EPITOPES IN THE RNA RECOGNITION MOTIF 1 (RRM1) OF TDP-43 AND MISFOLDING-SELECTIVE ANTIBODIES THERETO
The disclosure pertains to conformational epitopes in TDP-43, antibodies thereto and methods of making and using immunogens and antibodies specific thereto.
This application is a division of U.S. patent application Ser. No. 16/616,832, filed Nov. 25, 2019, which is a National stage entry of International Application No. PCT/CA2018/050634, filed May 30, 2018, which claims the benefit of 35 U.S.C. § 119 based on the priority of U.S. Provisional Patent Application Nos. 62/512,647, filed May 30, 2017; 62/570,582, filed Oct. 10, 2017; and 62/595,866, filed Dec. 7, 2017; each of these applications being incorporated herein in their entirety by reference.
INCORPORATION OF SEQUENCE LISTINGA computer readable form of the Sequence Listing “P51866US04_ST25” (29,299 bytes), submitted via EFS-WEB and created on Dec. 1, 2021, is herein incorporated by reference.
FIELDThe present disclosure relates to TDP-43 epitopes and antibodies thereto and more specifically to conformational TDP-43 epitopes that are predicted to be selectively accessible in misfolded TDP-43, and related antibody compositions.
BACKGROUNDTransactive response (TAR) element DNA binding protein of 43 kDa (TDP-43), is a 414 amino acid protein, and is comprised of an N-terminal ubiquitin like domain (NTD, residues 1-102), two RNA recognition motifs (RRMs) composed of residues 106-177 (RRM1), and residues 192-259 (RRM2), and a C-terminal domain (CTD, residues 274-414). The NTD contains a nuclear localization signal (NLS, residues 82-98). RRM2 includes a nuclear export signal (NES) from residue 239 to 250.
TDP-43 is predominantly a nuclear protein that plays a central role in RNA metabolism. TDP-43 has become a focal point of research in the amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) disease spectrum, since pathogenic inclusions within affected neurons can contain post-translationally modified TDP-43. The CTD of TDP-43 is particularly relevant to disease, as it is where nearly all familial ALS/FTD-associated mutations are found in TDP-43.
Other mutations include D169G which is located in RRM1 between beta strands 4 and 5, A90V which is a mutation in the NLS region, and the mutations K263E and N267S which are in the linker between RRM2 and the C-terminal domain.
RRM1 and RRM2 have been structurally determined by NMR. For example, RRM1 is available in the Protein Data Bank (PDB), a database of atomic resolution three dimensional structural data, as PDB entry 41UF, while RRM2 is available as PDB entry 1WF0, and the NTD is available as PDB entry 2N4P.
The structure of 41UF is reported in Kuo et al. [1]. The structure of 1WF0 is reported in He et al [2]. The structure of 2N4P is reported in Mompean et al. [3].
TDP-43 was found to be hyperphosphorylated, ubiquitinated, and fragmented in neuronal inclusions of patients with both sporadic and familial forms of ALS and FTD [4].
Aggregates of TDP-43 have now been found in nearly all (approx. 97%) cases of ALS and roughly half (approx. 45%) of the cases of FTD. TDP-43 is one of the main components of the cytoplasmic inclusions found in the motor neurons of ALS patients.
Pathological precursors of TDP-43 inclusions may have concentration far below that of functional TDP-43. The low concentration of misfolded TDP-43 makes this target elusive. Antibodies or drugs targeting healthy TDP-43 could be fatal for the cell. TDP-43 is an RNA regulatory protein that is essential for embryonic development [5].
Antibodies that bind TDP-43 have been described.
WO2012174666 titled METHODS FOR THE PROGNOSTIC AND/OR DIAGNOSTIC OF NEURODEGENERATIVE DISEASE, METHODS TO IDENTIFY CANDIDATE COMPOUNDS AND COMPOUNDS FOR TREATING NEURODEGENERATIVE DISEASE discloses methods for diagnosing neurodegenerative diseases such as ALS and FTD through assessing the interaction between TDP-43 and NF-κB p65 using an anti-TDP-43 antibody.
WO2016086320 titled TDP-43-BINDING POLYPEPTIDES USEFUL FOR THE TREATMENT OF NEURODEGENERATIVE DISEASES disclose antibodies that binds to the RRM1 domain of TDP-43 to disrupt its interaction with NF-κB for the treatment of ALS and FTD.
Antibodies that preferentially or selectively bind misfolded TDP-43 over natively folded TDP-43 are desirable.
SUMMARYDescribed herein is a conformational epitope in TDP-43 comprising and/or consisting of residues TTEQ (SEQ ID NO: 1) or a part thereof, and antibodies thereto. The epitope is identified as an epitope that may be selectively exposed in the misfolded species of TDP-43, in a conformation that distinguishes it from that in the native protein.
An aspect includes a compound, optionally a cyclic compound comprising: a TDP-43 peptide comprising 1) TTE 2) TTEQ (SEQ ID NO:1); 3) TEQ or a part thereof and up to 6 TDP-43 contiguous residues, and a linker, wherein the linker is covalently coupled to the TDP-43 peptide N-terminus residue and/or the peptide C-terminus residue, wherein at least one amino acid in the TDP-43 peptide is an alternate conformation than T, E, and/or Q in a corresponding linear and/or native TDP-43.
In an embodiment, the TDP-43 peptide is selected from TTEQ (SEQ ID NO: 1), TTE, TEQ, KTTE (SEQ ID NO: 10), KTTEQ (SEQ ID NO:12). TEQD (SEQ ID NO:8) or TTEQD (SEQ ID NO: 9), optionally wherein the cyclic compound is a compound selected from any one of SEQ ID NOs: 2, 3, 13, 22-39, and 42-44.
In an embodiment, the cyclic compound is selected from a cyclic structure described herein, preferably wherein the cyclic compound has a sequence selected from any one of SEQ ID NOs: 2, 3, 13, 22-39, and 42-44, more preferably SEQ ID NOs: 2, 3, 22, 23, 28, 29, 30, 31, 32, 33, 34, 35 and 42, optionally a sequence selected from any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34, 35 and 42 or a sequence selected from SEQ ID NO: 2, 3, 22, 23 and 42.
Also provided in another aspect is an immunogen comprising a cyclic compound described herein.
In an embodiment, the cyclic compound, is coupled to a carrier protein or immunogenicity enhancing component and/or formulated with an adjuvant.
In an embodiment, the carrier protein is bovine serum albumin (BSA) or the immunogenicity-enhancing component is keyhole limpet haemocyanin (KLH) and/or the adjuvant is selected from aluminum phosphate, aluminum hydroxide alum, monophosphoryl lipid A and QS21.
Another aspect provides an antibody that selectively binds an epitope in the TDP-43 peptide in the cyclic compound described herein compared to a corresponding linear compound and/or native TDP-43 polypeptide.
In an embodiment, the antibody selectively binds to a cyclic compound comprising 1) TTE, 2) TEQ, 3) TTEQ (SEQ ID NO:1), 4) KTTE, (SEQ ID NO: 10), 5) KTTEQ (SEQ ID NO:12). 6) TEQD (SEQ ID NO:8) and/or 7) TTEQD (SEQ ID NO: 9) compared to a corresponding linear compound and/or native TDP-43 polypeptide.
In another embodiment, the antibody is at least 2 fold, 3 fold, at least 5 fold, at least 10 fold or at least 20 fold, more selective for the cyclic compound compared to a corresponding linear compound and/or native TDP-43 polypeptide.
Also provided in an embodiment is an antibody that competes for binding misfolded TDP-43 or a cyclic peptide comprising the same or overlapping TDP-43 peptide as an antibody described herein, preferably one that shares at least 80%, or more sequence identity to a heavy chain and/or light chain variable region provided in Table 10.
In an embodiment, the antibody is raised or screened using a cyclic compound or an immunogen described herein.
In an embodiment, the antibody comprises a set of CDRs as described for example in Table 10.
In another embodiment, the antibody comprises a heavy chain variable region comprising: i) an amino acid sequence as set forth in Table 10; ii) an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to said heavy chain variable region sequence, wherein the CDR sequences are as set forth in said heavy chain variable region sequence, or iii) a conservatively substituted amino acid sequence of i); and/or wherein the antibody comprises a light chain variable region comprising i) an amino acid sequence as set forth in Table 10, ii) an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to said light chain variable region sequence wherein the CDR sequences are as set forth in said light chain variable region sequence, or iii) a conservatively substituted amino acid sequence of i); optionally wherein the heavy chain variable region amino acid sequence is encoded by a nucleotide sequence as set out in Table 10 or a codon degenerate or optimized version thereof and/or wherein the light chain variable region amino acid sequence is encoded by a nucleotide sequence as set out in Table 10 or a codon degenerate or optimized version thereof.
In another embodiment, the antibody is a monoclonal antibody, humanized antibody and/or a single chain antibody or a binding fragment of any of the foregoing.
Another aspect includes an immunoconjugate comprising the antibody described herein and a detectable label or a transport moiety, optionally a molecule that facilitates transport across the blood brain barrier and/or into a cell.
A further aspect provides a nucleic acid encoding the amino acid residues of the compound, immunogen, antibody or proteinaceous immunoconjugate described herein, or any part thereof.
Another aspect provides a cell expressing an antibody described herein, optionally wherein the cell is a hybridoma.
Another aspect is a composition comprising a cyclic compound, immunogen, antibody, immunoconjugate, nucleic acid or cell described herein, optionally with a diluent.
In an embodiment, the composition comprises a cyclic compound or immunogen described herein and an adjuvant.
In an embodiment, the adjuvant is aluminum phosphate, aluminum hydroxide aluminum hydroxide alum, monophosphoryl lipid A and/or QS21.
Also provided is a kit comprising one or more components described herein for example a compound, immunogen, antibody, immunoconjugate, nucleic acid, cell or composition described herein.
Also provides in another aspect is a method of making the antibody described herein, comprising administering an immunogen described herein or a composition comprising the immunogen to a subject and isolating antibody and/or cells expressing antibody selective or specific for the TDP43 peptide of the immunogen administered.
A further aspect includes a method of determining if a sample suspected of comprising misfolded TDP-43 polypeptides contains misfolded TDP-43 polypeptide the method comprising:
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- contacting the sample with an antibody described herein under conditions permissive for forming an antibody: misfolded TDP-43 polypeptide complex; and
- detecting the presence of any complex;
- wherein the presence of detectable complex is indicative that the sample may contain misfolded TDP-43 polypeptide.
In an embodiment, the sample comprises brain tissue extract, spinal cord tissue and/or CSF. In another embodiment, the sample is a human sample, optionally from a subject with or suspected of having ALS or FTD.
Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
An embodiment of the present disclosure will now be described in relation to the drawings in which:
Generation of misfolding-specific antibodies can be accomplished through the identification of targets on the misfolded TDP-43 peptide that are not present, or present to a lesser degree, in the native structure. Misfolding-specific epitopes need not differ in primary sequence from the corresponding segment in the native structure, however they should be conformationally-distinct in the context of the misfolded ensemble. That is, the epitope would present distinct conformations in terms of backbone and/or sidechain conformation in the misfolded ensemble that would not be present (or would be unfavourable) in the native structure.
Antibodies raised to native protein regions tend not to be selective for misfolded protein, and thus bind to native functional protein as well.
As described herein, to develop antibodies that may be selective for misfolded forms of TDP-43, the inventors sought to identify a region of TDP-43 sequence that is prone to disruption in the context of the native fold, and that may be exposed as well on the surface of the misfolded protein.
As described in the Examples, computational simulations, using molecular dynamics with standardized force-fields, were employed. An experimentally-validated structural model of the folded structure is globally biased away from its “native” conformation to be partially unfolded, using molecular dynamics, to yield one or more regions of contiguous primary sequence that are prone to be disordered upon an external challenge in an anomalous cellular environment.
It was hypothesized that these weakly-stable regions may be exposed in nascent misfolded proteins, or misfolded pathogenic species, and that in this context they are present in an alternate conformation than they are in the native ensemble. They may thus constitute misfolded protein-specific epitope predictions.
Without wishing to be bound by theory, these sequence regions in misfolded proteins may be exposed in conformations distinct from that of the native fold—for example, being on the surface, they may be exposed in turn regions that have higher exposed surface area, or alternate side chain conformations, as seen by different dihedral angle distributions, and/or different overall conformation as measured by structural alignment, than the corresponding quantities exhibit in native TDP-43.
As described the Examples, the inventors have identified minimal epitopes in a region predicted to be prone to disruption in the context of the native structure, so that it would be likely to be exposed in a partially-formed native structure. The inventors designed cyclic compounds comprising the identified epitope to satisfy criteria of an alternate conformation such as a higher exposed surface area, and/or that did not readily align by root mean squared deviation (RMSD) to the native ensemble, but would align more favorably to a biased, partially disordered ensemble.
Antibodies raised to these epitopes are shown by immunofluorescence to recognize misfolded TDP-43 in cell culture and by immuno dot-blotting to bind to pathogenic TDP43 in spinal cord homogenates from ALS patients vs controls.
I. DefinitionsAs used herein, the term “TDP-43” alternately referred to as “TDP43”, or “TDP” as used herein means all forms of TDP-43 including wildtype TDP-43, native TDP-43, as well as misfolded forms including mutant forms and analogs thereof from all species, particularly human TDP-43 (i.e. hTDP-43). Human TDP-43 is a protein of typically 414 amino acid residues and the amino acid sequence (e.g. Uniprot Accession number Q13148) and the nucleotide sequence (e.g. Accession number HGNC:11571) have been previously characterized. TDP-43 comprises RRM1 and RRM2 domains. The RRM1 of TDP-43 refers to the first RNA-recognition motif of the protein, consisting of amino acids 106-177. A structure of the RRM1 domain of TDP-43 has been determined and is listed on the protein databank as PDB entry 41UF. The PDB 41UF structure can be equilibrated on a computer to obtain an equilibrium ensemble, which was used for all measurements of the native conformations of the epitopes in the native structure of TDP-43, referred to herein variably as “native structure of RRM1”, “equilibrium native ensemble of TDP-43”, “equilibrium native ensemble of RRM1 of TDP-43”, or “TDP-43 native structural ensemble”.
“Wild type” as used herein refers to the primary amino acid sequence of non-mutant or naturally occurring protein.
“Native” as used herein refers to the normal three dimensional structure of a specific protein or part thereof (e.g. the atomic level coordinates of the crystal structure of native TDP-43 RRM1 domain is available at Protein Data Bank Accession Number 41UF). Native TDP-43 is optionally referred to as “natively folded” TDP-43 “normally folded” TPD-43 and/or “healthy” TDP-43. Similarly native RRM1 domain of TDP-43 is optionally referred to as “natively folded” RRM1 domain of TDP-43 or “normally folded” RRM1 domain of TPD-43. Accordingly the term “native TDP-43”, or “natively folded TDP-43”, herein refers to TDP-43 as natively folded after nascent translation and/or dimeric TDP-43 as folded in non-disease states (e.g. healthy cells) with a molecular structure that comprises a non-covalently associated, individual TDP-43 peptide which shows native structure under in x-ray crystallography or as reconstructed from a nuclear magnetic resonance measurement. The native structure of RRM1 has an alpha/beta structure, consisting of both alpha helices and beta sheets.
“Misfolded” as used herein refers to the secondary and tertiary structure of a polypeptide or part thereof, and indicates that the polypeptide has adopted a conformation that is not normal for that polypeptide in its properly functioning state. Although misfolding can be caused by mutations in a protein, such as amino acid deletion, substitution, or addition, wild-type sequence protein can also be misfolded in disease, and expose disease-specific epitopes for instance, as a result of microenvironmental conditions and/or amino acid modification such as nitration, oxidation, carbonylation or other modification. Other post-translational modifications include aberrant ubiquitination, phosphorylation, acetylation, sumoylation, and cleavage into C-terminal fragments ubiquitylation. Accordingly, “misfolded TDP-43 polypeptide”, or “misfolded TDP-43” when referring to the polypeptide herein refers to TDP-43 polypeptide that displays a plurality of conformations of TDP-43 wherein the conformations are partially-ordered, containing parts of the native structure, and partially-disordered, containing polymer segments of amino acids that have alternate conformations than native TDP-43, and often show an increase in SASA, and sample a more diverse conformational ensemble than that explored in the native equilibrium ensemble.
Misfolded TDP-43 is prone to the formation of aggregates resulting in a loss of protein function, toxicity and propagation of pathogenic aggregates.
The term “mutant TDP-43” refers to forms of TDP-43, and particularly endogenous forms of TDP-43 that occur as a result of genetic mutation that result for instance in amino acid substitution, such as those substitutions characteristic for instance of FTD or familial ALS including for example the mutations described in the bioinformatics tool described in [6].
The term “TTEQ (SEQ ID NO: 1)” means the amino acid sequence: threonine, threonine, glutamic acid, glutamine; as shown in SEQ ID NO: 1. Similarly TTE, TEQ, KTTE (SEQ ID NO:10), KTTEQD (SEQ ID NO: 7), TTEQD (SEQ ID NO: 9), TTEQDL (SEQ ID NO: 11), TEQD (SEQ ID NO: 8) refer to the amino acid sequences identified by the 1-letter amino acid code. Depending on the context, the reference of the amino acid sequence can refer to a sequence in TDP-43 or an isolated peptide, such as the amino acid sequence of a cyclic compound. The sequence TTEQ (SEQ ID NO: 1) consists of residues 115-118 in the amino acid primary sequence.
The term “TTEQ (SEQ NO: 1) or a related epitope and/or part of any of the foregoing” as used herein minimally comprises amino acid T115 and/or T116 and/or E117, and includes for example TTE, KTT or TEQ. Reference to TTEQ (SEQ NO: 1) or a related epitope and/or part of any of the foregoing can refer to the region on TDP-43 that is bound by an antibody raised for example by a cyclic compound comprising a TDP-43 peptide sequence. For example the antibody may selectively or specifically bind T115, T116 or E117, a particular part of T115 and/or T116 and/or E117, or a combination of any of the foregoing. Alternatively, it can refer to the TDP-43 peptide sequence that is comprised in a cyclic compound for making antibodies.
The term “alternate conformation than occupied by 115T, 116T, 117E and/or 118Q in the native” as used herein means having one or more differing conformational properties selected from solvent accessibility, (e.g. in the context of a peptide comprising TTEQ (SEQ ID NO: 1) as measured for example in the cyclic peptide described in the examples, RMSD structural alignment, and dihedral angle of one or more backbone or side chain dihedral angles compared to said property for 115T, 116T, 117E and/or 118Q in the TDP-43 native structural ensemble, as shown for example in PDB 41UF, and shown in the Figures, and/or in the Tables. Similarly, the term “alternate conformation” than occupied by one or more of the T's, E and/or Q in the native structure as used herein means having one or more differing conformational properties selected from solvent accessibility, (e.g. in the context of a corresponding linear peptide comprising TTEQ (SEQ ID NO: 1) or TTE, and dihedral angle of one or more backbone or side chain dihedral angles compared to said property for one or more of T, E and/or Q, for example T, E and/or Q in native TDP-43 as shown for example in PDB 41UF and shown in
An “epitope” as used herein means a region of a protein that is recognized by a B cell or T-cell receptor, or an antibody or a binding fragment thereof. The epitope is optionally represented herein by a linear amino acid sequence or the region of the protein recognized by the antibody. An epitope can comprise one or more antigenic determinants. For example an antibody generated against an isolated peptide corresponding to a misfolded epitope recognizes part or all of said epitope sequence.
As used herein, the term “misfolded epitope” or “conformational epitope” refers to a sequence of amino acids or an antigenic determinant thereof that have a particular three-dimensional structure wherein at least an aspect of the three-dimensional structure not present in a corresponding native structure is recognized by the cognate antibody. The “misfolded- or conformational epitope” becomes exposed or accessible in the misfolded protein (e.g. as present in ALS and FTD). Antibodies which selectively bind a misfolded epitope recognize the spatial arrangement of one or more of the amino acids of that conformation-specific epitope. For example, a TTEQ (SEQ ID NO: 1) conformational epitope refers to an epitope of TTEQ (SEQ ID NO: 1) that is recognized by antibodies selectively, for example at least 2 fold, 3 fold, 5 fold, 10 fold, 50 fold, 100 fold, 250 fold, 500 fold or 1000 fold or greater more selectivity as compared to the epitope on native TDP-43 or for example antibodies raised using a linear peptide comprising TTEQ (SEQ ID NO: 1).
The term “analog” as used herein includes parts, extensions, substitutions, variants, modifications or chemical equivalents and derivatives thereof of the amino acid and nucleotide sequences of the present invention that perform substantially the same function as the peptide, protein or nucleic acid molecules of described herein in substantially the same way. Analogs of the cyclic compounds such as the cyclic peptides also include additions and deletions to the TDP-43 peptides. Analogs of nucleic acids include degenerate nucleotide substitutions that encode an isolated peptide of the invention. In addition, analog peptides and analog nucleotide sequences include derivatives thereof.
The term “amino acid” includes all of the naturally occurring amino acids as well as modified L-amino acids. The atoms of the amino acid can for example include different isotopes. For example, the amino acids can comprise deuterium substituted for hydrogen, nitrogen-15 substituted for nitrogen-14, and carbon-13 substituted for carbon-12 and other similar changes.
A “conservative amino acid substitution” as used herein, is one in which one amino acid residue is replaced with another amino acid residue without abolishing the protein's desired properties. Suitable conservative amino acid substitutions can be made by substituting amino acids with similar hydrophobicity, polarity, and R-group size for one another. Examples of conservative amino acid substitution include:
The term “sequence identity” as used herein refers to the percentage of sequence identity between two polypeptide sequences or two nucleic acid sequences. To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions/total number of positions.times.100%). In one embodiment, the two sequences are the same length. The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, modified as in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches can be performed with the NBLAST nucleotide program parameters set, e.g., for score=100, word length=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present application. BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, word length=3 to obtain amino acid sequences homologous to a protein molecule described herein. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) can be used (see, e.g., the NCBI website). Another preferred non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4:11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used. The percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
For antibodies, percentage sequence identities can be determined when antibody sequences maximally aligned by IMGT or other (e.g. Kabat numbering convention). After alignment, if a subject antibody region (e.g., the entire mature variable region of a heavy or light chain) is being compared with the same region of a reference antibody, the percentage sequence identity between the subject and reference antibody regions is the number of positions occupied by the same amino acid in both the subject and reference antibody region divided by the total number of aligned positions of the two regions, with gaps not counted, multiplied by 100 to convert to percentage.
The term “antibody” as used herein is intended to include monoclonal antibodies, polyclonal antibodies, single chain, humanized and other chimeric antibodies as well as binding fragments thereof. The antibody may be from recombinant sources and/or produced in transgenic animals. Also included are human antibodies that can be produced through using biochemical techniques or isolated from a library. Humanized or chimeric antibody may include sequences from one or more than one isotype or class.
The phrase “isolated antibody” refers to antibody produced in vivo or in vitro that has been removed from the source that produced the antibody, for example, an animal, hybridoma or other cell line (such as recombinant cells that produce antibody). The isolated antibody is optionally “purified”, which means at least: 80%, 85%, 90%, 95%, 98% or 99% purity.
The term “binding fragment” as used herein to a part or portion of an antibody or antibody chain comprising fewer amino acid residues than an intact or complete antibody or antibody chain and which binds the antigen or competes with intact antibody. Exemplary binding fragments include without limitations Fab, Fab′, F(ab′)2, scFv, dsFv, ds-scFv, dimers, nanobodies, minibodies, diabodies, and multimers thereof. Fragments can be obtained via chemical or enzymatic treatment of an intact or complete antibody or antibody chain. Fragments can also be obtained by recombinant means. For example, F(ab′)2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab′)2 fragment can be treated to reduce disulfide bridges to produce Fab′ fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab′ and F(ab′)2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be constructed by recombinant expression techniques.
The terms “IMGT numbering” or “ImMunoGeneTics database numbering”, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e. hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or antigen binding portion thereof.
The CDR sequences referred to herein are based IGBlast identification which searches sequences with BLAST against the IMGT or NCBI germline V gene database. The sequences were also confirmed to correspond to IMGT numbering. As the full sequences for the variable regions are provided, it is possible to identify the CDRs based on other conventions such as Kabat as well.
When an antibody is said to bind to an epitope within specified residues, such as TTEQ (SEQ ID NO:1), what is meant is that the antibody selectively or specifically binds to a polypeptide containing the specified residues or a part thereof for example at least 1 residue or at least 2 residues in a conformation-selective manner. Such an antibody does not necessarily contact every residue of TTEQ (SEQ ID NO:1), and every single amino acid substitution or deletion within said epitope does not necessarily significantly affect or equally affect binding affinity.
The term “detectable label” as used herein refers to moieties such as peptide sequences, fluorescent proteins that can be appended or introduced into a peptide or compound described herein and which is capable of producing, either directly or indirectly, a detectable signal. For example, the label may be radio-opaque, positron-emitting radionuclide (for example for use in PET imaging), or a radioisotope, such as 3H, 13N, 14C, 18F, 32F, 35S, 123I, 125I, 131I; a fluorescent (fluorophore) or chemiluminescent (chromophore) compound, such as fluorescein isothiocyanate, rhodamine or luciferin; an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase; an imaging agent; or a metal ion. The detectable label may be also detectable indirectly for example using secondary antibody.
The term “epitope selectively presented or accessible in misfolded TDP-43” as used herein refers to an epitope that is selectively presented or accessible on misfolded TDP-43 as present in ALS or FTD (e.g. disease associated misfolded TDP-43) whether in monomeric, dimeric or aggregated forms, but not on the molecular surface of the native, correctly folded, homodimeric form of TDP-43.
The phrase as used herein “epitope consists of TTEQ”, indicates an epitope, optionally a conformational epitope, that is bound specifically and preferentially by an antibody that preferentially binds TTEQ (SEQ ID NO: 1) peptide compared to a mutated TTEQ (SEQ ID NO: 1) peptide where any one or more of the residues are mutated for example to alanine. Similarly, the phrase as used herein of “conformational epitope consists of TTEQ” indicates an epitope that is bound specifically by an antibody that preferentially binds TTEQ (SEQ ID NO: 1) in a particular conformation (e.g. misfolded protein, cyclic compound or some other constrained conformation) over another conformation (e.g. native) and optionally over, when at least one or more of the residues, are mutated to for example alanine (e.g cyclic conformation of a peptide comprising TTEQ (SEQ ID NO: 1), versus cyclic conformation of a peptide wherein one or more of the residues are mutated).
The phrase “epitope consisting of TTEQ or a part thereof” indicates an epitope that is bound specifically by an antibody that specifically and preferentially binds TTEQ (SEQ ID NO: 1) peptide compared to a peptide when at least one or more of the residues, optionally 115T, 116T, 117E and/or 118Q, are mutated to alanine or absent. Similarly, the phrase as used herein of “conformational epitope consists of TTEQ or a part thereof” indicates an epitope that is bound specifically by an antibody that preferentially binds TTEQ (SEQ ID NO: 1) or a part thereof when it is in a particular conformation (e.g. misfolded protein, cyclic, or some other constrained conformation) over another conformation (e.g. native conformation) and optionally over, when at least one or more of the residues, optionally 115T, 116T, 117E and/or 118Q, are mutated to alanine or absent (e.g cyclic conformation of a peptide comprising TTEQ (SEQ ID NO: 1), versus cyclic conformation of a peptide wherein one or more of the residues are mutated or absent).
The term “greater affinity” as used herein refers to a degree of antibody binding where an antibody X binds to target Y more strongly (Kon) and/or with a smaller dissociation constant (Koff) than to target Z, and in this context antibody X has a greater affinity for target Y than for Z. Likewise, the term “lesser affinity” herein refers to a degree of antibody binding where an antibody X binds to target Y less strongly and/or with a larger dissociation constant than to target Z, and in this context antibody X has a lesser affinity for target Y than for Z. The affinity of binding between an antibody and its target antigen, can be expressed as KA equal to 1/KD where KD is equal to kon/koff. The kon and koff values can be measured using surface plasmon resonance (measurable for example using a Biacore system).
Also as used herein, the term “immunogenic” refers to substances which elicit the production of antibodies, activate T-cells and other reactive immune cells directed against an antigenic portion of the immunogen.
An “immunogen” as used herein means a substance which provokes an immune response and/or causes production of an antibody. In addition to immunogenic compounds, conjugates and fusions described herein, including for example the isolated compounds conjugated to KLH, peptide mimetics which elicit cross-reactive antibodies to the epitopes identified, e.g. TTEQ and/or related epitopes such as TTE can be employed. To serve as a useful immunogen, the TDP-43 peptide desirably incorporates a minimum of about 3, 4, 5, 6, or 7 TDP-43 residues, comprising minimally T115 and/or T116 and/or E117 and optionally incorporates an immunogenicity enhancing agent such as KLH. As the number of residues in the cyclic peptide increases, the construct becomes more similar conformationally to the linear peptide. The optimal degree of similarity to misfolded states as compared to native structures occurs around 7 to 9 residues (see Table 6C and Table 8A).
The term “corresponding linear compound” with regard to a cyclic compound refers to a compound, optionally a peptide, comprising or consisting of the same sequence or chemical moieties as the cyclic compound but in linear (non-cyclized) form.
The term “nucleic acid sequence” as used herein refers to a sequence of nucleoside or nucleotide monomers consisting of naturally occurring bases, sugars and intersugar (backbone) linkages. The term also includes modified or substituted sequences comprising non-naturally occurring monomers or portions thereof. The nucleic acid sequences of the present application may be deoxyribonucleic acid sequences (DNA) or ribonucleic acid sequences (RNA) and may include naturally occurring bases including adenine, guanine, cytosine, thymidine and uracil. The sequences may also contain modified bases. Examples of such modified bases include aza and deaza adenine, guanine, cytosine, thymidine and uracil; and xanthine and hypoxanthine. The nucleic acid can be either double stranded or single stranded, and represents the sense or antisense strand. Further, the term “nucleic acid” includes the complementary nucleic acid sequences as well as codon optimized or synonymous codon equivalents. The term “isolated nucleic acid sequences” as used herein refers to a nucleic acid substantially free of cellular material or culture medium when produced by recombinant DNA techniques, or chemical precursors, or other chemicals when chemically synthesized. An isolated nucleic acid is also substantially free of sequences which naturally flank the nucleic acid (i.e. sequences located at the 5′ and 3′ ends of the nucleic acid) from which the nucleic acid is derived.
Operatively linked” is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid. Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes. Selection of appropriate regulatory sequences is dependent on the host cell chosen and may be readily accomplished by one of ordinary skill in the art. Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.
The term “vector” as used herein comprises any intermediary vehicle for a nucleic acid molecule which enables said nucleic acid molecule, for example, to be introduced into prokaryotic and/or eukaryotic cells and/or integrated into a genome, and include plasmids, phagemids, bacteriophages or viral vectors such as retroviral based vectors, Adeno Associated viral vectors and the like. The term “plasmid” as used herein generally refers to a construct of extrachromosomal genetic material, usually a circular DNA duplex, which can replicate independently of chromosomal DNA.
By “at least moderately stringent hybridization conditions” it is meant that conditions are selected which promote selective hybridization between two complementary nucleic acid molecules in solution. Hybridization may occur to all or a portion of a nucleic acid sequence molecule. The hybridizing portion is typically at least 15 (e.g. 20, 25, 30, 40 or 50) nucleotides in length. Those skilled in the art will recognize that the stability of a nucleic acid duplex, or hybrids, is determined by the Tm, which in sodium containing buffers is a function of the sodium ion concentration and temperature (Tm=81.5° C.−16.6 (Log10 [Na+])+0.41(% (G+C)−600/1), or similar equation). Accordingly, the parameters in the wash conditions that determine hybrid stability are sodium ion concentration and temperature. In order to identify molecules that are similar, but not identical, to a known nucleic acid molecule a 1% mismatch may be assumed to result in about a 1° C. decrease in Tm, for example if nucleic acid molecules are sought that have a >95% identity, the final wash temperature will be reduced by about 5° C. Based on these considerations those skilled in the art will be able to readily select appropriate hybridization conditions. In preferred embodiments, stringent hybridization conditions are selected. By way of example the following conditions may be employed to achieve stringent hybridization: hybridization at 5× sodium chloride/sodium citrate (SSC)/5×Denhardt's solution/1.0% SDS at Tm—5° C. based on the above equation, followed by a wash of 0.2×SSC/0.1% SDS at 60° C. Moderately stringent hybridization conditions include a washing step in 3×SSC at 42° C. It is understood, however, that equivalent stringencies may be achieved using alternative buffers, salts and temperatures. Additional guidance regarding hybridization conditions may be found in: Current Protocols in Molecular Biology, John Wiley & Sons, N.Y., 2002, and in: Sambrook et al., Molecular Cloning: a Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001.
As used herein “specifically binds” in reference to an antibody means that the antibody recognizes its target antigen and binds its target with greater affinity than it does to a structurally different antigen and/or to an antigen with modified or mutated sequence. For example a multivalent antibody binds its target with KD of at least 1e-6, at least 1e-7, at least 1e-8, at least 1e-9 or at least 1e-10. Affinities greater than at least 1e-8 are preferred. An antigen binding fragment such as Fab fragment comprising one variable domain, may find its target with a 10 fold or 100 fold less affinity than a multivalent interaction with a non-fragmented antibody.
The term “selective” as used herein with respect to an antibody that preferentially binds a form of TDP-43 (e.g. native, or misfolded protein) means that the binding protein binds the form with at least 3 fold, or at least 5 fold, at least 10 fold, at least 20 fold, at least 100 fold, at least 250 fold, at least 500 fold or at least 1000 fold or more greater affinity. Accordingly an antibody that is more selective for a particular conformation (e.g. misfolded protein, cyclic peptide) preferentially binds the particular form of TDP-43 with at least 3 fold etc greater affinity compared to another form.
The term “linker” as used herein means a chemical moiety that can be covalently linked to the peptide comprising TTEQ (SEQ ID NO:1) epitope peptide, optionally linked to TTEQ (SEQ ID NO:1) peptide N- and C-termini to produce a cyclic compound. The linker can comprise a spacer and/or one or more functionalizable moieties such as a cysteine residue. The linker can be linked via the functionalizable moieties to a carrier protein or an immunogen enhancing component such as keyhole limpet hemocyanin (KLH). The linker can be for example 1 to 9 amino acids.
The term “spacer” as used herein means any non-immunogenic or poorly immunogenic chemical moiety that can be covalently-linked directly or indirectly to a peptide N- and C-termini to produce a cyclic compound of longer length than the peptide itself, for example the spacer can be linked to the N- and C-termini of a peptide consisting of TTEQ (SEQ ID NO:1) to produce a cyclic compound of longer backbone length than the TTEQ (SEQ ID NO:1) sequence itself. That is, when cyclized the peptide with a spacer (for example of 3 amino acid residues) makes a larger closed circle than the peptide without a spacer. The spacer may include, but is not limited to, non-immunogenic moieties such as G, A, or PEG repeats, e.g. when in combination with the peptide being GTTEQG (SEQ ID NO: 4) TTEQG (SEQ ID NO: 5), GTTEQ (SEQ ID NO: 6), etc. The spacer may comprise or be coupled to one or more functionalizing moieties, such as one or more cysteine (C) residues, which can be interspersed within the spacer or covalently linked to one or both ends of the spacer. Where a functionalizable moiety such as a C or D residue is covalently linked to one or more termini of the spacer, the spacer is indirectly covalently linked to the peptide. The spacer can also comprise the functionalizable moiety in a spacer residue as in the case where a biotin molecule is introduced into an amino acid residue.
The term “functionalizable moiety” as used herein refers to a chemical entity with a “functional group” which as used herein refers to a group of atoms or a single atom that will react with another group of atoms or a single atom (so called “complementary functional group”) to form a chemical interaction between the two groups or atoms. In the case of cysteine, the functional group can be —SH which can be reacted to form a disulfide bond. Accordingly the linker can for example be CCC. The reaction with another group of atoms can be covalent or a strong non-covalent bond, for example as in the case of biotin-streptavidin bonds, which can have Kd˜1e-14. A strong non-covalent bond as used herein means an interaction with a Kd of at least 1e-9, at least 1e-10, at least 1e-11, at least 1e-12, at least 1e-13 or at least 1e-14.
Proteins and/or other agents may be functionalized (e.g. coupled) to the cyclic compound, either to aid in immunogenicity, or to act as a probe in in vitro studies. For this purpose, any functionalizable moiety capable of reacting (e.g. making a covalent or non-covalent but strong bond) may be used. In one specific embodiment, the functionalizable moiety is a cysteine residue which is reacted to form a disulfide bond with an unpaired cysteine on a protein of interest, which can be, for example, an immunogenicity enhancing component such as Keyhole limpet hemocyanin (KLH), or a carrier protein such as Bovine serum albumin (BSA) used for in vitro immunoblots or immunohistochemical assays.
The term “reacts with” as used herein generally means that there is a flow of electrons or a transfer of electrostatic charge resulting in the formation of a chemical interaction.
The term “animal” or “subject” as used herein includes all members of the animal kingdom including mammals, optionally including or excluding humans.
In understanding the scope of the present disclosure, the term “consisting” and its derivatives, as used herein, are intended to be close ended terms that specify the presence of stated features, elements, components, groups, integers, and/or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and/or steps.
The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term “about.” Further, it is to be understood that “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. The term “about” means plus or minus 0.1 to 50%, 5-50%, or 10-40%, preferably 10-20%, more preferably 10% or 15%, of the number to which reference is being made.
Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.
II. Epitopes and Binding ProteinsThe inventors have identified epitopes in TDP-43 including TTEQ (SEQ ID NO: 1) at amino acids 115 to 118 on TDP-43 protein and TTE at amino acids 115 to 117 on TDP-43 protein. They have further identified that these epitopes or parts thereof are conformational epitopes, and that TTE, TEQ or TTEQ (SEQ ID NO: 1) or a part thereof may be selectively accessible to antibody binding in misfolded proteinic species of TDP-43.
Based on one or more conformational differences identified between the epitopes identified in native and biased TDP-43 ensembles, the inventors have designed conformationally restricted compounds and immunogens for producing antibodies that selectively or specifically binds misfolded TDP-43.
Antibodies raised using said immunogens may be useful for detecting misfolded TDP-43.
As described in the Examples, cyclic compounds such as cyclic peptides cyclo (CGGTTEQGG) (SEQ ID NO: 2), cyclo (CGTTEQG) (SEQ ID NO: 3), cyclo (CGTTEG) (SEQ ID NO: 28) and cyclo (CGTTEGG) (SEQ ID NO: 29) may capture the conformational differences of the epitope in misfolded TDP-43 relative to the native species. For example, solvent accessible surface area, RMSD structural alignment, and the dihedral angle distributions for amino acids in the cyclic 9-mer cyclo (CGGTTEQGG) (SEQ ID NO: 2) were found to be significantly different than the corresponding quantities in the native ensemble. This suggests that the cyclic compound may provide for a conformational epitope that is conformationally-distinct from the sequence presented in the native ensemble.
III. TTEQ (SEQ ID NO:1) and TTE “Epitope” CompoundsAccordingly, the present disclosure identifies a conformational epitope in TDP-43 consisting of amino acids TTEQ (SEQ ID NO: 1) or TTE corresponding to amino acids residues 115-117 on TDP-43 and TEQ corresponding to amino acids 116-118. As demonstrated in the Examples, TTEQ (SEQ ID NO: 1) and TTE were identified as regions prone to disorder in TDP-43. The residues TTE and TTEQ (SEQ ID NO:1) emerged in computational predictions.
There are differences in the conformation occupied by the native RRM1 and RRM1 biased to identify regions that may be exposed in misfolded TDP-43. For example, Table 5 shows that E117 is more exposed in the biased protein than in the native protein, and more exposed still in the cyclic peptide cyclo(CGGTTEQGG) (SEQ ID NO: 2). In the native protein this residue participates in a salt bridge with Lys 137 which is disrupted in the biased configuration.
An aspect includes a compound comprising a TDP-43 peptide comprising or consisting of TTEQ (SEQ ID NO: 1), a related epitope and/or part of any of the foregoing such as TTE, wherein if the peptide is TTEQ (SEQ ID NO: 1), the peptide is in a conformation that is distinct in at least one feature from TTE and/or TTEQ (SEQ ID NO: 1) in native TDP-43. In an embodiment, the TDP-43 peptide is selected from TTE, TTEQ (SEQ ID NO: 1), KTTE, (SEQ ID NO: 10), KTTEQ (SEQ ID NO:12), TEQ, TEQD (SEQ ID NO:8) or TTEQD (SEQ ID NO: 9). The TDP-43 peptides TTEQD (SEQ ID NO:9), TEQD (SEQ ID NO:8), KTTEQ (SEQ ID NO:12), TTE, and TEQ can be used to raise antibodies that are included in the epitopes collectively referred to herein as TTEQ (SEQ ID NO: 1) and related epitopes. In an embodiment, the related epitope comprises or consists of TTE, TEQD (SEQ ID NO: 8), KTTE (SEQ ID NO: 10) and epitopes that comprise 1, 2 or 3 amino acids in TDP-43 either N-terminal or C-terminal to TTE.
In some embodiments, the peptide, a conformational peptide, comprising TTE, TEQ or TTEQ (SEQ ID NO: 1) can include 1, or 2 additional residues in TDP-43 N- and/or C-terminus of TTEQ (SEQ ID NO: 1) for example TTEQD (SEQ ID NO:9) or KTTEQD (SEQ ID NO:7). For example, the 3 amino acids N-terminal to TTEQ (SEQ ID NO:1) in TDP-43 are PWK and the 3 amino acids C-terminal to TTEQ (SEQ ID NO:1) are DLK. In an embodiment, the TDP-43 peptide is a maximum of 6 TDP-43 residues. In an embodiment, the TDP-43 peptide is a maximum of 5 TDP-43 residues. In yet another embodiment, the TDP-43 peptide (e.g. in the compound such as a cyclic compound) is 4 TDP-43 residues, optionally TTEQ (SEQ ID NO: 1).
In an embodiment, the compound further includes a linker. The linker comprises a spacer and/or one or more functionalizable moieties. The linker can for example comprise 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids and/or equivalently functioning molecules such as polyethylene glycol (PEG) moieties, and/or a combination thereof. In an embodiment, the spacer amino acids are selected from non-immunogenic or poorly immunogenic amino acid residues such as G and A, for example the spacer can be GGG, GAG, G(PEG)G, PEG-PEG (also referred to as PEG2)-GG and the like. One or more functionalizable moieties e.g. amino acids with a functional group may be included for example for coupling the compound to an agent or detectable tag or a carrier such as BSA or an immunogenicity enhancing component such as KLH.
In an embodiment the linker comprises GC-PEG, PEG-GC, GCG or PEG2-CG.
In an embodiment, the linker comprises 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids.
In embodiments wherein the peptide comprising TTE or TTEQ (SEQ ID NO: 1) includes 1, 2 or 3 additional residues found in TDP-43 that are N- and/or C-terminal to TTEQ (SEQ ID NO: 1) the linker in the cyclized compound is covalently linked to the N- and/or C-termini of the TDP-43 residues (e.g. where the peptide is KTTEQ (SEQ ID NO:12), the linker is covalently linked to K and Q residues). Similarly, where the TDP-43 peptide is TTEQ (SEQ ID NO: 1), the linker is covalently linked to residues T and Q and where the TDP-43 peptide is TTEQD (SEQ ID NO: 9), the linker is covalently linked to residues T and D.
Proteinaceous portions of compounds (or the compound wherein the linker is also proteinaceous) may be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis or synthesis in homogenous solution.
In an embodiment, the compound is a cyclic compound e.g the TDP-43 peptide comprising TTE or TTEQ (SEQ ID NO: 1) is comprised in a cyclic compound. Reference to the “cyclic peptide” herein can refer to a fully proteinaceous compound (e.g. wherein the linker is 2, 3, 4, 5, 6, 7, 8 or 9 amino acids). It is understood that properties described for the cyclic peptide determined in the examples can be incorporated in other compounds (e.g. cyclic compounds) comprising non-amino acid linker molecules.
An aspect therefore provides a cyclic compound comprising peptide TTEQ (SEQ ID NO:1) (or a part thereof such as TTE) and a linker, wherein the linker is covalently coupled directly or indirectly to the peptide comprising TTE or TTEQ (SEQ ID NO:1), optionally wherein at least the one of the T115, the T116, the E117, and/or Q118 residues is in an alternate conformation than the T, T, E, and Q residues in a native ensemble comprising TTEQ (SEQ ID NO:1), as may be manifest in native TDP-43, and optionally wherein at least T, T, E, and/or Q, is in either a more solvent exposed conformation, or an alternative conformation, than the conformation occupied in a native ensemble comprising TTEQ (SEQ ID NO:1), as may be manifest for example in TDP-43 dimer.
In an embodiment, the cyclic compound comprises a TDP-43 peptide comprising TTE or TTEQ (SEQ ID NO:1) and up to 6 TDP-43 residues (e.g. 1 or 2 (or 3 in the case of TTE) amino acids N and/or C terminus to TTE or TTEQ (SEQ ID NO:1)) and a linker, wherein the linker is covalently coupled directly or indirectly to the peptide N-terminus residue and the C-terminus residue of the TDP-43 peptide and optionally wherein at least T115, T116, E or Q is in an alternate conformation than T115, T116, E, or Q in the native ensemble comprising TTEQ (SEQ ID NO: 1), and/or the conformation of T115, T116, E, or Q in TTEQ (SEQ ID NO: 1) in the native and optionally wherein at least T115, T116, E, or Q, has more surface exposure than the conformation occupied in the native ensemble comprising TTEQ (SEQ ID NO: 1).
The cyclic compound can be synthesized as a linear molecule with the linker covalently attached to the N-terminus or C-terminus of the peptide comprising the TDP-43 peptide, optionally TTEQ (SEQ ID NO: 1) or related epitope, prior to cyclization. Alternatively part of the linker is covalently attached to the N-terminus and part is covalently attached to the C-terminus prior to cyclization. In either case, the linear compound is cyclized for example in a head to tail cyclization (e.g. amide bond cyclization).
In an embodiment the cyclic compound comprises a peptide comprising or consisting of TTEQ (SEQ ID NO: 1) and a linker, wherein the linker is coupled to the N- and C-termini of the peptide (e.g. the T and the Q residues when the peptide consists of TTEQ (SEQ ID NO: 1). In an embodiment, at least one of the T, E and/or Q residues is in an alternate conformation in the cyclic compound than occupied by at least one of the T, E and/or Q residues in a native ensemble comprising TTEQ (SEQ ID NO: 1).
In an embodiment, at least one of the T, E, and/or Q residues is in an alternate conformation in the cyclic compound than occupied by a residue, optionally by T, E, and/or Q, in the native ensemble.
In an embodiment, at least one of the T, E, and/or Q residues is in an alternate conformation in the cyclic compound than occupied by a residue in the native.
In an embodiment, the alternate conformation is a more solvent-exposed conformation.
In an embodiment, at least T115, optionally alone or in combination with T116, is in an alternate conformation than the conformation occupied in a native ensemble comprising TTEQ (SEQ ID NO: 1).
For example, the alternate conformation can include one or more differing dihedral angles in residue T115, differing from the dihedral angles in the native ensemble.
For example, the alternate conformation can include one or more differing backbone dihedral angles (Ramachandran angles) in residue T115, differing from the dihedral angles in the native ensemble.
In an embodiment, the cyclic compound comprises a minimum average side-chain/backbone dihedral angle difference between the cyclic compound and native ensemble.
In an embodiment, the cyclic compound comprises a residue selected from T, E, and Q, wherein one or more side-chain or backbone dihedral angles are at least 30 degrees, at least 40 degrees, at least 50 degrees, at least 60 degrees, at least 70 degrees, at least 80 degrees, at least 90 degrees, at least 100 degrees, at least 110 degrees, at least 120 degrees, at least 130 degrees, at least 140 degrees, at least 150 degrees, at least 160 degrees or at least 170 degrees, different in the cyclic compound, than the corresponding dihedral angle in the context of the native ensemble.
As shown in
According to the peak values of Ramachandran angles given in Table 4A, the most-likely Ramachandran ϕ and ψ values are different between the cyclic and native ensembles for residues T115, T116, E117, and Q118, as also presented in Table 4B. The respective differences Δϕ between the cyclic and native peak ϕ values are 50, −65, −40, and −35 degrees, and the respective differences Δψ between the ψ values of the cyclic and native peaks are −45, 30, 40, and 50 degrees. Overall the ϕ, ψ values are significantly different between cyclic and native peptides. Table 3A gives the overlap of the distributions of ϕ, ψ values in the cyclic and native ensembles. The average overlap of ϕ and ψ distributions for T115, T116, E117, and Q118 is 28%, 63%, 55% and 23% respectively. Together these numbers indicate different distributions of dihedral angles for the cyclic and native ensembles.
In an embodiment, the cyclic compound comprises a Q comprising an Ramachandran backbone angle that is at least 30 degrees, at least 40 degrees different, at least 50 degrees or at least 60 degrees different than the corresponding Ramachandran angle in the context of the native compound.
The angle difference can for example be positive or negative, (+) or (−).
The alternate conformation can comprise an alternate backbone orientation. For example, the backbone orientation that the cyclic epitope exposes for an antibody differs compared to the native form.
In an embodiment, T, T, E, Q, TT, TE, EQ, TTE, TEQ, and/or TTEQ (SEQ ID NO: 1) are in an alternate conformation, for example as compared to what is occupied by these residues in a non-misfolded proteinic conformation, such as the native ensemble.
The cyclic compound in some embodiments that comprises a peptide comprising TTEQ (SEQ ID NO: 1), TTE or KTTE (SEQ ID NO:10) can include 1, or 2 or more residues in TDP-43 upstream and/or downstream of one of the foregoing, for example of TTEQ (SEQ ID NO: 1). In such cases the spacer is covalently linked to the N- and C-termini of the ends of the corresponding residues of the TDP-43 sequence.
In an embodiment, the cyclic compound has a sequence selected from any one of SEQ ID NOs: 2, 3, 22, 23, 28, 29, 30, 31, 32, 33, 34, 35 and 42 or any subset thereof. In an embodiment, the cyclic compound comprises a sequence selected from any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34 and 35. In another embodiment, the cyclic compound comprises a sequence selected from SEQ ID NO: 2, 3, 22, 23 and 42. In yet other embodiments, the cyclic compound comprises a sequence selected from any one of SEQ ID NOs: 29, 32 and 34. In yet other embodiments, the cyclic compound comprises a sequence selected from any one of SEQ ID NOs: 2, 3, 22 and 23.
Methods for making cyclized peptides are known in the art and include SS-cyclization or amide cyclization (head-to-tail, or backbone cyclization). Methods are further described in in the Example section. For example, a peptide with “C” residues at its N- and C-termini, e.g. CGTTEQGC (SEQ ID NO: 13), can be reacted by SS-cyclization to produce a cyclic peptide. As described in the Examples, cyclic compounds were assessed for their relatedness to the conformational epitopes identified. A cyclic compound comprising TTEQ (SEQ ID NO: 1) or TTE TDP-43 peptide for example may be used to raise antibodies selective for misfolded TDP-43.
The epitope TTEQ (SEQ ID NO: 1) and/or a part thereof, as described herein may be a potential target in misfolded propagating strains of TDP-43, and antibodies that recognize the conformational epitope may for example be useful in detecting such propagating strains.
Another aspect includes an immunogen comprising a compound comprising a TTE, TTEQ (SEQ ID NO: 1) or related epitope, optionally a cyclic compound described herein. In an embodiment, the immunogen comprises an immunogenicity enhancing component such as Keyhole Limpet Hemocyanin (KLH) and/or is formulated for co-injection with an adjuvant (e.g. alum, monophosphoryl lipid A or QS21). Other similar components are known in the art and additional adjuvants are described below. The adjuvant is typically formulated in a composition with the compound as is further described below. The immunogenicity enhancing component can be coupled to the compound either directly, such as through an amide bond, disulfide bond, or indirectly through a chemical linker. In another embodiment, the immunogen is a multi-antigenic peptide (MAP). For example, the MAP can be synthesized by preparing a linear compound to be cyclized, cyclizing the peptide, optionally using head to tail cyclization and conjugating the cyclized peptide to a MAP resin through an amino acid in the linker, optionally through a C or D residue in the linker. MAPs have been constructed for cyclic structures (see for example Misumi et al J. Virol. December 2001 vol. 75 no. 23 11614-11620) and similar methods to those described therein can be used.
The immunogen with an immunogenicity enhancing component can be produced by conjugating the cyclic compound containing the constrained epitope peptide and a linker comprising a functionalizable moiety such as cysteine to an immunogenicity enhancing component such as Keyhole Limpet Hemocyanin (KLH) or a carrier such bovine serum albumin (BSA) using for example the method described in Lateef et al 2007, herein incorporated by reference. In an embodiment, the method described in Example 3 is used.
A further aspect is an isolated nucleic acid encoding the proteinaceous portion of a compound or immunogen described herein.
IV. Antibodies, Cells and Nucleic AcidsAccordingly, the compounds and particularly the cyclic compounds comprising epitope TTEQ (SEQ ID NO: 1) or a related epitope described above can be used to raise antibodies. Cyclic compounds comprising TTEQ (SEQ ID NO: 1) or a related epitope for example KTTE (SEQ ID NO:10), TTE, TEQ, TTEQD (SEQ ID NO:9), KTTEQD (SEQ ID NO: 7), KTTEQDL (SEQ ID NO:14) and/or other related epitope sequences described herein can be used to raise antibodies that selectively bind misfolded TDP-43.
Accordingly, the compounds and particularly the cyclic compounds described herein including for example those comprising epitope or cyclic compound sequences listed in Table 11 can be used to raise antibodies that specifically or selectively bind the epitope in TDP-43 that they comprise and/or which recognize specific conformations of these residues in misfolded TDP-43, including one or more differential features described herein.
Accordingly, an aspect includes an antibody that specifically or selectively bind an epitope on TDP-43, the epitope comprising or consisting TTEQ (SEQ ID NO: 1), a related epitope thereof or a part thereof or a conformational epitope of any of the foregoing. In an embodiment, wherein when the epitope consists of TTEQ (SEQ ID NO: 1) it is a conformational epitope.
In an embodiment the antibody is isolated.
In an embodiment, the antibody does not specifically bind and/or is not selective for native TDP-43 e.g. wherein the conformation of TTEQ (SEQ ID NO:1) or a related epitope such as TTE is as present in native TDP-43. Selective binding can be measured using an ELISA or surface plasmon resonance measurement, as described herein.
Accordingly a further aspect is an antibody which specifically or selectively binds an epitope present on TDP-43, wherein the epitope comprises or consists of at least one amino acid residue predominantly involved in binding to the antibody, wherein at least one amino acid is T115, T116, E, or Q embedded within the sequence TTEQ (SEQ ID NO:1), TEQ, or KTTE (SEQ ID NO:10), optionally wherein the epitope when consisting of TTEQ (SEQ ID NO:1) is a conformational epitope (e.g. selectively binds a peptide in an alternate optionally solvent-exposed conformation relative to the corresponding native ensemble, for example where at least one amino acid of the epitope is more solvent-exposed). In an embodiment, the epitope comprises or consists of at least two consecutive amino acid residues predominantly involved in binding to the antibody, wherein the at least two consecutive amino acids are TE, or EQ embedded within TTEQ (SEQ ID NO:1), TTE or TEQ
In another embodiment, the epitope is a conformational epitope and consists of TTEQ (SEQ ID NO:1), TTE or TEQ. In an embodiment, the antibody selectively binds TTEQ (SEQ ID NO:1), TTE or TEQ or other related epitope in a cyclic compound, optionally cyclic peptide, optionally cyclo(CGTTEQG) (SEQ ID NO: 3) or a cyclic peptide having a sequence of any one SEQ ID NOs: 2, 3, 22, 23, 28, 29, 30, 31, 32, 33, 34, 35 and 42, or any subset thereof, relative to a corresponding linear peptide and/or a native ensemble.
In an embodiment, the antibody specifically binds a cyclic compound comprising an epitope peptide described herein comprising at least one alternate conformational feature described herein (e.g. of the epitope in a cyclic compound compared to a native structural ensemble). For example an antibody that binds a particular epitope conformation can be referred to as a conformation specific antibody. The conformation specific antibody can differentially recognize a particular misfolded TDP-43 species, and can have a higher affinity for one species or group of species compared to the native species.
For example, in an embodiment, the antibody specifically binds a cyclic compound comprises a residue selected from T, E, and Q, wherein at least one dihedral angle is at least 30 degrees, at least 40 degrees, at least 50 degrees, at least 60 degrees, at least 70 degrees, at least 80 degrees, at least 90 degrees, at least 100 degrees, at least 110 degrees, at least 120 degrees, at least 130 degrees, at least 140 degrees or at least 150 degrees different in the cyclic compound, than the corresponding dihedral angle in the context of the native structure.
In an embodiment, the antibody selectively binds a cyclic compound comprising TTEQ (SEQ ID NO:1) or a part thereof, optionally in the context of cyclo (CGGTTEQGG) (SEQ ID NO:2) relative to a native ensemble comprising TTEQ (SEQ ID NO:1), optionally in the context of linear cyclo (CGTTEQG) (SEQ ID NO: 3) relative to a native ensemble comprising TTEQ (SEQ ID NO:1). For example, in an embodiment the antibody selectively binds TTEQ (SEQ ID NO:1) or TTE in a cyclic conformation and has at least 2 fold, at least 3 fold, at least 5 fold, at least 8 fold, at least 10, fold, at least 15 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 100 fold, at least 500 fold or at least 1000 fold more selective greater selectivity (e.g. binding affinity) for TTEQ (SEQ ID NO:1) or TTE in the cyclic conformation compared to TTEQ (SEQ ID NO:1) or TTE in a native ensemble, for example as measured by ELISA, immunohistochemistry or surface plasmon resonance, optionally using a method described herein.
In an embodiment, the cyclic compound selectively bound and/or used to produce antibodies is a compound of Table 11. In another embodiment, said cyclic compound is a cyclic compound selected from SEQ ID NOs: 2, 3, 22, 23, 28, 29, 30, 31, 32, 33, 34, 35 and 42, or any subset thereof. In an embodiment, the cyclic compound comprises a sequence selected from any one of SEQ ID NOs: 28, 29, 30, 31, 32, 33, 34 and 35. In another embodiment, the cyclic compound comprises a sequence selected from SEQ ID NO: 2, 3, 22, 23 and 42. In yet other embodiments, the cyclic compound comprises a sequence selected from any one of SEQ ID NOs: 29, 32 and 34. In yet other embodiments, the cyclic compound comprises a sequence selected from any one of SEQ ID NOs: 2, 3, 22 and 23.
In an embodiment, the cyclic compound selectively bound is a compound of Table 11. In an embodiment, the selectivity is at least 2 fold, at least 3 fold, at least 5 fold, at least 10 fold, at least 20 fold, at least 30 fold, at least 40 fold, at least 50 fold, at least 100 fold, at least 500 fold or at least 1000 fold more selective for the cyclic compound and/or Misfolded TDP-43 polypeptide over a species of TDP-43 selected from native TDP-43.
In yet another aspect, the disclosure provides an antibody that competes for selectively binds to a cyclic peptide having a sequence selected from any one of SEQ ID NOs: 2, 3, 22, 23, 28, 29, 30, 31, 32, 33, 34, 35 and 42.
To produce monoclonal antibodies, antibody producing cells (lymphocytes) can be harvested from a subject immunized with an immunogen described herein, and fused with myeloma cells by standard somatic cell fusion procedures thus immortalizing these cells and yielding hybridoma cells. Such techniques are well known in the art, (e.g. the hybridoma technique originally developed by Kohler and Milstein (Nature 256:495-497 (1975)) as well as other techniques such as the human B-cell hybridoma technique (Kozbor et al., Immunol. Today 4:72 (1983)), the EBV-hybridoma technique to produce human monoclonal antibodies (Cole et al., Methods Enzymol, 121: 140-67 (1986)), and screening of combinatorial antibody libraries (Huse et al., Science 246:1275 (1989)). Hybridoma cells can be screened immunochemically for production of antibodies specifically reactive with the desired epitopes and the monoclonal antibodies can be isolated.
Specific antibodies, or antibody fragments, reactive against particular antigens or molecules, may also be generated by screening expression libraries encoding immunoglobulin genes, or portions thereof, expressed in bacteria with cell surface components. For example, complete Fab fragments, VH regions and FV regions can be expressed in bacteria using phage expression libraries (see for example Ward et al., Nature 41:544-546 (1989); Huse et al., Science 246:1275-1281 (1989); and McCafferty et al., Nature 348:552-554 (1990)).
The humanization of antibodies from non-human species has been well described in the literature. See for example EP-B1 0 239400 and Carter & Merchant 1997 (Curr Opin Biotechnol 8, 449-454, 1997 incorporated by reference in their entirety herein). Humanized antibodies are also readily obtained commercially (e.g. Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, Great Britain).
Humanized forms of rodent antibodies are readily generated by CDR grafting (Riechmann et al. Nature, 332:323-327, 1988). In this approach the six CDR loops comprising the antigen binding site of the rodent monoclonal antibody are linked to corresponding human framework regions. CDR grafting often yields antibodies with reduced affinity as the amino acids of the framework regions may influence antigen recognition (Foote & Winter. J Mol Biol, 224: 487-499, 1992). To maintain the affinity of the antibody, it is often necessary to replace certain framework residues by site directed mutagenesis or other recombinant techniques and may be aided by computer modeling of the antigen binding site (Co et al. J Immunol, 152: 2968-2976, 1994).
Humanized forms of antibodies are optionally obtained by resurfacing (Pedersen et al. J Mol Biol, 235: 959-973, 1994). In this approach only the surface residues of a rodent antibody are humanized.
Human antibodies specific to a particular antigen may be identified by a phage display strategy (Jespers et al. Bio/Technology, 12: 899-903, 1994). In one approach, the heavy chain of a rodent antibody directed against a specific antigen is cloned and paired with a repertoire of human light chains for display as Fab fragments on filamentous phage. The phage is selected by binding to antigen. The selected human light chain is subsequently paired with a repertoire of human heavy chains for display on phage, and the phage is again selected by binding to antigen. The result is a human antibody Fab fragment specific to a particular antigen. In another approach, libraries of phage are produced where members display different human antibody fragments (Fab or Fv) on their outer surfaces (Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047). Phage displaying antibodies with a desired specificity are selected by affinity enrichment to a specific antigen. The human Fab or Fv fragment identified from either approach may be recloned for expression as a human antibody in mammalian cells.
Human antibodies are optionally obtained from transgenic animals (U.S. Pat. Nos. 6,150,584; 6,114,598; and 5,770,429). In this approach the heavy chain joining region (JH) gene in a chimeric or germ-line mutant mouse is deleted. Human germ-line immunoglobulin gene array is subsequently transferred to such mutant mice. The resulting transgenic mouse is then capable of generating a full repertoire of human antibodies upon antigen challenge.
Humanized or human antibodies are selected from any class of immunoglobulins including: IgM, IgG, IgD, IgA or IgE; and any isotype, including: IgG1, IgG2, IgG3 and IgG4. The humanized or human antibody may include sequences from one or more than one isotype or class. Further, these antibodies are typically produced as antigen binding fragments such as Fab, Fab′ F(ab′)2, Fd, Fv and single domain antibody fragments, or as single chain antibodies in which the heavy and light chains are linked by a spacer. Also, the human or humanized antibodies may exist in monomeric or polymeric form. The humanized antibody optionally comprises one non-human chain and one humanized chain (i.e. one humanized heavy or light chain).
Additionally, antibodies specific for the epitopes described herein are readily isolated by screening antibody phage display libraries. For example, an antibody phage library is optionally screened by using a disease specific epitope of the current invention to identify antibody fragments specific for the disease specific epitope. Antibody fragments identified are optionally used to produce a variety of recombinant antibodies that are useful with different embodiments of the present invention. Antibody phage display libraries are commercially available, for example, through Xoma (Berkeley, Calif.) Methods for screening antibody phage libraries are well known in the art.
As indicated below, several antibodies that were positive for detecting misfolded, disease-associated TDP-43 in transfected cells or ALS spinal cord homogenates were sequenced.
Accordingly, in another embodiment, the antibody described herein comprises a light chain variable region and a heavy chain variable region, optionally fused, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2 and CDR-H3, the light chain variable region comprising complementarity determining region CDR-L1, CDR-L2 and CDR-L3 and with the amino acid sequences of said CDRs comprising the sequences:
In another embodiment, the antibody described herein comprises a light chain variable region and a heavy chain variable region, optionally fused, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2 and CDR-H3, the light chain variable region comprising complementarity determining region CDR-L1, CDR-L2 and CDR-L3 and with the amino acid sequences of said CDRs comprising the sequences:
In another embodiment, the antibody described herein comprises a light chain variable region and a heavy chain variable region, optionally fused, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2 and CDR-H3, the light chain variable region comprising complementarity determining region CDR-L1, CDR-L2 and CDR-L3 and with the amino acid sequences of said CDRs comprising the sequences:
In another embodiment, the antibody described herein comprises a light chain variable region and a heavy chain variable region, optionally fused, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2 and CDR-H3, the light chain variable region comprising complementarity determining region CDR-L1, CDR-L2 and CDR-L3 and with the amino acid sequences of said CDRs comprising the sequences:
In yet another embodiment, the antibody described herein comprises a light chain variable region and a heavy chain variable region, optionally fused, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2 and CDR-H3, the light chain variable region comprising complementarity determining region CDR-L1, CDR-L2 and CDR-L3 and with the amino acid sequences of said CDRs comprising the sequences:
In yet another embodiment, the antibody described herein comprises a light chain variable region and a heavy chain variable region, optionally fused, the heavy chain variable region comprising complementarity determining regions CDR-H1, CDR-H2 and CDR-H3, the light chain variable region comprising complementarity determining region CDR-L1, CDR-L2 and CDR-L3 and with the amino acid sequences of said CDRs comprising the sequences:
As shown in
In an embodiment, the antibody is a monoclonal antibody. In an embodiment, the antibody is a chimeric antibody such as a humanized antibody comprising the CDR sequences as recited in Table 10.
Also provided in another embodiment, is an antibody comprising the CDRs in Table 10 and a light chain variable region and a heavy chain variable region, optionally in the context of a single chain antibody.
In yet another aspect, the antibody comprises a heavy chain variable region comprises: i) an amino acid sequence as set forth in SEQ ID NO: 46; ii) an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to SEQ ID NO: 46, wherein the CDR sequences are as set forth in SEQ ID NO: 67, 68 and 69, or iii) a conservatively substituted amino acid sequence i). In another aspect the antibody comprises a light chain variable region comprising i) an amino acid sequence as set forth in SEQ ID NO: 48, ii) an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to SEQ ID NO: 48 wherein the CDR sequences are as set forth in SEQ ID NO: 70, 71 and 72, or iii) a conservatively substituted amino acid sequence of i). In another embodiment, the heavy chain variable region amino acid sequence is encoded by a nucleotide sequence as set out in SEQ ID NO: 45 or a codon degenerate or optimized version thereof. In another embodiment, the antibody comprises a light chain variable region amino acid sequence encoded by a nucleotide sequence as set out in SEQ ID NO: 47 or a codon degenerate or optimized version thereof. In an embodiment, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 46. In an embodiment, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 48.
In yet another aspect, the antibody comprises a heavy chain variable region comprises: i) an amino acid sequence as set forth in SEQ ID NO: 50; ii) an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to SEQ ID NO: 50, wherein the CDR sequences are as set forth in SEQ ID NO: 73, 74 and 75, or iii) a conservatively substituted amino acid sequence i). In another aspect the antibody comprises a light chain variable region comprising i) an amino acid sequence as set forth in SEQ ID NO: 52, ii) an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to SEQ ID NO: 52 wherein the CDR sequences are as set forth in SEQ ID NO: 76, 77 and 78, or iii) a conservatively substituted amino acid sequence of i). In another embodiment, the heavy chain variable region amino acid sequence is encoded by a nucleotide sequence as set out in SEQ ID NO: 49 or a codon degenerate or optimized version thereof. In another embodiment, the antibody comprises a light chain variable region amino acid sequence encoded by a nucleotide sequence as set out in SEQ ID NO: 51 or a codon degenerate or optimized version thereof. In an embodiment, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 50. In an embodiment, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 52.
In yet another aspect, the antibody comprises a heavy chain variable region comprises: i) an amino acid sequence as set forth in SEQ ID NO: 54; ii) an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to SEQ ID NO: 54, wherein the CDR sequences are as set forth in SEQ ID NO: 73, 79 and 80, or iii) a conservatively substituted amino acid sequence i). In another aspect the antibody comprises a light chain variable region comprising i) an amino acid sequence as set forth in SEQ ID NO: 56, ii) an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to SEQ ID NO: 56 wherein the CDR sequences are as set forth in SEQ ID NO: 76, 77 and 81, or iii) a conservatively substituted amino acid sequence of i). In another embodiment, the heavy chain variable region amino acid sequence is encoded by a nucleotide sequence as set out in SEQ ID NO: 53 or a codon degenerate or optimized version thereof. In another embodiment, the antibody comprises a light chain variable region amino acid sequence encoded by a nucleotide sequence as set out in SEQ ID NO: 55 or a codon degenerate or optimized version thereof. In an embodiment, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 54. In an embodiment, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 56.
In yet another aspect, the antibody comprises a heavy chain variable region comprises: i) an amino acid sequence as set forth in SEQ ID NO: 58; ii) an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to SEQ ID NO: 58, wherein the CDR sequences are as set forth in SEQ ID NO: 67, 68 and 82, or iii) a conservatively substituted amino acid sequence i). In another aspect the antibody comprises a light chain variable region comprising i) an amino acid sequence as set forth in SEQ ID NO: 60, ii) an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to SEQ ID NO: 60 wherein the CDR sequences are as set forth in SEQ ID NO: 83, 84 and 85, or iii) a conservatively substituted amino acid sequence of i). In another embodiment, the heavy chain variable region amino acid sequence is encoded by a nucleotide sequence as set out in SEQ ID NO: 57 or a codon degenerate or optimized version thereof. In another embodiment, the antibody comprises a light chain variable region amino acid sequence encoded by a nucleotide sequence as set out in SEQ ID NO: 59 or a codon degenerate or optimized version thereof. In an embodiment, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 58. In an embodiment, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 60.
In yet another aspect, the antibody comprises a heavy chain variable region comprises: i) an amino acid sequence as set forth in SEQ ID NO: 62; ii) an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to SEQ ID NO: 62, wherein the CDR sequences are as set forth in SEQ ID NO: 86, 87 and 88, or iii) a conservatively substituted amino acid sequence i). In another aspect the antibody comprises a light chain variable region comprising i) an amino acid sequence as set forth in SEQ ID NO: 64, ii) an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to SEQ ID NO: 64 wherein the CDR sequences are as set forth in SEQ ID NO: 76, 77 and 92, or iii) a conservatively substituted amino acid sequence of i). In another embodiment, the heavy chain variable region amino acid sequence is encoded by a nucleotide sequence as set out in SEQ ID NO: 61 or a codon degenerate or optimized version thereof. In another embodiment, the antibody comprises a light chain variable region amino acid sequence encoded by a nucleotide sequence as set out in SEQ ID NO: 63 or a codon degenerate or optimized version thereof. In an embodiment, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 62. In an embodiment, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 64.
In yet another aspect, the antibody comprises a heavy chain variable region comprises: i) an amino acid sequence as set forth in SEQ ID NO: 66; ii) an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to SEQ ID NO: 66, wherein the CDR sequences are as set forth in SEQ ID NO: 89, 90 and 91, or iii) a conservatively substituted amino acid sequence i). In another aspect the antibody comprises a light chain variable region comprising i) an amino acid sequence as set forth in SEQ ID NO: 64, ii) an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80% or at least 90% sequence identity to SEQ ID NO: 64 wherein the CDR sequences are as set forth in SEQ ID NO: 76, 77 and 92, or iii) a conservatively substituted amino acid sequence of i). In another embodiment, the heavy chain variable region amino acid sequence is encoded by a nucleotide sequence as set out in SEQ ID NO: 65 or a codon degenerate or optimized version thereof. In another embodiment, the antibody comprises a light chain variable region amino acid sequence encoded by a nucleotide sequence as set out in SEQ ID NO: 63 or a codon degenerate or optimized version thereof. In an embodiment, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 66. In an embodiment, the light chain variable region comprises an amino acid sequence as set forth in SEQ ID NO: 64.
Another aspect is an antibody that specifically binds a same epitope as the antibody with CDR sequences as recited in Table 10.
Another aspect includes an antibody that competes for binding to a cyclic compound described herein and/or to human misfolded TDP43 with an antibody described herein such as an antibody comprising the CDR sequences as recited in Table 10.
Competition between antibodies can be determined for example using an assay in which an antibody under test is assessed for its ability to inhibit specific binding of a reference antibody to the common antigen. A test antibody competes with a reference antibody if an excess of a test antibody (e.g., at least a 2 fold, 5, fold, 10 fold or 20 fold) inhibits binding of the reference antibody by at least 50%, at least 75%, at least 80%, at least 90% or at least 95% as measured in a competitive binding assay.
A further aspect is an antibody conjugated to a detectable label. In an embodiment, the detectable label is a positron-emitting radionuclide. A positron-emitting radionuclide can be used for example in PET imaging. In an embodiment, the antibody is conjugated to a transport moiety that permits transport across the blood brain barrier and/or into a cell. For example the antibody can be covalently linked to the iron-transport protein melanotransferrin (p97) or fused to antibody fragments specific for BBB receptors such as the transferrin receptor, insulin receptor, lipoprotein receptor, basigin, Glut1 or CD98hc. Another example is fusion to the BBB-permeable single domain antibody FC5 or single domain antibodies directed against other BBB surface receptors. In an embodiment, the antibody is conjugated to a transport moiety that facilitates entry into a cell for diagnostic detection of intracellular aggregated TDP43. For example, the antibody can be chemically linked or recombinantly fused to cell-penetrating peptides such as trans-activating transcriptional activator (TAT) and TAT derivatives, penetratin or transportan, and the like.
A further aspect relates to an antibody complex comprising an antibody described herein and/or a binding fragment thereof and misfolded TDP-43. A further aspect is an isolated nucleic acid encoding an antibody or part thereof described herein.
Nucleic acids encoding a heavy chain or a light chain are also provided, for example encoding a heavy chain comprising CDR-H1, CDR-H2 and/or CDR-H3 regions described herein or encoding a light chain comprising CDR-L1, CDR-L2 and/or CDR-L3 regions described herein.
The present disclosure also provides variants of the nucleic acid sequences that encode for the antibody and/or binding fragment thereof disclosed herein. For example, the variants include nucleotide sequences that hybridize to the nucleic acid sequences encoding the antibody and/or binding fragment thereof disclosed herein under at least moderately stringent hybridization conditions or codon degenerate or optimized sequences In another embodiment, the variant nucleic acid sequences have at least 50%, at least 60%, at least 70%, most preferably at least 80%, even more preferably at least 90% and even most preferably at least 95% sequence identity to nucleic acid sequences encoding any one of SEQ ID NOs: 45, 47, 49, 51, 53, 55, 57, 59, 61, 63 and 65.
Another aspect is an expression cassette or vector comprising the nucleic acid herein disclosed. In an embodiment, the vector is an isolated vector.
The vector can be any vector, including vectors suitable for producing an antibody and/or binding fragment thereof or expressing a peptide sequence described herein.
The nucleic acid molecules may be incorporated in a known manner into an appropriate expression vector which ensures expression of the protein. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g. replication defective retroviruses, adenoviruses and adeno-associated viruses). The vector should be compatible with the host cell used. The expression vectors are “suitable for transformation of a host cell”, which means that the expression vectors contain a nucleic acid molecule encoding the peptides corresponding to epitopes or antibodies described herein.
In an embodiment, the vector is suitable for expressing for example single chain antibodies by gene therapy. The vector can be adapted for specific expression in neural tissue, for example using neural specific promoters and the like. In an embodiment, the vector comprises an IRES and allows for expression of a light chain variable region and a heavy chain variable region. Such vectors can be used to deliver antibody in vivo.
Suitable regulatory sequences may be derived from a variety of sources, including bacterial, fungal, viral, mammalian, or insect genes.
Examples of such regulatory sequences include: a transcriptional promoter and enhancer or RNA polymerase binding sequence, a ribosomal binding sequence, including a translation initiation signal. Additionally, depending on the host cell chosen and the vector employed, other sequences, such as an origin of replication, additional DNA restriction sites, enhancers, and sequences conferring inducibility of transcription may be incorporated into the expression vector.
In an embodiment, the regulatory sequences direct or increase expression in neural tissue and/or cells.
In an embodiment, the vector is a viral vector.
The recombinant expression vectors may also contain a marker gene which facilitates the selection of host cells transformed, infected or transfected with a vector for expressing an antibody or epitope peptide described herein.
The recombinant expression vectors may also contain expression cassettes which encode a fusion moiety (i.e. a “fusion protein”) which provides increased expression or stability of the recombinant peptide; increased solubility of the recombinant peptide; and aid in the purification of the target recombinant peptide by acting as a ligand in affinity purification, including for example tags and labels described herein. Further, a proteolytic cleavage site may be added to the target recombinant protein to allow separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Typical fusion expression vectors include pGEX (Amrad Corp., Melbourne, Australia), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the recombinant protein.
Systems for the transfer of genes for example into neurons and neural tissue both in vitro and in vivo include vectors based on viruses, most notably Herpes Simplex Virus, Adenovirus, Adeno-associated virus (AAV) and retroviruses including lentiviruses. Alternative approaches for gene delivery include the use of naked, plasmid DNA as well as liposome-DNA complexes. Another approach is the use of AAV plasmids in which the DNA is polycation-condensed and lipid entrapped and introduced into the brain by intracerebral gene delivery (Leone et al. US Application No. 2002076394).
Accordingly, in another aspect, the compounds, immunogens, nucleic acids, vectors and antibodies described herein may be formulated in vesicles such as liposomes, nanoparticles, and viral protein particles, for example for delivery of antibodies, compounds, immunogens and nucleic acids described herein. In particular synthetic polymer vesicles, including polymersomes, can be used to administer antibodies.
Also provided in another aspect is a cell expressing an antibody or part thereof described herein. In an embodiment, the cell is an isolated and/or recombinant cell, expressing an antibody described herein or comprising a vector herein disclosed. In an embodiment, the cell is a fused cell such as a hybridoma.
The recombinant cell can be generated using any cell suitable for producing a polypeptide, for example suitable for producing an antibody and/or binding fragment thereof. For example to introduce a nucleic acid (e.g. a vector) into a cell, the cell may be transfected, transformed or infected, depending upon the vector employed.
Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins described herein may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells.
In an embodiment, the cell is a eukaryotic cell selected from a yeast, plant, worm, insect, avian, fish, reptile and mammalian cell.
In another embodiment, the mammalian cell is a myeloma cell, a spleen cell, or a hybridoma cell.
In an embodiment, the cell is a neural cell.
Yeast and fungi host cells suitable for expressing an antibody or peptide include, but are not limited to Saccharomyces cerevisiae, Schizosaccharomyces pombe, the genera Pichia or Kluyveromyces and various species of the genus Aspergillus. Examples of vectors for expression in yeast S. cerivisiae include pYepSec1, pMFa, pJRY88, and pYES2 (Invitrogen Corporation, San Diego, Calif.). Protocols for the transformation of yeast and fungi are well known to those of ordinary skill in the art.
Mammalian cells that may be suitable include, among others: COS (e.g., ATCC No. CRL 1650 or 1651), BHK (e.g. ATCC No. CRL 6281), CHO (ATCC No. CCL 61), HeLa (e.g., ATCC No. CCL 2), 293 (ATCC No. 1573) and NS-1 cells. Suitable expression vectors for directing expression in mammalian cells generally include a promoter (e.g., derived from viral material such as polyoma, Adenovirus 2, cytomegalovirus and Simian Virus 40), as well as other transcriptional and translational control sequences. Examples of mammalian expression vectors include pCDM8 and pMT2PC.
In an embodiment, the cell is a fused cell such as a hybridoma cell, the hybridoma cell producing an antibody specific and/or selective for an epitope or epitope sequence described herein, including for example that selectively binds TDP-43 pathological oligomers over TDP43 native structures, selectively binds an epitope sequence presented in a cyclic compound relative to a linear compound or lacks or has negligible plaque binding.
A further aspect is a hybridoma cell line producing an antibody specific for an epitope described herein.
V. CompositionsA further aspect is a composition comprising a compound, immunogen, nucleic acid, vector or antibody described herein.
In an embodiment, the composition comprises a diluent.
Suitable diluents for nucleic acids include but are not limited to water, saline solutions and ethanol.
Suitable diluents for polypeptides, including antibodies or fragments thereof and/or cells include but are not limited to saline solutions, pH buffered solutions and glycerol solutions or other solutions suitable for freezing polypeptides and/or cells.
In an embodiment comprising a compound or immunogen described herein, the composition comprises an adjuvant.
In an embodiment, the adjuvant is selected from. alum, monophosphoryl lipid A and QS21.
Adjuvants that can be used for example, include Intrinsic adjuvants (such as lipopolysaccharides) normally are the components of killed or attenuated bacteria used as vaccines. Extrinsic adjuvants are immunomodulators which are typically non-covalently linked to antigens and are formulated to enhance the host immune responses. Aluminum hydroxide, aluminum sulfate and aluminum phosphate (collectively commonly referred to as alum) are routinely used as adjuvants. A wide range of extrinsic adjuvants can provoke potent immune responses to immunogens. These include saponins such as Stimulons (QS21, Aquila, Worcester, Mass.) or particles generated therefrom such as ISCOMs and (immunostimulating complexes) and ISCOMATRIX, complexed to membrane protein antigens (immune stimulating complexes), pluronic polymers with mineral oil, killed mycobacteria and mineral oil, Freund's complete adjuvant, bacterial products such as muramyl dipeptide (MDP) and lipopolysaccharide (LPS), as well as lipid A, and liposomes.
In an embodiment, the adjuvant is aluminum hydroxide. In another embodiment, the adjuvant is aluminum phosphate. Oil in water emulsions include squalene; peanut oil; MF59 (WO 90/14387); SAF (Syntex Laboratories, Palo Alto, Calif.); and Ribi™ (Ribi Immunochem, Hamilton, Mont.). Oil in water emulsions may be used with immunostimulating agents such as muramyl peptides (for example, N-acetylmuramyl-L-threonyl-D-isoglutamine (thr-MDP), -acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP), N-acetylmuramyl-L-alanyl-D-isoglutamyl-L-alanine-2-(1′-2′dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine (MTP-PE), N-acetylglucsaminyl-N-acetylmuramyl-L-Al-D-isoglu-L-Ala-dipalmitoxy propylamide (DTP-DPP) theramide (TM)), or other bacterial cell wall components.
The adjuvant may be administered with an immunogen as a single composition.
Alternatively, an adjuvant may be administered before, concurrent and/or after administration of the immunogen.
In an embodiment, the composition comprises an antibody or part thereof described herein. In another embodiment, the composition comprises an antibody or part thereof described herein and a diluent. In an embodiment, the composition is a sterile composition.
In an embodiment, the composition is for a method described herein such as detecting misfolded TDP-43.
VI. KitsA further aspect relates to a kit comprising i) an antibody and/or binding fragment thereof, ii) a nucleic acid, iii) peptide or immunogen, iv) composition or v) recombinant cell described herein, comprised in a vial such as a sterile vial or other housing and optionally a reference agent and/or instructions for use thereof.
In an embodiment, the kit is an ELISA.
In an embodiment, the kit comprises an antibody or binding fragment described herein contained in a container such as a sterile vial.
VII. MethodsIncluded are methods for making the compounds, immunogens and antibodies described herein.
In particular, provided are methods of making an antibody selective for a conformational epitope of TTEQ (SEQ ID NO:1) or related epitope. In an embodiment, the method comprises administering an immunogen described herein to a subject and isolating antibodies that selectively bind the TDP-43 peptide of the immunogen and/or misfolded TDP-43.
A further aspect provides a method of detecting whether a sample comprises misfolded TDP-43, for example misfolded TDP-43 comprising TTE, TTEQ (SEQ ID NO:1) or related conformational epitope and/or wherein at least one of the residues T115, T116, E, or Q is in an alternate conformation than occupied by T, E, and/or Q in a non-misfolded proteinic conformation.
In an embodiment, the method comprises:
a. contacting the sample with the antibody described herein under conditions permissive to produce an antibody:misfolded TDP-43 polypeptide complex; and
b. detecting the presence of any complex;
wherein the presence of detectable complex is indicative that the sample may contain misfolded TDP-43 polypeptide.
In another embodiment, the method comprises:
(a) contacting a test sample of said subject with an antibody described herein, under conditions permissive to produce an antibody-antigen complex;
(b) measuring the amount of the antibody-antigen complex in the test sample; and
(c) comparing the amount of antibody-antigen complex in the test sample to a control;
wherein detecting antibody-antigen complex in the test sample as compared to the control indicates that the sample comprises TDP-43 comprising TTEQ (SEQ ID NO:1) or related epitope such as TTE (e.g. misfolded TDP-43).
In an embodiment, the sample is a biological sample. In an embodiment, the sample comprises brain tissue, spinal cord tissue or an extract thereof and/or CSF. In an embodiment, the sample is obtained from a human subject.
In an embodiment, the sample is from a subject with ALS. In another embodiment, the sample is from a subject with FTD.
A number of methods can be used to determine if misfolded TDP-43 polypeptides is present in a sample using the antibodies described herein, including immunoassays such as flow cytometry, dot or slot blots, Western blots, ELISA, and immunoprecipitation followed by SDS-PAGE immunocytochemistry. In an embodiment, the method used comprises one or more of the steps described in Example 7, 8 or 10.
Surface plasmon resonance can be used to assess conformation specific binding.
A labelled antibody described herein can also be administered to a subject detect location of misfolded TDP-43.
A further aspect includes a method of inducing an immune response in a subject, comprising administering to the subject a compound, immunogen and/or composition comprising a compound described herein; and optionally isolating cells and/or antibodies that specifically bind the compound or immunogen administered. The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.
The following non-limiting examples are illustrative of the present disclosure:
EXAMPLES Example 1 TDP-43 Epitope PredictionsAlthough most ALS/FTD mutations are in the C-terminus, the effects of mutation can lead to pathological aggregates of TDP-43, and these multimeric aggregates can induce the disorder of the structured domains. Accordingly, the RRM1 domain was assessed for the presence of conformation specific epitopes present in misfolded TDP-43.
Putative misfolded epitopes in TDP-43 were predicted with the aid of a method referred to as “Collective Coordinates biasing” which is described in WO/2017/079836, SYSTEMS AND METHODS FOR PREDICTING MISFOLDED PROTEIN EPITOPES BY COLLECTIVE COORDINATE BIASING filed Nov. 9, 2016. As described therein, the method uses molecular-dynamics-based simulations which impose a global coordinate bias on a protein (or peptide-aggregate) to force the protein (or peptide-aggregate) to misfold and then predict the most likely unfolded regions of the partially unstructured protein (or peptide aggregate). Biasing simulations were performed and the solvent accessible surface area (SASA) corresponding to each residue index (compared to that of the initial structure of the protein under consideration). SASA represents a surface area that is accessible to H2O. A positive change in SASA (compared to that of the initial structure of the protein under consideration) may be considered to be indicative of unfolding in the region of the associated residue index. Two other methods were used in addition to SASA to identify candidate epitopes. These were the loss of native contacts, defined by non-hydrogen atoms within a cut-off length, and root mean squared fluctuations (RMSF), measuring the extent of deviations about the average in a structural ensemble; here an increase in RMSF for some amino acids indicates an increase in the dynamics of those amino acids.
The methods were applied to the natively folded RRM1 domain of TDP-43 (PDB entry 4IUF).
Simulations were performed for each initial structure using the a method as described in WO/2017/079836 and the CHARMM force-field parameters described in: K. Vanommeslaeghe, E. Hatcher, C. Acharya, S. Kundu, S. Zhong, J. Shim, E. Darian, 0. Guvench, P. Lopes, I. Vorobyov, and A. D. Mackerell. Charmm general force field: A force field for drug-like molecules compatible with the charmm all-atom additive biological force fields. Journal of Computational Chemistry, 31(4):671-690, 2010; and P. Bjelkmar, P. Larsson, M. A. Cuendet, B. Hess, and E. Lindahl. Implementation of the CHARMM force field in GROMACS: analysis of protein stability effects from correlation maps, virtual interaction sites, and water models. J. Chem. Theo. Comp., 6:459-466, 2010, both of which are hereby incorporated herein by reference, with TIP3P water as solvent.
I. Conformation Specific EpitopesThis disclosure pertains to antibodies that may be selective for misfolded TDP-43.
A prerequisite for the generation of misfolding-specific antibodies is the identification of targets on TDP-43 peptide that are not present (e.g. not accessible to binding) in the context of the native structure. These misfolding-specific epitopes would not differ in primary sequence from the corresponding segment in native TDP-43, however they would be conformationally distinct in the context of the misfolded protein. That is, they would present a distinct conformation in the misfolded protein that would not be present in the natively folded protein.
Antibodies directed either against native ensemble regions tend not to be selective for misfolded protein, and thus bind to healthy protein as well. Because the concentration of normal protein may be substantially higher than that of misfolded protein, such antibodies would likely suffer from “target distraction”, primarily binding to healthy protein and promoting clearance of functional TDP-43, rather than selectively targeting and clearing misfolded proteinic species. This may interfere with critical RNA binding and stress response functions vital to the survival of the cell.
To develop antibodies selective for misfolded proteinic forms of TDP-43, a region in the native protein that is likely to be disrupted upon application of external perturbing forces was identified, using the Collective Coordinates algorithm. Without wishing to be bound to theory, it was hypothesized that disruptions in the context of the native may be exposed as well on the surface of the misfolded protein. On misfolded proteins however, these sequence regions may be exposed in conformations distinct from native TDP-43. For example, being on the surface, they may be exposed in turn regions that have higher exposed surface area, different dihedral angle distribution and/or overall different conformational geometry as determined by structural alignment than the corresponding quantities exhibit in either the native ensemble.
Cyclic compounds comprising TTEQ (SEQ ID NO: 1) and TTE are described herein. The cyclic compounds have been designed to satisfy one or more of the above criteria.
II. Epitope PredictionsThe epitopes TTEQ (SEQ ID NO: 1) and TTE emerge as predicted epitopes.
The TTEQ (SEQ ID NO: 1) epitope emerges as a prediction for PDB structure 41UF when considering loss of native contacts. (
For the plots in
Further computational support for the identification of a misfolded protein-selective epitope, is provided by both the side chain dihedral angle distributions, and the Ramachandran, and distributions for the backbone dihedral angles in the cyclic peptide, a proxy for an exposed epitope in the misfolded protein. Some angles have substantially different distributions than the corresponding distributions in native TDP-43.
The side-chain and backbone dihedral distributions were examined for the four residues T115, T116, E117, and Q118. Percent overlap of distribution e.g. “native” in distribution “cyclic” is obtained by dividing the angles into elements of 5°, then decreasing a cutoff in probability amplitude from infinity, until 90% of the cyclic distribution is above the cutoff, and 10% remains below. This defines one or more regions in the allowable angles. Percent of the native distribution within this region was then found. The recipe is non-reciprocal and generally yields different numbers between pairs of distributions. The average of the overlaps, e.g. average of both native in cyclic and cyclic in native, is generally considered.
As shown in
According to
From the dihedral distributions shown in
The accumulation of relatively small differences in individual dihedral angles can result in a large and significant difference in global conformation of the peptide, and thus significant deviations in the structural alignment, as described further below.
Based on
Table 1B is derived from the numbers in Table 1A. The numbers in Table 1B show that the average overlap of the dihedral angle distributions between the cyclic and native forms is less than the average overlap of the dihedral angle distributions between the cyclic and biased forms, specifically for residues T115 and T116. This implies that residues T115 and T116 may be residues which confer the largest conformational selectivity against the native form.
Based on the data shown in
The backbone orientation that the epitope exposes to an antibody differs depending on whether the peptide is in the cyclic, biased, or native form. This discrepancy can be quantified by plotting the Ramachandran angles phi and psi (or ϕ and ψ), along the backbone, for each residue T115, T116, E117 and Q118 in the above 3 ensembles.
The overlap of the Ramachandran angle distributions, as defined above, is given in Table 3A. Table 3A shows the overlap probabilities of Ramachandran angles of the residues T115, T116, E117, and Q118 presented in
Table 3B shows the overlap probabilities, averaged over both Ramachandran angles, and averaged for example in column 2 for cyclic in native, and native in cyclic; likewise column 3 averages over phi, psi, cyclic in biased and biased in cyclic of Table 3A, to achieve a net overlap percentage for a given residue Table 3B shows that the Ramachandran angles of T115 and T116 confer the greatest conformational selectivity against the native form, and towards biased, misfolded forms.
Table 4A gives the peak (most-likely) values of the Ramachandran Cy angles plotted in
Table 5 shows the SASA of residues T115, T116, E117, and Q118 in the context of the cyclic cyclo(CGGTTEQGG) (SEQ ID NO: 2) ensemble, the biased ensemble, and the native ensemble.
Definitive evidence that the sequence TTEQ (SEQ ID No: 1) displays a different conformation in the context of the cyclic peptide than in the native ensemble can be seen by using standard structural alignment metrics between conformations, and then implementing clustering analysis. Equilibrium ensembles of conformations are obtained for the native RRM1 (PDB 41UF), biased RRM1, and cyclic peptides cyclo(CGGTTEQGG) (SEQ ID No: 2). Snapshots of conformations from these ensembles for residues TTEQ (SEQ ID NO: 1) are collected and then structurally aligned to the centroids of the largest cluster of the cyclic peptide ensemble, the largest cluster of the native ensemble, and the largest cluster of TTEQ (SEQ ID NO: 1) in the biased ensemble; the three values of the root mean squared deviation (RMSD) are then recorded and plotted. The clustering is performed here by the maxcluster algorithm. The 3 corresponding RMSD values for the cyclic, biased, and native ensembles are plotted as a 3-dimensional scatter plot in
The other number that is important is the overlap between either the cyclic or linear structural ensembles with the native structural ensemble. This number should be low. The lower this number, the less that antibodies raised to the epitope scaffold will target the native structure. The numeric overlapping percentages are given in Table 6A. It is evident from
Table 6A gives the overlap percentages in the RMSD clustering scatter plot of the cyclic, biased, and native (41UF) peptide conformations, as presented in
The overlap of the cyclic peptide in the non-native biased ensemble gives the fraction of scaffolds in the equilibrium ensemble that accurately represent misfolded non-native states. Likewise, the overlap of the cyclic peptide in the native biased ensemble gives the fraction of scaffolds in the equilibrium ensemble that accurately represent native states. The overlap of the non-native biased ensemble in the cyclic peptide ensemble gives the similarity of non-native misfolded states to the cyclic ensemble, and likewise, the overlap of the native biased ensemble in the cyclic peptide ensemble gives the similarity of native misfolded states to the cyclic ensemble. This latter quantity is not of particular interest, however the reciprocal overlaps between biased ensemble and cyclic ensemble are both measures quantifying the appropriateness of using the cyclic scaffold. In Table 6B we take an average of these two measures, divided by the cyclic in native ensemble overlap as a measure of the goodness in using a given cyclic scaffold to target non-native misfolded states vs. the native to provide a goodness ratio for cyclic peptides in targeting non-native misfolded conformations vs. native conformations. Column 1 gives the sequence, column 2 gives the average of the overlap of the cyclic peptide in the non-native biased ensemble and the overlap of the non-native biased ensemble in the cyclic peptide ensemble, Column 3 gives the overlap of the cyclic peptide in the native ensemble, Column 4 gives the difference of Column 2 and Column 3, and Column 5 gives the ratio of Column 2 divided by Column 3, which is defined as the goodness ratio for the cyclic peptide construct.
Table 6C lists the goodness ratio as defined in Table 6B—the ability of cyclic peptides to target non-native misfolded conformations vs. native conformations—for several candidate cyclic scaffolds as a function of several different cyclic radii, sorted in decreasing order of goodness ratio. Two cyclic scaffolds analyzed, constructs CGTTEQG (SEQ ID NO: 3) and CGGTTEQGG (SEQ ID NO: 2) have the largest goodness ratio, and so would be predicted to be the best proxies for non-native misfolded structures using these measures.
Table 6D lists the overlap percentage between the cyclic and linear conformational ensembles. The ensembles are different as seen by their small to moderate overlap percentage; this indicates that the cyclic peptide ensemble is conformationally distinct from the linear peptide ensemble, and also likely to be conformationally distinct from the nascent unfolded peptide chain.
The overlap between the ensembles was calculated as follows. The fraction (percent) of the biased ensemble that overlaps with the cyclic ensemble is obtained by first dividing the volume of this 3-dimensional RMSD space up into cubic elements of length 0.1 Angstrom. Then a “cutoff density” of points in the cyclic distribution is found such that the cubes with cyclic distribution density equal to or higher than the cutoff density contain 90% of the cyclic distribution. This defines a volume (which may be discontiguous) that gives the characteristic volume containing the cyclic distribution and removes any artifacts due to outliers. Then the fraction of points from the biased distribution that are within this region is found. With this method, it is possible to find the overlapping percentages for cyclic in native, cyclic in biased, etc.
A schematic of the atomic structure of the most-representative conformation of TTEQ (SEQ ID NO: 1) from the cyclic peptide ensemble of CGGTTEQGG (SEQ ID NO: 2), constituting the centroid of the largest cluster from the cyclic peptide ensemble of structures, is shown in
Table 7 lists values of the Ramachandran backbone and side chain dihedral angles occupied by residues T115, T116, E117, and Q118 in the centroid structure of the cyclic CGGTTEQGG (SEQ ID NO: 2) peptide ensemble, the centroid structure of the native ensemble, and the centroid structure of the non-native biased ensemble; cyclic and native centroid conformations are plotted in
A similar set of experiments were performed for TTE in the cyclic compounds CGTTEG (SEQ ID NO: 28) and CGTTEGG (SEQ ID NO: 29).
RMSD was analysed as in Example 1 for TTE cyclic compounds. Clustering plots by root mean squared deviation (RMSD) are shown in
Table 8A shows the overlapping percentage of the RMSD clustering between the cyclic in native, cyclic in non-native biased, and non-native biased in cyclic forms of the cyclopeptides as presented in
Additional computational simulations were performed to measure the similarity bewen various ensembles by measuring the Jensen-Shannon distance [Lindorff-Larsen K, Ferkinghoff-Borg J (2009) Similarity Measures for Protein Ensembles. PLOS ONE 4(1): e4203] between the cyclic-native, cyclic-biased, and cyclic-(unfolded monomer) ensemble pairs. Applying weights to these measurement criteria along with uncertainties in the weights allows the use of multiple criteria decision-making analysis [Hwang C L, Yoon K. Multiple attribute decision making: methods and applications a state-of-the-art survey. vol. 186. Springer Science & Business Media; (2012)] to select viable candidates that differentiate misfolded/biased TDP-43 from native TDP-43 as well as pre-folded TDP-43 monomer (i.e. transient unfolded TDP-43 monomer). A sequence of length 77 residues in RRM1 was used to model an unfolded monomer of the RRM1 domain, starting from amino acid 103 and ending at amino acid 179. The analysis was performed and used to assess the similarity of TTE and TTEQ (SEQ ID NO:1) epitopes in the context of biased TDP-43 RRM1 domain and various cyclic peptides comprising said epitopes, in the context of different scaffolds consisting of variable numbers of glycine spacers. The ensemble overlap was also measured, for the same simulation data, in terms of the number of standard deviations between two separated Gaussian distributions. All of the ensemble overlap data is shown in Table 8B.
Cyclic peptides which overlapped significantly with TTE and TTEQ (SEQ ID NO:1) epitopes in the context of biased TDP-43 RRM1 domain as measured by the JSD data in Table 8B, but had small overlap with either natively folded TDP-43 or transient unfolded TDP-43, included scaffolds (linker) with up to 9 amino acids (SEQ ID NO: 35) and are given in Table 8C.
Peptides comprising TTEQ (SEQ ID NO: 1) or TTE such as cyclo (CGTTEQG) (SEQ ID NO:3) or cyclo(CGTTEG) (SEQ ID NO: 28) can be cyclized head to tail.
A native ensemble comprising TTEQ (SEQ ID NO:1) or TTE and a linker, preferably comprising 2, 3, or 4 amino acids and/or PEG units, can be synthesized using known methods such as Fmoc based solid phase peptide synthesis alone or in combination with other methods. PEG molecules can be coupled to amine groups at the N terminus for example using coupling chemistries described in Hamley 2014 [7] and Roberts et al. 2012 [8], each incorporated herein by reference. The native ensemble compound may be cyclized by covalently bonding 1) the amino terminus and the carboxy terminus of the peptide+linker to form a peptide bond (e.g. cyclizing the backbone), 2) the amino or carboxy terminus with a side chain in the peptide+linker or 3) two side chains in the peptide+linker.
The bonds in the cyclic compound may be all regular peptide bonds (homodetic cyclic peptide) or include other types of bonds such as ester, ether, amide or disulfide linkages (heterodetic cyclic peptide).
Peptides may be cyclized by oxidation of thiol- or mercaptan-containing residues at the N-terminus or C-terminus, or internal to the peptide, including for example cysteine and homocysteine. For example two cysteine residues flanking the peptide may be oxidized to form a disulphide bond. Oxidative reagents that may be employed include, for example, oxygen (air), dimethyl sulphoxide, oxidized glutathione, cystine, copper (II) chloride, potassium ferricyanide, thallium (III) trifluro acetate or other oxidative reagents such as may be known to those of skill in the art and used with such methods as are known to those of skill in the art.
Methods and compositions related to cyclic peptide synthesis are described in US Patent Publication 2009/0215172. US Patent publication 2010/0240865, US Patent Publication 2010/0137559 and U.S. Pat. No. 7,569,541 describe various methods for cyclization. Other examples are described in PCT Publication WO01/92466, and Andreu et al., 1994. Methods in Molecular Biology 35:91-169.
More specifically, a cyclic peptide comprising the TTEQ (SEQ ID NO: 1) epitope, TEQ epitope or TTE epitope can be constructed by adding a linker comprising a spacer with cysteine residues flanking and/or inserted in the spacer. The peptide can be structured into a cyclic conformation by creating a disulfide linkage between the non-native cysteines residues added to the N- and C-termini of the peptide. It can also be synthesized into a cyclic compound by forming a peptide bond between the N- and C-termini amino acids (e.g. head to tail cyclization).
Peptide synthesis is performed by CPC Scientific Inc. (Sunnyvale Calif., USA) following standard manufacturing procedures. The structure of the cyclic peptide was designed to mimic the conformation and orientation of the amino acid backbone and side chains of TTEQ (SEQ ID NO: 1) in misfolded TDP-43 polypeptide.
Cyclo (CGTTEQG) (SEQ ID NO: 3)Cyclo (CGTTEQG) (SEQ ID NO: 3) and cyclo (CGGTTEQGG) (SEQ ID NO: 2) may be synthesized using the following method (CPC Scientific Inc, Sunnyvale Calif.). The protected native ensemble was synthesized by standard conventional Fmoc-based solid-phase peptide synthesis on 2-chlorotrityl chloride resin, followed by cleavage from the resin with 30% HFIP/DCM. Protected native ensemble was cyclized to the corresponding protected cyclic peptide by using EDC. HCl/HOBt/DIEA in DMF at low concentration. The protected cyclic peptide was deprotected by TFA to give crude cyclic peptide and the crude peptide was purified by RP HPLC to give pure cyclic peptide after lyophilize.
Cyclo (CGTTEQG) (SEQ ID NO: 3) and cyclo (CGGTTEQGG) (SEQ ID NO: 2) can be prepared by amide condensation of the linear peptide CGTTEQG (SEQ ID NO: 3) or CGGTTEQGG (SEQ ID NO: 2) respectively.
Cyclo (C-PEG2-TTEQG) (SEQ ID NO: 24) and cyclo (C-PEG2-TTEQGG) (SEQ ID NO: 25) can be prepared by amide condensation of the linear compound C-PEG2-TTEQG (SEQ ID NO: 24) or C-PEG2-TTEQGG (SEQ ID NO: 25) respectively.
Cyclo (CGTTEQ-PEG2) (SEQ ID NO: 26) and cyclo (CGGTTEQ-PEG2) (SEQ ID NO: 27) can be prepared by amide condensation of the linear compound CGTTEQ-PEG2 (SEQ ID NO: 26) or CGGTTEQ-PEG2 (SEQ ID NO: 27) respectively.
Linear (CGTTEQG) (SEQ ID NO: 3) or Linear (CGGTTEQGG) (SEQ ID NO: 2) may be prepared (CPC Scientific Inc, Sunnyvale Calif.). The protected native ensemble was synthesized by standard conventional Fmoc-based solid-phase peptide synthesis on Fmoc-Gly-Wang resin, then the protected peptide was cleaved by TFA to give crudepeptide and the crude peptide was purified by RP HPLC to give pure peptide after lyophilize, and which was used to conjugate BSA.
Similarly, cyclo (CGTTEG) (SEQ ID NO: 28) and Cyclo(CGTTEGG) (SEQ ID NO: 29) can be prepared by amide condensation of the linear peptide CGTTEG (SEQ ID NO: 28) or CGTTEGG (SEQ ID NO: 29) respectively.
Cyclo(C-PEG2-TTEG) (SEQ ID NO: 36) and Cyclo(C-PEG2-TTEGG) (SEQ ID NO: 37) can be prepared by amide condensation of the linear compound C-PEG2-TTEG (SEQ ID NO: 36) or C-PEG2-TTEGG (SEQ ID NO: 37) respectively.
Cyclo(CGTTE-PEG2) (SEQ ID NO: 38) and Cyclo(CGGTTE-PEG2) (SEQ ID NO: 39) can be prepared by amide condensation of the linear compound CGTTE-PEG2 (SEQ ID NO: 38) or CGGTTE-PEG2 (SEQ ID NO: 39) respectively.
Cyclic and Linear (CGTTEG) (SEQ ID NO: 28) or Linear (CGGTTEGG) (SEQ ID NO: 30) peptides may be prepared (CPC Scientific Inc, Sunnyvale Calif.). The protected was synthesized by standard conventional Fmoc-based solid-phase peptide synthesis on Fmoc-Gly-Wang resin, then the protected peptide was cleaved by TFA to give crudepeptide and the crude peptide was purified by RP HPLC to give pure peptide after lyophilize, and which was used to conjugate BSA.
Immunogen ConstructionThe cyclic compounds cyclo (CGTTEQG) (SEQ ID NO: 3), cyclo (CGGTTEQGG) (SEQ ID NO: 2), cyclo (CGTTEG) (SEQ ID NO: 28) and cyclo (CGTTEGG) (SEQ ID NO: 29) are synthesized as described above and then conjugated to KLH (for immunizing) or BSA (for screening) (CPC Scientific Inc, Sunnyvale Calif.). BSA or KLH is re-activated by SMCC in PBS buffer, then a solution of the pure peptide in PBS buffer is added to the conjugation mixture, the conjugation mixture is stirred at room temperature for 2 h. Then the conjugation mixture is lyophilized after dialysis to give the conjugation product.
Peptides can also be conjugated to KLH (for immunizing) and BSA (for screening) using a trifluoroacetate counter ion protocol. Peptides are desalted and checked by MS and HPLC to confirm at least 95% pure.
Example 4 Antibody Generation and SelectionA conformationally constrained compound optionally a cyclic compound such as a cyclic peptide comprising TTEQ (SEQ ID NO: 1) or TTE such as cyclo (CGTTEQG) (SEQ ID NO: 3), cyclo(CGGTTEQGG) (SEQ ID NO: 2), cyclo (CGTTEG) (SEQ ID NO: 28) or cyclo (CGTTEGG) (SEQ ID NO: 29) is linked to Keyhole Limpet Hemocyanin (KLH). The linked peptide or peptides are sent to Immunoprecise Antibodies LTD (Victoria BC, Canada) for mouse monoclonal antibody production, following protocols approved by the Canadian Council on Animal Care.
ImmunizationBriefly, female BALB/c mice (Charles River Laboratories, Quebec) are immunized. A series of subcutaneous aqueous injections containing antigen but no adjuvant are given over a period of 19 days. Mice are immunized with 100 μg per mouse per injection of a 0.5 mg/mL solution in sterile saline of cyclic peptide-KLH. All mice are euthanized on Day 19 and lymphocytes are harvested for hybridoma cell line generation.
Fusion/Hybridoma DevelopmentLymphocytes are isolated and fused with murine SP2/0 myeloma cells in the presence of poly-ethylene glycol (PEG 1500). Fused cells are cultured using HAT selection. This method uses a semi-solid methylcellulose-based HAT selective medium to combine the hybridoma selection and cloning into one step. Single cell-derived hybridomas grow to form monoclonal colonies on the semi-solid media. Approximately 10 days after the fusion event, resulting hybridoma clones are transferred to 96-well tissue culture plates and grown in HT containing medium until mid-log growth is reached (approximately 5 days).
Hybridoma Analysis (Screening)Tissue culture supernatants from the hybridomas can be tested by indirect ELISA on screening antigen (cyclic or linear peptide-BSA) and probed for both IgG and IgM antibodies using a Goat anti-IgG/IgM(H&L)-HRP secondary and developed with TMB substrate.
Clones >0.2 OD in this assay are taken to the next round of testing. Positive cultures are retested on screening antigen to confirm secretion and on an irrelevant antigen (Human Transferrin). Clones of interest are isotyped by antibody trapping ELISA to determine if they are IgG or IgM isotype and can be tested by indirect ELISA on other cyclic peptide-BSA conjugates as well as native-BSA conjugates to evaluate cross-reactivity.
Positive IgG-secreting clones are subjected to large-scale production.
Direct Binding AssaysBinding of clones to linear and cyclic peptides (conjugated to BSA) can be examined by surface plasmon resonance using a Biacore™ 3000 instrument (GE Healthcare).
Binding analysis is carried out using a high density (at least 1000 response units (RU)) of antigen immobilized on flow cells. Dilutions of a selected clone are sequentially injected over the surface to assess binding.
For affinity kinetics and specificity analysis, a conformational peptide comprising TTEQ (SEQ ID NO: 1) or TTE and BSA, for example a cyclic peptide having the sequence of SEQ ID NO:2, 3 28 or 29 conjugated to BSA via amine conjugation, are immobilized at low densities (50-100 RU) on adjacent flow cells. Serial 2-fold dilutions of a selected clone (4.7 nM to 75 nM) are then sequentially injected over the surfaces at 60 μl/minute for 3 minutes, followed by a dissociation phase. Following a double-reference subtraction, the sensorgrams are fitted to a Langmuir 1:1 binding model. Up to three separate analyses are performed on 3 consecutive days using the same sensorchip and the same conditions.
Binding analysis can be carried out also using Molecular Affinity Screening System (MASS-1) (Sierra Sensors GmbH, Hamburg, Germany). MASS-1 is a Surface Plasmon Resonance (SPR) Imaging analytical biosensor that employs high intensity laser light and high speed optical scanning to monitor binding interactions in real time. The peptide-BSA conjugates are covalently immobilized on separate flow cells of a High Amine Capacity (HAC) sensor chip, using standard amine-coupling chemistry, and unreacted sites blocked. Adjacent flow cells are similarly immobilized with BSA as a reference control surface.
Tissue culture supernatants are screened for the presence of antibody binding against their cognate cyclic peptide. Each sample is diluted and injected in duplicate over the immobilized peptide and BSA reference surfaces for 2 minutes, followed by injection of running buffer only for a 5-min dissociation phase. After every analytical cycle, the sensor chip surfaces are regenerated. Sensorgrams are double-referenced by subtracting out binding from the BSA reference surfaces and blank running buffer injections, and binding response report points measured at 20 seconds before the end of the injection in a 20 second window of data.
IsotypingThe hybridoma antibodies are isotyped using antibody trap experiments. Trap plates are coated with 1:10,000 Goat anti-mouse IgG/IgM(H&L) antibody at 100 uL/well carbonate coating buffer pH9.6 overnight at 4 C. Primary antibody (hybridoma supernatants) is added at 100 ug/mL. Secondary Antibody is added at 1:5,000. Goat anti-mouse IgGy-HRP or 1:10,000 Goat anti-mouse IgMp-HRP is added at 100 uL/well in PBS-Tween for 1 hour at 37 C with shaking. All washing steps are performed for 30 mins with PBS-Tween. The substrate TMB is added at 50 uL/well, developed in the dark and stopped with equal volume 1M HCl.
Example 5Monoclonal antibodies were made using the methods in Example 4. Specifically cyclo(CGGTTEQGG) (SEQ ID NO: 2) conjugated to KLH or cyclo(CGTTEQG) (SEQ ID NO:3) conjugated to KLH via the cysteine residues were injected into mice and monoclonal antibodies were generated.
Antibody-containing hybridoma tissue culture supernatants were typed for immunoglobulin type and screened against negative control peptide and BSA. IgG producing clones that did not bind the negative control peptide or BSA were tested by ELISA for binding to the immunizing cyclic peptides, to corresponding linear peptides and related cyclic peptides cyclo(CGGTTEGGG) (SEQ ID NO: 32), cyclo (CGTTEGG) (SEQ ID NO: 29) as described below.
ELISA Conditions:ELISA plates were coated with 0.1 μg/well with peptide cyclo(CGGTTEQGG) (SEQ ID NO: 2) or cyclo(CGTTEQG) (SEQ ID NO: 3) or the linear versions thereof at 100 μL/well in carbonate Coating Buffer (pH 9.6) overnight at 4 C. In other assays, cyclo(CGGTTEGGG) (SEQ ID NO: 32) or cyclo (CGTTEGG) (SEQ ID NO: 29) were also used to coat the ELISA plates.
The plates were blocked with 3% skim milk powder in PBS for 1 hour at room temperature. The primary antibody, hybridoma tissue culture supernatant neat at 100 uL/well, was incubated for 1 hour at 37 C with shaking. The secondary antibody, goat anti-mouse IgGy-HRP at a 1:10,000 dilution was added at 100 uL/well in PBS-Tween and incubated for 1 hour at 37 C with shaking. All washing steps were performed for 30 mins with PBS-Tween. TMB Substrate was added at 50 uL/well, developed in the dark and stopped with equal volume 1M HCl. The development time was 1-2 min and the plate was read at 450 nm.
The ELISA results are presented in Table 9 A and B for which shows the binding affinity of monoclonal antibodies generated against the immunizing peptide versus the corresponding linear peptide. As shown therein, IgG antibodies were produced that showed preferential binding to the immunizing cyclic peptides compared to the linear peptide.
In addition antibodies were tested for binding to related cyclic peptides cyclo(CGGTTEGGG) (SEQ ID NO: 32) and cyclo (CGTTEGG) (SEQ ID NO: 29) comprising the TTE epitope. Fifteen of 17 antibodies, had greater affinity for a related cyclic peptide compared to the linear peptide corresponding to the immunogen and 14 of the 17 antibodies tested had about a 2 fold or greater selectivity for a related cyclic peptide compared to the linear peptide corresponding to the immunogen.
Example 6 Misfolded TDP-43 CharacterizationAntibodies will be tested for their ability to bind native TDP-43 polypeptide as well as misfolded TDP-43 polypeptide using surface plasmon resonance and immunohistochemistry.
Surface Plasmon Resonance (Biacore) of Biological Samples.Homogenization: Human and mouse neurological tissue samples are weighed and subsequently submersed in a volume of fresh, ice cold TBS (supplemented with 5 mM EGTA, 5 mM EDTA, (both from Sigma) and EDTA-free protease inhibitor cocktail from Roche Diagnostics, Laval QC, Canada) such that the final concentration of tissue is 20% (w/v). Tissue is homogenized in this buffer using a mechanical probe homogenizer (3×30 sec pulses with 30 sec pauses in between, all performed on ice). TBS homogenized samples are then subjected to ultracentrifugation (70,000×g for 90 min). Supernatants are collected, aliquoted and stored at −80° C. The protein concentration of TBS homogenates is determined using a BCA protein assay (Pierce Biotechnology Inc, Rockford Ill., USA).
Surface Plasmon Resonance Analysis: CSF samples and neurological tissue samples from ALS patients and/or FTD patients and age-matched controls are analyzed. Test antibodies and an IgG1 isotype control are directly immobilized at high densities (10,000 RU) on 2 separate flow cells of a sensor chip. Using a Biacore 3000, diluted neurological tissue samples, homogenized in TBS are injected sequentially over the surfaces for 300 seconds, followed by 150 seconds of dissociation in buffer and surface regeneration. Binding responses are double-referenced by subtraction of IgG1 reference surface binding and normalized with assay buffer, and the different groups of samples compared.
Example 7Human embryonic kidney cells (HEK293FT) were transiently transfected with a modified form of TDP-43 with an HA-tag and lacking a nuclear localization signal (TDP-43ΔNLS). This form of the protein accumulates in the cytoplasm and forms misfolded aggregates. Empty vector was used as a negative control.
The cells were stained overnight at 4° C. with 1 μg/ml polyclonal rabbit antibody against the HA tag (to detect misfolded cytoplasmic TDP-43) or with 10 μg/ml of test antibody. Bound antibodies were then detected by staining for 1 hour at room temperature with fluorescently-labeled anti-rabbit (red Alexa Fluor 647) or anti-mouse (green Alexa Fluor 488) secondary antibody. Nuclear DNA was stained with Hoeschst 33342 dye (blue). Micrographs were acquired on a confocal Leica SP8 microscope in a Z-stack fashion with 0.3 μm steps. Included images represent a Z-plane projection of the entire cell thickness. Z-stacked images (20-30 steps) of single fluorescence channels and merged signals were captured.
The results are presented in
Human spinal cord homogenates were prepared from control and sporadic ALS patients and were analysed by dot blot with selected antibodies.
Tissue from a human without ALS was used as the control and two different sporadic ALS tissue samples were assessed. One was a tissue sample obtained from a patient carrying the C90rf72 mutation and another was a tissue sample obtained from a patient with unknown mutation status.
PVDF membranes were dotted with 10 micrograms of homogenate in duplicate and the test antibodies and control antibody (mIgG1) were used at a 500 fold dilution of 2 microgram/microliter stock solution. Rabbit polyclonal TDP-43 antibody (ProteinTech, Rosemont Ill.) was used as a positive control.
As shown in
Selective staining of sALS samples was seen using the test antibodies. Antibody 1 is clone IG10, antibody 2 is clone 2H10, antibody 3 is clone 11F3, antibody 4, is clone 3H5, antibody 5 is 4G5 and antibody 6 is clone 9C5. Antibody 6C5 also showed selective staining of sALS samples.
Example 9Several of the antibodies positive for binding aggregated TDP-43 were sequenced. Immunoglobulin gene transcripts expressed by the hybridomas were amplified with sets of proprietary primers from cDNA generated from the hybridoma cells using standard RT-PCR protocol and sequenced using a standard dye-terminator capillary sequencing method (Immunoprecise, Victoria BC Canada).
Complementarity determining regions (CDRs) CDR1, CDR2 and CDR3 are highlighted are bolded and underlined, and were identified according to IgBLAST (available using ncbi tools (Ye et al., Nucelic Acids Research 2013, Vol. 41, Web Server Issue doi:10:1093|nar|gkt382). Using this tool a query is searched with BLAST against the IMGT or NCBI germline V gene database (the sequences in such databases have been pre-annotated for the FR/CDR boundaries). The top database sequence hit is used to map the pre-annotated FR/CDR boundary information to the query sequence. The BLAST search parameters are Expect cut-off, 20; word size 9; mismatch penalty, −1; Dust filtering, off.
Two heavy chains were identified for antibody 2H10 which are labelled VH1 and VH2.
Whether one or both heavy chains produce antibodies selective for its cyclic peptide immunogen, can be confirmed by expressing each heavy chain (or variable region) with the light chain (or variable region) and assessing target binding.
Antibody 2H10-VH1 heavy chain alignment is more closely related to the heavy chains for 6C5 and 9C5 compared to 2H10-V2.
Example 10 Immunofluorescent Staining of TDP-43 Using Antibody 9C5Misfolded TDP-43 was recombinantly expressed using the construct and protocol described in Example 7 with the following modifications to reduce background. The primary and secondary antibodies were spun for 10 min at 10000 RPM prior to use, and the sections were blocked with 10% NGS for 1 hour at RT prior to primary addition. DAPI stain is used to identify the nucleus.
As shown in
While the present application has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the application is not limited to the disclosed examples. To the contrary, the application is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Specifically, the sequences associated with each accession numbers provided herein including for example accession numbers and/or biomarker sequences (e.g. protein and/or nucleic acid) provided in the Tables or elsewhere, are incorporated by reference in its entirely.
The scope of the claims should not be limited by the preferred embodiments and examples, but should be given the broadest interpretation consistent with the description as a whole.
CITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATION
- [1] Kuo P H, Chiang C H, Wnag Y T, Doudeva L G, Yuan H S, The Crystal Structure of TDP-43 RRM1-DNA Complex Reveals the Specific Recognition for UG- and TG-Rich Nucleic Acids. Nucleic Acids Res., 2014, vol 42, 4712.
- [2] DOI: 10.2210/pdb1wf0/pdb (No publication).
- [3] Mompean, M., Romano, V., Pantoja-Uceda, D., Stuani, C., Baralle, F. E., Buratti, E., and Laurents, D. V. The TDP-43 N-Terminal Domain Structure at High Resolution. FEBS J., 2016, 283, 1242.
- [4] Arai, T., Hasegawa, M., Akiyama, H., Ikeda, K., Nonaka, T., Mori, H., Mann, D., Tsuchiya, K., Yoshida, M., Hashizume, Y., and Oda, T. TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochem. Biophys. Res. Commun., 2006, 351, 602-611.
- [5] Chantelle F. Sephton, Shannon K. Good, Stan Atkin, Colleen M. Dewey, Paul Mayer III, Joachim Herz, and Gang Yu J. Biol. Chem. 2010, vol. 285, No. 9, 6826-6834.
- [6] Abel, O., Powell, J. F., Andersen, P. M., and Al-Chalabi, A. Hum Mutat, 2012, 33:1345-51.
- [7] Hamley, I. W. PEG-Peptide Conjugates 2014; 15, 1543-1559; dx.doi.org/10.1021/bm500246w
- [8] Roberts, M J et al Chemistry for peptide and protein PEGylation 64: 116-127.
Claims
1. A cyclic compound comprising: a TDP-43 peptide comprising 1) TTE; 2) TTEQ (SEQ ID NO:1) or 3) TEQ and up to 6 TDP-43 contiguous residues, and a linker, wherein the linker is covalently coupled to the TDP-43 peptide N-terminus residue and the peptide C-terminus residue, wherein at least one amino acid in the TDP-43 peptide is an alternate conformation than T, E, and/or Q in a corresponding linear and/or native TDP-43.
2. The cyclic compound of claim 1, wherein the TDP-43 peptide is selected from TTEQ (SEQ ID NO: 1), TTE, TEQ, KTTE (SEQ ID NO: 10), KTTEQ (SEQ ID NO:12), TEQD (SEQ ID NO:8) or TTEQD (SEQ ID NO: 9) optionally wherein the cyclic compound is a compound selected from any one of SEQ ID NOs: 2, 3, 13, 22-39, and 42-44.
3. The cyclic compound of claim 1, wherein the TDP-43 peptide is TTEQ (SEQ ID NO: 1), TEQ or TTE.
4. The cyclic compound of claim 1, wherein the linker comprises or consists of 1-8 amino acids and/or one or more functionalizable moieties.
5. The cyclic compound of claim 4, wherein the linker amino acids are selected from A and G, and/or wherein the functionalizable moiety is C.
6. The cyclic compound of claim 1, wherein the linker comprises or consists of GGCGG (SEQ ID NO: 40), GCGG (SEQ ID NO: 41) or GCG.
7. The cyclic compound of claim 1, wherein the linker comprises one or more PEG molecules.
8. The cyclic compound of claim 1, wherein the cyclic compound has a sequence selected from any one of SEQ ID NOs: 2, 3, 13, 22-39, and 42-44.
9. An immunogen comprising a cyclic compound: comprising a TDP-43 peptide comprising 1) TTE; 2) TTEQ (SEQ ID NO:1) or 3) TEQ and up to 6 TDP-43 contiguous residues, and a linker, wherein the linker is covalently coupled to the TDP-43 peptide N-terminus residue and the peptide C-terminus residue, wherein at least one amino acid in the TDP-43 peptide is an alternate conformation than T, E, and/or Q in a corresponding linear and/or native TDP-43.
10. The immunogen of claim 9, wherein the cyclic compound, is coupled to a carrier protein or immunogenicity enhancing component and/or formulated with an adjuvant.
11. The immunogen of claim 10, wherein the carrier protein is bovine serum albumin (BSA) or the immunogenicity-enhancing component is keyhole limpet haemocyanin (KLH).
12. The immunogen of claim 11, wherein the adjuvant is selected from aluminum phosphate, aluminum hydroxide alum, monophosphoryl lipid A and QS21.
13.-41. (canceled)
42. A composition comprising the cyclic compound of claim 1 or an immunogen comprising the cyclic compound, optionally with a diluent.
43.-45. (canceled)
46. A kit comprising the cyclic compound of claim 1, an immunogen comprising the cyclic compound or a composition comprising the cyclic compound or immunogen.
47.-50. (canceled)
51. The cyclic compound of claim 1, wherein the cyclic compound has a sequence selected from any one of SEQ ID NOs: 2, 3, 22, 23, 28, 29, 30, 31, 32, 33, 34, 35 and 42.
52. The cyclic compound of claim 1, wherein the cyclic compound has a sequence selected from any one of SEQ ID NOs: 2, 3, 22, 23 and 42.
53. The immunogen of claim 9, wherein the TDP-43 peptide is selected from TTEQ (SEQ ID NO: 1), TTE, TEQ, KTTE (SEQ ID NO: 10), KTTEQ (SEQ ID NO:12), TEQD (SEQ ID NO:8) or TTEQD (SEQ ID NO: 9), optionally wherein the TDP-43 peptide is TTEQ (SEQ ID NO: 1), TEQ or TTE, and/or wherein the linker comprises or consists of 1-8 amino acids, optionally selected from A and G, and/or one or more functionalizable moieties, optionally selected from C.
54. The immunogen of claim 9, wherein the cyclic compound has a sequence selected from any one of SEQ ID NOs: 2, 3, 13, 22-39, and 42-44.
55. The immunogen of claim 9, wherein the cyclic compound has a sequence selected from any one of SEQ ID NOs: 2, 3, 22, 23, 28, 29, 30, 31, 32, 33, 34, 35 and 42.
56. The immunogen of claim 9, wherein the cyclic compound has a sequence selected from any one of SEQ ID NOs: 2, 3, 22, 23 and 42.
Type: Application
Filed: Dec 3, 2021
Publication Date: May 26, 2022
Inventors: Steven S. Plotkin (Vancouver), Neil R. Cashman (Vancouver), Xubiao Peng (Beijing)
Application Number: 17/541,812
