were incorporated as intervening sequence between the mask and either the TCR or anti-CD3 N-termini to provide the TCR and anti-CD3 targeted binding only when preferentially cleaved and subsequently activated within an opportunistic tumor environment. In additional constructs either of these masks were extended at their respective N-termini to contain an albumin binding single domain antibody to extend the systemic half-life of the resulting proteins.
Each of the individual T cell receptor chains necessary for the above constructs were overexpressed in E. coli and recovered from inclusion bodies. Specifically, genes encoding the alpha or beta subunits with or without additional peptide or protein fusions were synthesized using E. coli codon optimization. Additionally, the C-terminus of the alpha subunit has appended a poly histidine epitope for protein purification purposes and to the C-terminus of the beta subunit a BirA biotinylation substrate (“Avitag”) was appended for enzymatic site specific biotin conjugation. Following protein expression, inclusion bodies were isolated and then dissolved in solubilization buffer (8 M urea, 25 mM MES pH 6.0, 10 mM EDTA, 0.1 mM DTT), while TCRs were dissolved in the solubilization buffer containing 6 M guanidine hydrochloride (GnHCl). Thirty milligrams each of TCR alpha and TCR beta were diluted into 500 mL refolding buffer [3 M urea, 0.2 M Arg-HCl, 150 mM Tris-HCl pH 8.0, 1.5 mM reduced glutathione, 0.15 mM oxidized glutathione and stirred at 4° C. for 72 h. Refolded TCR was dialyzed at 4° C. for 24 h in 4 L dialysis buffer (10 mM Tris pH 8.5, 50 mM NaCl) and then for an additional 24 h in fresh 4 L dialysis buffer. The resultant TCR complexes are concentrated and purified using Ni-NTA, and size-exclusion chromatography. The resulting proteins were analyzed by SDS-PAGE under reducing and non-reducing conditions. Final protein concentrations quantitated by A280 analysis. Purified proteins were stored in aliquots at -80 degrees until used.
After expression, purification, and reconstitution of the heterodimeric masked, half-life extended, bispecific T-cell recruiting T cell receptor, the resulting protein was analyzed to determine the quality and integrity of the preparation. To do so TCR-19 was examined by determining intact mass through mass spectroscopy, aggregate content by HPLC size exclusion chromatography (SEC), and finally a qualitative analysis of dimeric integrity and purity by SDS-PAGE analysis. Specifically, TCR-19 is composed of the extracellular domains of the alpha and beta subunits of the MAGE-A3 affinity optimized T cell receptor, IC-3. To the amino terminus of the IC-3 extracellular alpha subunit (SEQ ID NO: 5) a pMHC competitive mask corresponding to a Peptide-5 was recombinantly fused via a split flexible linker containing an intervening protease cleavage site (SEQ ID NO: 4). To the amino terminus of the Peptide-5 mask (SEQ ID NO: 3) an anti-human serum albumin single domain antibody was additionally fused to enable half-life extension properties (SEQ ID NO: 2). For purification purposes a poly histidine extension was appended to the C-terminus. For the complementary IC-3 beta subunit an anti-human CD3 scFv (SEQ ID NO: 8) was recombinantly appended via a short flexible linker to the amino terminus. Finally, a biotin accepting substrate (Avitag) for birA was recombinantly appended to the C-terminus (SEQ ID NO: 10). Again, following the reconstitution as described above, the assembled resulting protein complex accurate correct mass and complete disulfide bond formation was examined via intact mass spectroscopy (
TCR-19 alpha subunit with N-terminal HSA and Peptide-5 cleavable mask, and C-terminal His-tag (SEQ ID NO: 1)
Anti-albumin SDA (SEQ ID NO: 2)
P MAGE-A3 peptide mask (SEQ ID NO: 3)
Protease substrate (SEQ ID NO: 4)
IC-3 alpha subunit (SEQ ID NO: 5)
Polyhistidine (SEQ ID NO: 6)
TCR-19 beta subunit with N-terminal anti-CD3 scFv and C-terminal AviTag (SEQ ID NO: 7)
Anti-CD3 scFv (SEQ ID NO: 8)
IC-3 beta subunit (SEQ ID NO: 9)
Avitag (SEQ ID NO: 10)
BLI based kinetic binding of masked bispecific TCR constructs to relevant tumor antigen pMHC was measured using a ForteBio Octet RED96 instrument. Biotinylated pMHC was first captured on streptavidin biosensors. Sensors were quenched using excess biocytin and then baselined in buffer. Masked bispecific TCRs were unmasked with protease where indicated. TCRs were associated onto the pMHC loaded biosensor. Association signal was monitored in real-time. Biosensors were then transferred to buffer and the dissociation of TCR was measured in real-time. Kinetic rates of association and dissociation were calculated using a one to one binding model within the instrument software (
Masked bispecific TCRs were also evaluated in an ELISA format. Briefly, biotinylated pMHC was captured on neutravidin coated plates. Masked bispecific TCRs were diluted in bovine serum albumin buffer or human serum albumin buffer, titrated onto the pMHC captured plates, washed, and incubated with secondary antibody. The His-tag present at the C-terminus of the TCRs allowed for anti-His-tag horse radish peroxidase conjugated secondary antibody recognition. When indicated masked bispecific TCRs were unmasked with protease before binding to pMHC coated plates. Plates were then developed using tetramethylbenzidine (TMB) and stopped using acid. Absorbance at 450 nm was measured and plotted versus log-scale TCR concentration. The concentration of TCR required for half maximal saturation signal was calculated in Graphpad Prism software and reported as EC50 (
Masked bispecific TCR binding to albumin was evaluated in an ELISA format. Briefly, high binding plates were coated with albumin overnight. Masked bispecific TCRs were titrated onto the plates, washed, and incubated with secondary antibody. The His-tag present at the C-terminus of the TCRs allowed for anti-His-tag horse radish peroxidase conjugated secondary antibody recognition. Plates were then developed using tetramethylbenzidine (TMB) and stopped using acid. Absorbance at 450 nm was measured and plotted versus log-scale TCR concentration. The concentration of TCR required for half maximal saturation signal was calculated in Graphpad Prism software and reported as EC50 (
Masked bispecific TCRs ability to bind CD3 was evaluated on the surface of human T cells. T cells were thawed from frozen stock and diluted in buffer. 200,000 cells per well were loaded onto a 96 deep well round bottom polypropylene plate. Masked bispecific TCRs were unmasked with protease where indicated and serially diluted into buffer. Bispecific TCRs were diluted in bovine serum albumin buffer or human serum albumin buffer and incubated with CD8+ T cells for one hour on ice in a total volume of 100 uL. Cells were then pelleted, supernatant removed, and washed with 2 mL of buffer. Cells were then pelleted again, supernatant removed, and resuspended in 100 uL of cold diluted MAGE-A3 pMHC PE labeled tetramer. Cells were incubated on ice for 30 min with the fluorescent MAGE-A3 pMHC tetramer dilution before washing. Cells were pelleted, supernatant removed, and washed with 2 mL buffer. Cells were pelleted again, resuspended in buffer, and immediately run on a NovoCyte flow cytometer. The mean fluorescence intensity (MFI) was measured for each cell staining distribution, normalized to the maximum signal (100%), and plotted against the log concentration of bispecific TCR. The concentration of bispecific TCR that resulted in half maximal signal was calculated in GraphPad Prism and reported as EC50 (
Tumor cells were seeded onto 96 well tissue culture treated flat bottom plates and allowed to adhere overnight. The following day, culture medium was removed from the cells, and replaced with medium containing serially diluted masked bispecific TCRs and CD8+ T cells. Masked bispecific TCRs were unmasked with protease when indicated. CD8+ T cells were added in at an effector cell: target cell ratio of 2:1 using the number of target cells seeded the day prior. CD8+ T cells and bispecific TCRs were co-cultured with target cells for 48 hours. Plates were gently spun down to collect cells at the bottom of the plate and the clarified supernatants collected. Amount of interferon gamma (IFNγ) was quantified using an ELISA kit and a human IFNγ protein standard following manufacturer’s instructions. Briefly, plates were coated with an anti IFNγ capture antibody, washed, and protein standard or diluted test supernatants were added to the plate and incubated overnight at 4° C. Plates were washed, and a secondary biotinylated detection antibody was added to the plate for one hour at room temperature. Plates were washed, streptavidin HRP loaded, washed again, and developed using TMB for 10 min. Plates were stopped in acid and absorbance was measured at 450 nm. The amount of IFNγ in test samples was quantified using a calibration curve generated using known amounts of IFNγ protein standard. The concentration of TCR bispecific required to generate half maximal IFNγ production was calculated using Graphpad Prism and reported as EC50 (
Tumor cells were seeded onto 96 well tissue culture treated flat bottom plates and allowed to adhere overnight. The following day, culture medium was removed from the cells, and replaced with medium containing serially diluted bispecific TCRs, and CD8+ T cells. Masked bispecific TCRs were treated with protease when indicated. CD8+ T cells were added in an effector cell: Target cell ratio of 2:1 using the number of target cells seeded the day prior. CD8+ T cells and TCR bispecifics were co-cultured with target cells for 48 hours. Plates were gently spun down to collect cells at the bottom of the plate and the clarified supernatants collected. The relative amount of lactate dehydrogenase (LDH) present in the supernatants was quantified using the Promega LDH-Glo assay kit following the manufacturer’s instructions. Briefly, supernatants were diluted 500× in LDH storage buffer (200 mM Tris, 10% glycerol, 1% BSA, pH 7.3) of which 50 uL was added to white opaque 96 well assay plates. LDH detection enzyme mix was prepared with reductase substrate and added in equal volume of 50 uL per well. Luminescence was then measured on a luminometer. Signals were corrected for spontaneous LDH release from tumor cells alone and T cells alone. Maximum tumor cell lysis was measured in cells treated with lysis buffer for 45 min prior to supernatant harvest. Percent tumor cell lysis was calculated as corrected signal divided by corrected target maximum signal. The concentration of TCR bispecifics required to generate half maximal percent tumor cell lysis was calculated in Graphpad Prism and reported as EC50 (
Masked bispecific TCRs in vivo efficacy is evaluated in human tumor bearing immunodeficient mice. Female NOD/SCID (NSG) mice (N=10 per group were) are subcutaneously implanted with a mixture of human donor PBMCs and target tumor cells. HCT116 (5 × 106 viable cells per inoculum) are injected s.c. together with PBMCs from healthy human donors at an E:T cell ratio of 1:2 in the right dorsal flank of female NSG mice. As indicated, different conditions are tested for their influence on tumor outgrowth: vehicle alone, non-masked bispecific TCR, and various masked bispecific TCRs. TCRs are injected every day for 10 days starting the day of implantation. External calipers are used to measure tumor volumes twice weekly for five weeks.
BALB/c mice weighing 25-30 g are dosed intravenously with a single dose at 3 mg/kg bodyweight. Serial blood samples (35 uL) are collected via lateral tail sampling at 0.5, 4, 7, 24, 48, 72 and 96 h (anti-albumin binding fusion containing constructs) and at 0.033, 0.25, 0.75 and 1.66 h (non anti-albumin binding fusion constructs). To obtain sera, blood samples are centrifuged for 5 min at 10,000 rpm at room temperature. The presence of bispecific TCR in mouse serum samples are analyzed in a quantitative ELISA format. Bispecific TCR in serum is captured on CD3 coated plates, followed by detection using an anti-TCR variable beta antibody that recognizes the beta chain of bispecific TCR of interest. A final horseradish peroxidase conjugated secondary antibody is then added followed by development using TMB. ELISAs are stopped in acid and measured for OD 450 nm. Amount of bispecific TCR in mouse serum is calculated relative to a standard curve using relevant bispecific TCR spiked into mouse serum at known concentrations. Standard pharmacokinetic parameters are calculated based upon these quantitative measurements.
Cynomolgus monkeys are dosed intravenously with a single dose at 0.3 mg/kg bodyweight. Serial blood samples are collected at 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours and 8 hours post dose, then daily for three additional days, followed by weekly until study termination. The presence of bispecific TCR in serum samples is analyzed in a quantitative ELISA format. Bispecific TCR in cyno serum is captured on CD3 coated plates, followed by detection using an anti-TCR variable beta antibody that recognizes the beta chain of bispecific TCR of interest. A final horseradish peroxidase conjugated secondary antibody is then added followed by development using TMB. ELISAs are stopped in acid and measured for OD 450 nm. Amount of bispecific TCR in cyno serum is calculated relative to a standard curve using relevant bispecific TCR spiked into cyno serum at known concentrations. Standard pharmacokinetic parameters are calculated based upon these quantitative measurements.
Expression plasmids encoding the TCR alpha and beta chains or the TCR gamma and delta chains were produced using standard molecular biology techniques. Plasmids were transformed into chemically-competent cells and grown overnight at 37° C. Protein expression was induced by the addition of Isopropyl -D-1 -thiogalactopyranoside (IPTG) to 1 mM and bacteria were grown for a further 3 hours at 37° C. Bacteria were harvested by centrifugation at 4000 × g for 15 minutes and lysed in a protein extraction reagent containing DNAse. Lysis proceeded for 1 hour at room temperature with agitation before inclusion bodies were harvested by centrifugation at 10,000 × g for 5 minutes. Pellets were washed twice with a detergent buffer containing 1% Triton X100 and resuspended in a buffered saline solution.
Soluble TCRs were prepared by dissolving alpha and beta inclusion bodies in 6 M guanidine- HCI containing 10 mM dithiothreitol and incubating at 37° C. for 30 minutes. Samples were diluted into 1ml urea folding buffer (5 M urea; 0.4 ML-arginine; 0.1 M Tris-CI, pH 8.1; 2 mM EDTA; 6.5 mM -mercapthoethylamine; 1.9 mM cystamine) and dialysed against eight volumes of water overnight at 4° C., followed by dialysis for a further 24 hours in eight volumes of 10 mM Tris (8.1), with one buffer change. Dialysate (30 ml) was concentrated to 1 ml. Concentrated protein was diluted to 5 ml in phosphate-buffered saline and concentrated to 0.5 ml.
The resulting soluble TCRs were tested for their biochemical integrity by three methods. First, portions of the resulting TCRs were tested by heating in loading buffer in the presence or absence of reducing agent. Several concentrations of total protein were then examined by SDS- PAGE analysis to insure consistent results (
TCR fusion constructs were either produced in E. coli cells similar to methods described above or transiently produced in suspension mammalian HEK293 cells according to known methods.
A representative example of the preparation of a MAGE-A3 TCR (TCR-1) is shown in Fig. TBD.
Kinetic binding of soluble TCR to pMHC was measured using a ForteBio Octet RED96 instrument. Biotinylated pMHC was first captured on streptavidin biosensors. Sensors were quenched using excess biocytin and then baselined in buffer. TCR was titrated in a 2-fold dilution series starting from 50 nM and was associated onto the pMHC loaded biosensor. Association signal was monitored in real-time. Biosensors were then transferred to buffer and the dissociation of TCR was measured in real-time. Data was background corrected, fit to a classic 1:1 binding model, and used to calculate kinetic rate constants.
A representative example of the binding verification of a prepared MAGE-A3 TCR (TCR-1) is shown in
Peptides with the ability to bind to a T cell receptor (TCR) of interest are identified by biopanning a phagemid-display libraries of candidate peptides (
Biopanning of m13 phagemid p3 displayed peptide libraries is performed with biotin conjugated TCR immobilized on streptavidin coated paramagnetic beads. Following binding to the target at pH 7.4 and subsequent washing steps, specifically bound phage are recovered by elution at pH 2.2, or at pH 11.0. Though individual clones can be sequenced or tested after a single round, enrichment of specific binding clones is typically accomplished by 2-4 rounds of successive biopanning and amplification. Following the enrichment of pools, phage biopanning phage pools are infected into TG1 cells and plated out on LB-ampicillin/agar plates for subsequent clonal isolation, DNA sequencing, and characterization (
For hit identification, individual colonies were grown in 96-deep well plates for 2-4 hours and infected with helper phage to produce peptide displayed phagemid following an overnight growth. The next day the deep well plates were centrifuged to separate the soluble phagemid from the E. coli cells. The phagemid containing supernatants were then combined with PBS-Tween 20 (0.05%) + BSA (1%) pH neutral blocking buffer and incubated in previously TCR coated and blocked wells. After binding at 4° C. the plates were washed and specifically bound phage were detected by anti-m13 HRP conjugated antibodies using standard TMB-based chromogenic ELISA procedures (
Phagemid peptide clones were next tested to determine whether they bound within the cognate pMHC binding space of the TCR, by target-based competition assay. We prepared biotin conjugated TCR immobilized and blocked 96-well ELISA plates similar to above. Next we added cognate pMHC to the well to block the antigen binding site. After a brief incubation period, phagemid supernatants were next added to the wells. Following an incubation at 4° C. the plates were washed and specifically bound phage were detected by anti-ml3 HRP conjugated antibodies using standard TMB-based chromogenic ELISA procedures. Phagemid clones binding within the pMHC binding pocket of the TCR would be blocked and be identified by a decreased ELISA signal, compared to a well lacking previous antigen blockade. A representative example of the phagemid competition ELISA is seen in
Peptides expressed on clonal phage that exhibit TCR specific binding and inhibition were chosen for further characterization. Exemplary phagemid peptides that bind to MAGE-A3 TCR (TCR-1) are listed in Table 2A and selected for peptide synthesis. Peptides selected for additional evaluation were first chemically synthesized and then evaluated for TCR binding, pMHC competition, and TCR selectivity.
Peptides were synthesized via standard peptide chemistry. Peptides were synthesized as linear or cyclic as appropriate. A C-terminal linker consisting of Gly4Ser (SEQ ID NO: 139), PEG4, and Lys(Biotin) was added to the phagemid peptide sequence identified from panning and DNA sequencing. The C-terminal acids were also capped via amidation. Peptides were purified by HPLC to ≥95% purity and verified by liquid chromatography assisted mass spectrometry (LC-MS). Peptides were lyophilized prior to dissolution in DMSO.
Synthetic peptides were initially screened for binding to their panning target. As an example, peptides listed bind to MAGE-A3 TCR, TCR-1. Peptide binding was evaluated using both kinetic measurements via Bio-layer Interferometry (BLI) or equilibrium measurements using enzyme linked immunosorbent assays (ELISAs).
BLI based kinetic binding of TCR to peptides was measured using a ForteBio Octet RED96 instrument. Biotinylated peptides were first directly captured on streptavidin biosensors. Sensors were quenched using excess biocytin and then baselined in buffer. A dilution series of TCR was made and associated onto the peptide loaded biosensor. Association and dissociation signals were monitored in real-time. Signals were fit to a 1:1 binding model in order to derive binding constants, kon and koff, as well as KD. Exemplary kinetic binding sensorgrams are shown in
Peptide binding was also examined in an ELISA format. Biotinylated peptides were captured on neutravidin coated plates. TCR was then prepared in a half-log dilution series starting from 10 uM and titrated onto the peptide captured plates. A secondary horse radish peroxidase (HRP) antibody conjugate that recognizes 6x histidine tag (SEQ ID NO: 99) was used to detect bound TCR-1. The concentration of TCR required to observe half maximal binding signal (EC50) was then calculated using Graphpad Prism. Example binding of MAGE-A3 TCR (TCR-1) to captured peptide or MAGE-A3 pMHC is shown in
Peptides that bind do not necessarily exhibit desired function. For a peptide to function as a mask it must by definition inhibit the TCR of interest from binding its cognate pMHC. Therefore, peptides that bind the example MAGE-A3 TCR, TCR-1, were progressed into competitive inhibition studies designed to test the inhibitory function of each peptide. Multiple peptides were evaluated for masking function via BLI and ELISA.
Dose dependent kinetic inhibition of TCR-1 binding to MAGE-A3 pMHC using the identified peptide binders was measured via BLI using a ForteBio Octet RED96 instrument. First, biotinylated pMHC was captured on streptavidin biosensors. Sensors were quenched using excess biocytin and then baselined in buffer. Inhibitory peptide was titrated in a two fold dilution series starting from 100 uM and pre-incubated with a constant concentration of 50 nM TCR. Peptide and TCR mixtures were then associated onto the pMHC loaded biosensor. Zero concentration of inhibitory peptide or zero concentration of TCR were used as controls. Association and dissociation signals were monitored in real-time. The maximal association signal was normalized from 100% (0 uM inhibitory peptide control) to 0% (0 nM TCR control) and plotted versus log-scale inhibitory peptide concentration. Graphpad Prism was used to calculate the inhibitory concentration of peptide required to achieve 50% maximal signal (IC50) (Table 2).
Inhibition of TCR binding to its cognate pMHC was also measured in an ELISA format. Biotinylated pMHC was captured on neutravidin coated plates, quenched using excess biocytin, and washed. Inhibitory peptide was titrated in a half-log dilution series starting from 100 uM and pre-incubated with a constant concentration of 1 nM TCR. Inhibitory peptide and TCR mixtures were then incubated on the pMHC captured plates. A secondary HRP antibody conjugate that recognized the histag of the TCR was then used to detect the plate bound TCR. The ELISA signal was normalized from 100% (0 nM inhibitory peptide control) to 0% (0 nM TCR control) and plotted versus log-scale inhibitory peptide concentration (
Potent inhibitory peptides were tested for TCR specific binding. For example, the TCR specificity of Peptide-5 was tested using closely related TCRs, TCR-2, TCR-3, and TCR-4, of the same family that also recognize the cognate MAGE-A3 pMHC. This family of TCRs differ in sequence by point mutations in the CDR domains know to contribute to binding affinity and specificity for cognate MAGE-A3 pMHC. Similar to previous kinetic binding experiments, binding of TCRs to Peptide-5was evaluated by BLI at a loading concentration of Peptide-5 that saturated the streptavidin biosensor. TCRs were associated at the extreme concentration of 100 uM onto the inhibitory peptide loaded biosensor. In parallel, 100 uM TCRs were associated on a blank control sensor to establish background signal related to non-specific binding of the TCRs. Association and dissociation signals were monitored in real-time. While TCR-1 readily bound Peptide-5 with a high signal well above background, closely related A3a TCRs do not as their signal was identical to that of background TCR binding to a blank sensor (
Given the selective binding of Peptide-5and TCR-1 relative to TCR-2, TCR-3, and TCR-4 TCRs, it is likely that Peptide-5interacts with residues present in the CDR2α, CDR3α, or CDR3β domains of TCR-1. Since TCR CDR domains form key binding interactions with cognate pMHC, binding of Peptide-5 at or near the CDR2 and CDR3 domains could explain why Peptide-5 blocks TCR-1 recognition of MAGE-A3 pMHC. In order to understand the key residues within Peptide-5 that drive binding interaction with TCR-1, all residues of Peptide-5 were mutated one by one to alanine. Not surprisingly, Ala scan peptides revealed key residues corresponding to the consensus sequence identified from phage panning, “CXXXYDXXFC” (SEQ ID NO: 120), required for TCR-1 binding (
Ala mutated versions of Peptide-5 were evaluated in kinetic and equilibrium binding and inhibition studies as previously described in Example 22.
Peptide-5 was fused to the N-terminus of the alpha or beta chain of TCR-1 via a flexible linker to assess functional masking. In some instances, the flexible linker was of different lengths and incorporated of a tumor protease specific substrate between the inhibitory peptide mask (Peptide-5) and the TCR alpha or beta chain. The proteolytic substrate within the linker enables tumor actuated binding that requires tumor restricted protease activity for therapeutic activation. Masked TCRs were produced and qualified as described in Example 19.
Masked TCR constructs were designed using inhibitory peptide Peptide-5 fused to the N-terminal alpha or beta chain of the parental TCR, TCR-1, using a protease cleavable linker (
Recognition of Peptide-5 TCR fusions for cognate pMHC were evaluated in kinetics based BLI binding experiments similar to those described in Example 20. In some instances, TCRs were pre-treated with urokinase (uPa) or matriptase (MTSP1) where indicated. Briefly, biotinylated MAGE-A3 pMHC was loaded onto BLI streptavidin biosensors, quenched with excess biocytin and baselined in buffer. TCRs were then associated onto the pMHC loaded sensors at a single concentration of 50 nM. Sensors were transferred back to buffer to measure dissociation. Kinetic binding signals suggest that while the parental non-masked TCR-1 control exhibits full binding to its cognate pMHC, fusion of Peptide-5 to the N-terminal beta chain, TCR-8 and TCR-9, or the N-terminal alpha chain, TCR-10 and TCR-11, of the TCR completely blocked the TCR recognition of MAGE -A3 pMHC (
Recognition of Peptide-5 TCR fusions for cognate pMHC were also evaluated in equilibrium based ELISA binding experiments similar to those described in Example 20. In some instances the TCRs were treated with protease where indicated. Briefly, biotinylated pMHCs were captured on neutravidin coated plates followed by the addition of titrated TCRs. Plates were then incubated for a short time followed by a wash. A secondary anti-histag HRP conjugate antibody was used to detect bound TCR to the plate. The ELISA data in
The BLI and ELISA data for masked TCRs suggests that the masking ability of Peptide-5 was maintained regardless of fusion to the alpha or beta chain of the TCR using different length linkers. Likewise, the Peptide-5 peptide fusion was able to hinder TCR binding to cognate MAGE-A3 pMHC as well as the off target Titin pMHC known to contribute to toxicity in the clinic.
Importantly however, fusion of MAGE-A3 antigen peptide (EVDPIGHLY (SEQ ID NO: 45); Peptide 34) to TCR-1 TCR in the identical formats with and without the protease cleavage site, TCR-6 and TCR-7 respectively, did not inhibit the TCR kinetic binding of MAGE-A3 pMHC (
In order to further elucidate the specific interactions of Peptide-5 and MAGE-A3 TCR, the high resolution crystal structure of TCR-10 with Peptide-5 fused at the N-terminal alpha chain of MAGE-A3 TCR was determined via x-ray diffraction (
The PACT Premier crystallization screen was set at 22C with protein TCR-10 at 9 mg/ml. Several conditions yielded crystal hits. The PEG + NaNO3 condition was selected for optimization (15.5%peg3350, 0.2 M NaNO3). Several crystals were selected and frozen in different cryoprotectant for data collection. Cryoprotection with higher PEG concentration (25%) and the addition of 20% glycerol provided the best data set. A complete dataset was collected at the Advanced Light Source in Berkeley, CA USA on BCSB beamline 5.0.2 from a single crystal. Data was processed using XDS software and scaled with the CCP4 suite to a resolution of 2.3 Å resolution. The space group is P21 with cell dimension: 64.45 114.53 80.70 90 113 90 and 2 molecules in the asymmetric unit. Structure was solved by molecular replacement (MR) with Phaser (CCP4) using chains D and E of the 5BRZ structural model (100% sequence homology with the target sequence). The MR search provided a unique solution with an initial Rfactor of 48.6%. Automatic fitting followed by manual rebuilding of the model and refinement in Refmac5 decreased the Rfactor/Rfree. Clear density was visible for the cyclic peptide between the α and β TCR subunits (
Successfully masked, Peptide-5 fused TCRs were progressed into anti-CD3 bispecific T cell engager construction. Various constructs were made by fusing an anti-CD3 single-chain variable fragment (scFv) to the TCRs (
MAGE-A3 TCR anti-CD3 bispecifics were constructed by fusion of the anti-CD3 scFv at the N-terminus of the TCR alpha or beta chain with or without Peptide-5 fused to the N-terminus of the alternative TCR chain. Additional TCR bispecifics were explored with the anti-CD3 scFv fused to the C-terminal Anti-CD3 scFv was derived from published antibody UCHT1 and separated from the TCR chain by a short Gly4Ser linker (SEQ ID NO: 139). Peptide-5 on the other hand was fused to the alternative TCR chain at the N-terminus using the previously described linker (Example 24) containing the protease recognition sequence, LSGRSDNH (SEQ ID NO: 4), or a non-cleavable linker composed of GlySer repeats. Masked MAGE-A3 TCR anti-CD3 bispecific molecule designs are illustrated in
TCR bispecifics were first characterized for their ability to recognize cognate pMHC in BLI based binding experiments (
TCR bispecifics were also characterized for their ability to recognize cognate pMHC in ELISA based binding experiments (
All masked TCR bispecific constructs utilizing Peptide-5 N-terminal fusions required protease treatment and a cleavable linker in order to observe potent cognate pMHC recognition similar to the non-masked TCR bispecific controls. By comparison, replacement of LSGRSDNH (SEQ ID NO: 4) protease substrate sequence with non-cleavable GlySer repeats (TCR-19) eliminated the observed protease dependent binding of TCR bispecifics.
TCR bispecifics were further characterized for their ability to form a ternary complex on the surface of human CD8+ T cells via binding of cellular CD3 and subsequently stained using fluorescently labeled MAGE-A3 pMHC tetramer (
Briefly, 100,000 T cells per well were distributed in a 96 well plate, washed cold, followed by incubation with the indicated concentration of non-masked, masked, or protease treated TCR bispecifics. Cells were incubated cold for a few hours, then washed with cold buffer, followed by a short incubation with cold MAGE-A3 pMHC tetramer formed using fluorescently labeled streptavidin. Cells were washed cold, resuspended in cold buffer, and run on a Novocyte flow cytometer. Scattering signals were gated in the typical fashion to exclude debris of incorrect cellular shape and size. Mean fluorescent intensity was normalized, plotted against TCR concentration, and Graphpad Prism 6.0 was used to calculate the concentration of TCR bispecific required to achieve 50% maximal signal (EC50).
In general, all non-masked or protease treated TCR bispecifics were able to form a ternary complex in a dose dependent fashion on the surface of human CD8+ T cells. Closer examination suggests that N-terminal fusion of the anti-CD3 scFv on the TCR beta chain was most efficient and had the lowest EC50 for ternary complex formation. The masked TCR bispecifics had little to no observable binding at the highest concentrations tested. For example, the non-masked control, TCR-14, as well as urokinase treated TCR-15 bound to human CD8+ T cells with low nanomolar EC50s, whereas the fully masked TCR-15 and TCR-19 signals remained at background levels despite testing up to 3 uM. The flow cytometry data demonstrated a >300× shift in the ability of TCR-15 to bridge soluble MAGE-A3 pMHC tetramer and CD3 on the surface of human CD8+ T cells (
TCR bispecifics were next evaluated in functional in vitro tumor cell killing and related T cell activation studies (
In general, TCR bispecifics required >500× higher concentrations in order to register a 50% maximal cytotox or IFNy signal relative to those activated by protease. For example, parental non-masked TCR bispecific TCR-14, performed similarly to protease treated TCR bispecific with a cleavable linker substrate, TCR-15, indicating full activation post proteolysis. The differences in functional potency between cleavable masked (TCR-15) and non-cleavable masked (TCR-19) TCR bispecifics in the A375 and HCT116 cytotoxicity assays likely indicated differential proteolytic activity from the target cells (
S Bispecific T cell engagers typically have poor pharmacokinetics (PK) properties. We hypothesized that adding a half-life extension molecule in tandem with the proteolytically cleavable mask would exhibit crossover PK defined by a long half-life in systemic circulation but fast clearance after mask and PK extender cleavage at the tumor site due to specific proteolytic activity. Thus these cross over PK molecules would have an additional safety switch preventing accumulation in healthy tissue once activated at the tumor site.
The best configurations of masked TCR bispecifics were progressed into analogous crossover PK construction. Various constructs were made by fusing an anti-albumin single domain antibody (SDA) in tandem to the Peptide-5mask separated by a short GlySer linker. The SDA and Peptide-5 tandem mask was fused to the TCR bispecifics using the same cleavable or non-cleavable linkers previously described (Example 26). Functional binding, tumor cell killing, and T cell activation of the masked TCR anti-CD3 bispecifics were then evaluated. Masked MAGE-A3 TCR anti-CD3 bispecifics were tested against MAGE-A3 positive tumor cells lines A375 and HCT16 as well as the Titin positive human skeletal muscle myoblasts (HSMM). In addition, mouse and cynomologus monkey PK was evaluated for the constructs.
Generalized dual mask and single mask TCR bispecific molecule designs are shown in
Anti-albumin single domain antibody (SDA) was tethered in tandem to the TCR mask attached to the core TCR bispecific structure to form a complete TCR bispecific molecule of various formats (
TCR bispecific molecules were evaluated for their ability to bind the cognate MAGE-A3 pMHC via BLI. TCR bispecific binding kinetics of MAGE-A3 pMHC was measured before and after protease treatment in the presence of bovine albumin (BSA), human albumin (HSA), mouse serum (MS), bovine serum (BS), cynomolgus monkey serum (CS), or human serum (HS). Briefly, biotinylated MAGE-A3 pMHC was loaded onto streptavidin coated biosensors, quenched in biocytin, and baselined in buffer containing appropriate albumin or serum. The concentration of albumin or serum used was at a level expected to saturate the TCR bispecific albumin binding site. TCR BISPECIFIC molecules were treated with active matriptase (MTSP1) or urokinase (uPa) where indicated. TCR bispecific molecules diluted in albumin or serum supplemented buffer to 50 nM or 100 nM were then associated onto the MAGE-A3 pMHC loaded biosensors. Sensors were then transferred to the appropriate albumin or serum supplemented buffer where TCR bispecific molecules then dissociate from the sensors. Association and dissociation rates were measured in real time using an OCTET RED96 instrument. Example sensorgrams are shown in
TCR bispecifics were also characterized for their ability to recognize cognate pMHC in ELISA based binding experiments (
TCR bispecifics were further characterized for their ability to form a ternary complex on the surface of human CD8+ T cells via binding of cellular CD3 and subsequently stained using fluorescently labeled MAGE-A3 pMHC tetramer (
Briefly, 100,000 T cells per well were distributed in a 96 well plate, washed cold, followed by incubation with the indicated concentration of non-masked TCR bispecific (TCR-14), TCR bispecifics, or protease treated TCR bispecifics in human albumin buffer. Cells were incubated cold for a few hours, then washed with cold buffer, followed by a short incubation with cold MAGE-A3 pMHC tetramer formed using fluorescently labeled streptavidin. Cells were washed cold, resuspended in cold buffer, and run on a Novocyte flow cytometer. Scattering signals were gated in the typical fashion to exclude debris of incorrect cellular shape and size. Mean fluorescent intensity was normalized, plotted against TCR bispecific concentration, and the concentration of TCR bispecific required to achieve 50% maximal signal (EC50) was calculated in GraphPad Prism 6.0. In general, treatment of TCR bispecific molecules with protease enabled potent ternary complex formation equivalent to the non-masked TCR bispecific controls with low nanomolar EC50s. In contrast, minimal ternary complex formation could be detected using TCR bispecific molecules at the highest concentrations tested. Data suggests functional TCR bispecific ternary complex formation requires specific protease activity.
TCR bispecifics were evaluated in functional in vitro tumor cell killing and related T cell activation studies using the MAGE-A3 positive A375 (
TCR bispecific pharmacokinetics were determined in male 6-8 week old Balb/c mice. Briefly, animals were assessed for their general health by a member of veterinary staff or other designated personnel upon arrival and allowed to acclimate for at least 3 days before study commencement. Animals were group housed during acclimation and throughout the study. The animal room environment was controlled according to facility operation with temperature between 20 to 26° C. and relative humidity between 30 and 70%. Lighting was controlled on a 12 hour light dark cycle. Animals were fed certified pellet diet (Certified Rodent Diet #5002, LabDiet). Purified water (reverse osmosis) was provided to the animals ad libitum. Periodic analyses of water quality was performed.
Concentrated test articles were diluted to appropriate dosing volume in sterile phosphate buffered saline and administered intravenously via tail vein at 10 mL/kg. Dose volume was determined individually by body weight obtained immediately prior to dosing for each animal.
Blood samples were collected before and after dosing at the indicated time points. For each timepoint a subset of 3 mice were euthanized by carbon dioxide inhalation. Following confirmation of death, blood samples were collected through the inferior vena cava using a syringe. The blood samples were placed in pre-labeled serum separation tube and incubated for 30 min before being processed to serum. After 30 min, the blood samples were centrifuged cold at 3000×g for 10 min to separate clots from serum. The serum supernatant was harvested and stored frozen prior to analysis.
The concentration of TCR bispecifics in serum samples was determined by ELISA. Briefly, CD3 was captured on neutravidin coated plates, washed, blocked, quenched with biocytin, and washed again. Standard dilutions of TCR bispecifics in mouse serum were used to generate a calibration curve to which animal PK test samples could be compared. Standards and test samples were added to the plate and incubated cold overnight. Several different dilutions of test samples were used to make sure signals landed within appropriate dynamic range of the standard curve. Plates were washed and incubated with a FITC labeled anti-TCR secondary antibody for a brief time followed by another wash. A tertiary anti-FITC HRP conjugate antibody was used to detect bound TCR bispecifics. Plates were washed, developed, and stopped using standard ELISA techniques. Standard curves plotting absorbance at 450 nm versus known TCR bispecifics concentration were used to calculate the concentration of unknown test articles in each mouse PK serum sample. Concentration of TCR bispecifics were plotted versus time and fit to a standard two stage distribution and elimination pharmacokinetic model. The calculated pharmacokinetic parameters for TCR bispecific TCR-14 and TCR bispecific TCR-20, from Balb/c mice are shown in
TCR bispecific pharmacokinetics were determined in naive male cynomolgus monkeys weighing 2-3 kg. Briefly, two group housed monkeys were used per dosing group and allowed to acclimate to their surroundings prior to dosing. Animals were sedated with Ketamine HCL 10-20 mg/kg IM prior to dosing and bleeding. Concentrated test articles were diluted in sterile phosphate buffered saline and administered to animals at a quantity relative to the animals’ mass in kg. The dose for each test article was 0.2 mg/kg administered intravenously at 1 mL/kg dosing volume. For dosing, the left and right limbs were clipped and prepped with alcohol. The saphenous vein was identified and a standard catheter was placed for IV bolus infusion (in either the left or right limb). The test article dosing solution was attached to the catheter via syringe and the bolus infusion occurred via manual compression of the syringe.
For blood collections, animals were sedated using ketamine, the femoral triangle was prepared, and blood was collected from the femoral vein using a 22G 1.5 inch needle, vacutainer sheath, and collection tube. Following venipuncture, manual compression of the vein was maintained until hemostasis was achieved. Blood collections were based on weight of the animals and did not exceed AGI maximum bleeds as set forth by IACUC. Blood was collected in SST tubes and processed to serum. Serum samples were frozen prior to analysis.
The concentration of TCR bispecifics in serum samples was determined by ELISA. Briefly, CD3 was captured on neutravidin coated plates, washed, blocked, quenched with biocytin, and washed again. Standard dilutions of TCR bispecifics in cyno serum were used to generate a calibration curve to which animal PK test samples could be compared. Standards and test samples were added to the plate and incubated cold overnight. Several different dilutions of test samples were used to make sure signals landed within appropriate dynamic range of the standard curve. Plates were washed and incubated with a FITC labeled anti-TCR secondary antibody for a brief time followed by another wash. A third anti-FITC HRP conjugate antibody was used to detect bound TCR bispecifics. Plates were washed, developed, and stopped using standard ELISA techniques. Standard curves plotting absorbance at 450 nm versus known TCR bispecifics concentration were used to calculate the concentration of test articles in each PK serum sample. Concentration of TCR bispecifics were plotted versus time and fit to a standard two stage distribution and elimination pharmacokinetic model. The calculated pharmacokinetic parameters for TCR bispecific TCR-14 and TCR bispecific TCR-20, from cynomolgus monkey are shown in
While preferred embodiments of the present disclosure have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the disclosure. It should be understood that various alternatives to the embodiments of the disclosure described herein may be employed in practicing the disclosure. It is intended that the following claims define the scope of the disclosure and that methods and structures within the scope of these claims and their equivalents be covered thereby.
This application claims the benefit of U.S. Provisional Application No. 62/858,254, filed Jun. 6, 2019, and U.S. Provisional Application No. 62/978,662, filed Feb. 19, 2020, which applications are incorporated herein by reference.
Filing Document | Filing Date | Country | Kind |
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PCT/US2020/036489 | 6/5/2020 | WO |
Number | Date | Country | |
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62978662 | Feb 2020 | US | |
62858254 | Jun 2019 | US |